CRISPR plasmid construction All enzymes were obtained from NEB, unless otherwise noted. Vector maps were produced using SnapGene (www.Snapgene.com), and sequence views generated with Benchling (www.Benchling.com). Primers used in this study are listed in Table S2. Generation of pNOC-CRISPR-GFP: The Cas9 gene was PCR amplified from hCas9 (AddGene 41815) with the primers Cas9 NLS F+ and Cas9 linker R-, and GFP was amplified from pOCS-Ole-LDSP (3) with the primers GFP linker F+ and GFP NLS R- (Table S2). The vector backbone of pNOC 411 (containing the EF promoter (NannoCCMP1779_10181) and LDSP terminator (NannoCCMP1779_4188)) (2) was amplified with the primers 411 Cas9 F- and 411 GFP R+, and fused by Gibson Assembly (30) with Cas9 and GFP fragments to form pNOC-Cas9-GFP. A Hygromycin B resistance cassette, composed of the LDSP promoter, the HygR gene, and the 35S terminator was PCR amplified from pSELECT100 (3) with the primers LDSP NsiI F+ and LDSP BspQI R-. This PCR product and the vector pNOC-Cas9-GFP were digested with BspQI and NsiI and ligated to form pNOC-Cas9-GFP-HygC. The Ribi promoter (between NannoCCMP1779_9669 and NannoCCMP1779_9668) and the terminator of the N. oceanica cellulose synthase gene (CS, NannoCCMP1779_5780) were PCR amplified from genomic DNA with the primer pair Ribi CRISPR F+ and Ribi CRISPR R-, and CSterm sgRNA F+ and CSterm sgRNA R- respectively, and were combined with the XhoI/AgeI digested pNOC-Cas9-GFP backbone by Gibson Assembly, to form pNOC-stacked-Cas9-GFP. A smaller section of the CS terminator that did not contain BspQI sites was PCR amplified CSterm sgRNA F+ and CSterm sgRNA R2-, and used to replace the CS terminator. The gRNA scaffold with a HDV 3’ ribozyme sequence was synthesized (Integrated DNA Technologies) and placed into the ClaI and KpnI sites in pNOC-stacked-Cas9-GFP to form pNOC-CRISPR-GFP. Generation of pNOC-CRISPR: A C-terminal section of Cas9 was amplified with the addition of an HA tag-SV40 NLS by overlap extension PCR using the primers Cas9 3’ AscI F+, Cas9 3’ RE R1-, and Cas9 3’ RE R2-, and after digestion with AscI/MfeI ligated with AscI/MfeI digested pNOC-CRISPR-GFP to form pNOC-CRISPR-HA. N. oceanica codon optimized Nlux-HA was PCR amplified from pNOC-OX-Nlux (2) with primers Nlux HpaI F+ and Nlux NheI R-. This PCR product and the vector pNOC-CRISPR-HA were digested with HpaI/NheI and ligated to form pNOC-CRISPR. Generation of pNOC-ARS-CRISPR: The CEN/ARS region was PCR amplified from pDEST22 (Thermofisher) with the primers CEN/ARS F+ and CEN/ARS R-, and digested with AgeI. The vector pNOC-CRISPR was digested with AgeI, phosphatase (CIP) treated, and ligated with the ARS region to form the vector pNOC-ARS-CRISPR. Design and cloning of NR sgRNAs: Target sequences were chosen by identifying NGG motifs at the 5’ end of the NR coding sequence. The potential target sequences were submitted to the N. oceanica CCMP1779 BLAST (https://genome.jgi.doe.gov/pages/blast-query.jsf?db=Nanoce1779) to identify mismatches and nonspecific targets sharing PAM sequences were rejected. Corresponding guide sequences with a HH ribozyme sequence (21) were appended to the 5’ end of gRNA scaffold by PCR site directed mutagenesis. For this process a fragment of the pNOC-CRISPR including the gRNA scaffold, the CS terminator, the AmpR resistance gene and the origin of replication was amplified with the primers sgHH NR-1 ClaI F+ and sgHH NR-1 R-, and sgHH NR-2 ClaI F+ and sgHH NR-2 R-, for sgNR1 and sgNR2 respectively (Figures S2). These primers contain the HH ribozyme sequence that includes a section complementary to the guide sequence (Figure S2A). After PCR amplification, the product was ligated and cloned into E. coli to generate the sgHH-target plasmid (Figure S2B). The ClaI/KpnI fragment of sgHH-target was transferred to pNOC-ARS-CRISPR digested with ClaI/KpnI. Generation of pNOC-ARS-CRISPR-v2: This smaller vector was generated to allow HH-guide insertion on the gRNA scaffold using BspQI restriction sites. To remove the BspQI site originally present in the CEN/ARS region, the backbone of pDEST22 (Thermofisher) was amplified with the primers Crispr compact F+ and Crispr compact R-, religated, and amplified with CEN/ARS -bspq sdm F+ and CEN/ARS -bspq sdm R. This vector backbone and the CRISPR cassette (the Cas9 and sgRNA expression cassette under the control of the Ribi promoter, and CS and LDSP terminators) from pNOC-ARS-CRISPR were digested with NotI/AgeI and ligated together to form pNOC-ARS-CRISPR-compact. A 35S terminator was amplified from pSELECT100 (3) with the primers 35S terminator F+ and 35S terminator R- and digested with NotI/PmeI and ligated into pNOC-ARS-CRISPR-compact NotI/PmeI sites to form pNOC-ARS-CRISPR-35S. The Hygromycin B resistance cassette from pNOC-ARS-CRISPR, that includes the LDSP promoter and HygR gene, was amplified with the primers LDSPpro F+ and HygR R-, digested with NotI/NheI and ligated into the NotI/SpeI sites of pNOC-ARS-CRISPR-35S to form pNOC-ARS-CRISPR-v2. LOCUS Exported 9566 bp ds-DNA circular SYN 10-JUL-2017 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS pNOC-Cas9-GFP SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 9566) AUTHORS . TITLE Direct Submission JOURNAL Exported Thursday, Jul 13, 2017 from SnapGene Viewer 3.3.4 http://www.snapgene.com FEATURES Location/Qualifiers source 1..9566 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 110..1059 /label=EF promoter CDS 1073..1093 /codon_start=1 /product="nuclear localization signal of SV40 large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 1094..5194 /codon_start=1 /product="Cas9 (Csn1) endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system" /label=Cas9 /note="generates RNA-guided double strand breaks in DNA" /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD ATLIHQSITGLYETRIDLSQLGGD" CDS 5225..5941 /codon_start=1 /product="enhanced GFP" /label=EGFP /note="mammalian codon-optimized" /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTFTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITHGMDELYK" CDS 5942..5962 /codon_start=1 /product="nuclear localization signal of SV40 large T antigen" /label=SV40 NLS /translation="PKKKRKV" terminator 5994..6593 /label=LDSP terminator promoter complement(6691..6709) /label=SP6 promoter /note="promoter for bacteriophage SP6 RNA polymerase" primer_bind complement(6727..6743) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" protein_bind 6751..6767 /label=lac operator /bound_moiety="lac repressor encoded by lacI" /note="The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-beta-D-thiogalactopyranoside (IPTG)." promoter complement(6775..6805) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 6820..6841 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." rep_origin complement(7129..7717) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" CDS complement(7888..8748) /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" promoter complement(8749..8853) /gene="bla" /label=AmpR promoter rep_origin complement(8931..9386) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind 9527..9543 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" ORIGIN 1 gggcgaattg ggcccgacgt cgcatgctcc cggccgccat ggcggccgcg ggaattcgat 61 atcactagag tatagctaca tggtagctag tagtcgtcaa aggaaataaa gacatagcgt 121 aaagtataca aaatgacttc cgcggcgcgg gagcggcagg cagcacttgc aaaacgaagc 181 ccaatgggcc agattacggt caacctcgtc attttattac ggtcgtcagc aagagcggtt 241 tagaccgtcg ccggcacggg tggctcccga tattcttggc tttcgtgggt ggaggcggcg 301 tgtgactcct catctttaag tagtggtcgc ttttgctgca tgcttttgat cacctcgtga 361 ttgatggctg gattctcagc tgtcttctga catctttggg tgcatgggtt gaccggtgtc 421 gcgggaggca gaccctgttc cgtggaggtg tgtggctgcg gatgcggcga cacggaagaa 481 gggcgtgcat cacttctctc ttagtcgatt tacgatgcgc ttctgcgcac agctcattca 541 cccacccgcc accactcctc ttgcacatgt atgcaggtgt cgggccaagt tagtcgcgcg 601 ctgagtggcc cgaaccccgc atgtggtagt ggaatgcgtg gtcggcagaa gccttgagca 661 cagcgaaaga agaaaacaag ataagcaggg gatggctgcg gtttgcgcgt gcctttgccg 721 cgcagcccgt cacaacaata cgtatccctc atgggaatta cactcacctg tttcggcgcg 781 caggtgggac atgcctcgtt cagcatgaga cgcaaggctt ctcgttgttg ggccggacgg 841 cggcgtggct tctttgctgc actgccggcg ctgcaccacc tcgccctgcc agccaccggc 901 cagggtcgtg gcatcgatgg gcatgtacgc gcctcacgca cggcccgcgc cggcattttg 961 ttcataggac tttgataaac acctctggct cattctccct ctcctcctca cccatccacc 1021 acgagcacag cacccccacc cctcaaccct cacttcgtaa cactcgagca tgcccaagaa 1081 aaagcggaag gtggacaaga agtactccat tgggctcgat atcggcacaa acagcgtcgg 1141 ctgggccgtc attacggacg agtacaaggt gccgagcaaa aaattcaaag ttctgggcaa 1201 taccgatcgc cacagcataa agaagaacct cattggcgcc ctcctgttcg actccgggga 1261 gacggccgaa gccacgcggc tcaaaagaac agcacggcgc agatataccc gcagaaagaa 1321 tcggatctgc tacctgcagg agatctttag taatgagatg gctaaggtgg atgactcttt 1381 cttccatagg ctggaggagt cctttttggt ggaggaggat aaaaagcacg agcgccaccc 1441 aatctttggc aatatcgtgg acgaggtggc gtaccatgaa aagtacccaa ccatatatca 1501 tctgaggaag aagcttgtag acagtactga taaggctgac ttgcggttga tctatctcgc 1561 gctggcgcat atgatcaaat ttcggggaca cttcctcatc gagggggacc tgaacccaga 1621 caacagcgat gtcgacaaac tctttatcca actggttcag acttacaatc agcttttcga 1681 agagaacccg atcaacgcat ccggagttga cgccaaagca atcctgagcg ctaggctgtc 1741 caaatcccgg cggctcgaaa acctcatcgc acagctccct ggggagaaga agaacggcct 1801 gtttggtaat cttatcgccc tgtcactcgg gctgaccccc aactttaaat ctaacttcga 1861 cctggccgaa gatgccaagc ttcaactgag caaagacacc tacgatgatg atctcgacaa 1921 tctgctggcc cagatcggcg accagtacgc agaccttttt ttggcggcaa agaacctgtc 1981 agacgccatt ctgctgagtg atattctgcg agtgaacacg gagatcacca aagctccgct 2041 gagcgctagt atgatcaagc gctatgatga gcaccaccaa gacttgactt tgctgaaggc 2101 ccttgtcaga cagcaactgc ctgagaagta caaggaaatt ttcttcgatc agtctaaaaa 2161 tggctacgcc ggatacattg acggcggagc aagccaggag gaattttaca aatttattaa 2221 gcccatcttg gaaaaaatgg acggcaccga ggagctgctg gtaaagctta acagagaaga 2281 tctgttgcgc aaacagcgca ctttcgacaa tggaagcatc ccccaccaga ttcacctggg 2341 cgaactgcac gctatcctca ggcggcaaga ggatttctac ccctttttga aagataacag 2401 ggaaaagatt gagaaaatcc tcacatttcg gataccctac tatgtaggcc ccctcgcccg 2461 gggaaattcc agattcgcgt ggatgactcg caaatcagaa gagaccatca ctccctggaa 2521 cttcgaggaa gtcgtggata agggggcctc tgcccagtcc ttcatcgaaa ggatgactaa 2581 ctttgataaa aatctgccta acgaaaaggt gcttcctaaa cactctctgc tgtacgagta 2641 cttcacagtt tataacgagc tcaccaaggt caaatacgtc acagaaggga tgagaaagcc 2701 agcattcctg tctggagagc agaagaaagc tatcgtggac ctcctcttca agacgaaccg 2761 gaaagttacc gtgaaacagc tcaaagaaga ctatttcaaa aagattgaat gtttcgactc 2821 tgttgaaatc agcggagtgg aggatcgctt caacgcatcc ctgggaacgt atcacgatct 2881 cctgaaaatc attaaagaca aggacttcct ggacaatgag gagaacgagg acattcttga 2941 ggacattgtc ctcaccctta cgttgtttga agatagggag atgattgaag aacgcttgaa 3001 aacttacgct catctcttcg acgacaaagt catgaaacag ctcaagaggc gccgatatac 3061 aggatggggg cggctgtcaa gaaaactgat caatgggatc cgagacaagc agagtggaaa 3121 gacaatcctg gattttctta agtccgatgg atttgccaac cggaacttca tgcagttgat 3181 ccatgatgac tctctcacct ttaaggagga catccagaaa gcacaagttt ctggccaggg 3241 ggacagtctt cacgagcaca tcgctaatct tgcaggtagc ccagctatca aaaagggaat 3301 actgcagacc gttaaggtcg tggatgaact cgtcaaagta atgggaaggc ataagcccga 3361 gaatatcgtt atcgagatgg cccgagagaa ccaaactacc cagaagggac agaagaacag 3421 tagggaaagg atgaagagga ttgaagaggg tataaaagaa ctggggtccc aaatccttaa 3481 ggaacaccca gttgaaaaca cccagcttca gaatgagaag ctctacctgt actacctgca 3541 gaacggcagg gacatgtacg tggatcagga actggacatc aatcggctct ccgactacga 3601 cgtggatcat atcgtgcccc agtcttttct caaagatgat tctattgata ataaagtgtt 3661 gacaagatcc gataaaaata gagggaagag tgataacgtc ccctcagaag aagttgtcaa 3721 gaaaatgaaa aattattggc ggcagctgct gaacgccaaa ctgatcacac aacggaagtt 3781 cgataatctg actaaggctg aacgaggtgg cctgtctgag ttggataaag ccggcttcat 3841 caaaaggcag cttgttgaga cacgccagat caccaagcac gtggcccaaa ttctcgattc 3901 acgcatgaac accaagtacg atgaaaatga caaactgatt cgagaggtga aagttattac 3961 tctgaagtct aagctggtct cagatttcag aaaggacttt cagttttata aggtgagaga 4021 gatcaacaat taccaccatg cgcatgatgc ctacctgaat gcagtggtag gcactgcact 4081 tatcaaaaaa tatcccaagc ttgaatctga atttgtttac ggagactata aagtgtacga 4141 tgttaggaaa atgatcgcaa agtctgagca ggaaataggc aaggccaccg ctaagtactt 4201 cttttacagc aatattatga attttttcaa gaccgagatt acactggcca atggagagat 4261 tcggaagcga ccacttatcg aaacaaacgg agaaacagga gaaatcgtgt gggacaaggg 4321 tagggatttc gcgacagtcc ggaaggtcct gtccatgccg caggtgaaca tcgttaaaaa 4381 gaccgaagta cagaccggag gcttctccaa ggaaagtatc ctcccgaaaa ggaacagcga 4441 caagctgatc gcacgcaaaa aagattggga ccccaagaaa tacggcggat tcgattctcc 4501 tacagtcgct tacagtgtac tggttgtggc caaagtggag aaagggaagt ctaaaaaact 4561 caaaagcgtc aaggaactgc tgggcatcac aatcatggag cgatcaagct tcgaaaaaaa 4621 ccccatcgac tttctcgagg cgaaaggata taaagaggtc aaaaaagacc tcatcattaa 4681 gcttcccaag tactctctct ttgagcttga aaacggccgg aaacgaatgc tcgctagtgc 4741 gggcgagctg cagaaaggta acgagctggc actgccctct aaatacgtta atttcttgta 4801 tctggccagc cactatgaaa agctcaaagg gtctcccgaa gataatgagc agaagcagct 4861 gttcgtggaa caacacaaac actaccttga tgagatcatc gagcaaataa gcgaattctc 4921 caaaagagtg atcctcgccg acgctaacct cgataaggtg ctttctgctt acaataagca 4981 cagggataag cccatcaggg agcaggcaga aaacattatc cacttgttta ctctgaccaa 5041 cttgggcgcg cctgcagcct tcaagtactt cgacaccacc atagacagaa agcggtacac 5101 ctctacaaag gaggtcctgg acgccacact gattcatcag tcaattacgg ggctctatga 5161 aacaagaatc gacctctctc agctcggtgg agacagcagg gctgacgggt ccggcagcgg 5221 gtccatggtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct 5281 ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg gcgatgccac 5341 ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc 5401 caccctcgtg accaccttca cctacggcgt gcagtgcttc agccgctacc ccgaccacat 5461 gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat 5521 cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac 5581 cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg 5641 gcacaagctg gagtacaact acaacagcca caacgtctat atcatggccg acaagcagaa 5701 gaacggcatc aaggtgaact tcaagatccg ccacaacatc gaggacggca gcgtgcagct 5761 cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa 5821 ccactacctg agcacccagt ccgccctgag caaagacccc aacgagaagc gcgatcacat 5881 ggtcctgctg gagttcgtga ccgccgccgg gatcactcac ggcatggacg agctgtacaa 5941 gccgaaaaag aagaggaagg tctgacaatt ggaaagatcc aagagagacg agtagagatt 6001 ttttttttgg gattgatgtt tgtcgttctt tgagttgtcg tcgagttacg ccttttgtaa 6061 gaatgttccg caggagagga ggaggatggg catgagtgag ggtgagaggg cttgcccgct 6121 ttttttttta aaaacgctga agacgtggtt gtcaaacaaa ccccccatag aaacgatttt 6181 gttacggtgc ggtccagacg tcacttgaat ggctccgcgg aaaggccagg gagggaaggg 6241 gggagggagg aaacatgaaa catgttgaac ggctcaacag ggtttggggg acaagagagg 6301 tagcgccctg atggactgct ccctcccctc ctttccctca atgtctcatt catccatgct 6361 tcccccttct ctctctcccc tccgttccat cccccgcggg cgtggtagtg gcgtgatggg 6421 atccactaaa atgtacgtgt aagaaaagcc ggtgagctta cgcttttgtg aaagtgggag 6481 tacgagtgtt gtgtgtgtgt gtagtggttt cagaccccag acagaggcga agcagaaaaa 6541 gcagacgatg aagacgacga agaaatgagc agtctatttt tatcgtggaa acagaagagg 6601 tgatatcgtc tcctgcaggc atgcaagctg atccactaga ggccatggcg gccgcactag 6661 tggatccccc gggatgcata gcttgagtat tctatagtgt cacctaaata gcttggcgta 6721 atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat 6781 acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt 6841 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta 6901 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc 6961 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa 7021 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa 7081 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct 7141 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac 7201 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc 7261 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc 7321 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg 7381 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga 7441 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag 7501 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta 7561 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag 7621 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg 7681 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac 7741 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc 7801 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag 7861 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc 7921 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac 7981 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc 8041 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg 8101 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag 8161 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc 8221 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac 8281 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag 8341 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac 8401 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg 8461 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc 8521 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact 8581 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg 8641 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa 8701 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt 8761 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg 8821 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga 8881 tgcggtgtga aataccgcac agatgcgtaa ggagaaaata ccgcatcagg aaattgtaag 8941 cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca 9001 ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag 9061 tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg 9121 gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt 9181 tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag 9241 agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc 9301 gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc 9361 gcttaatgcg ccgctacagg gcgcgtccat tcgccattca ggctgcgcaa ctgttgggaa 9421 gggcgatcgg tgcgggcctc ttcgctatta cgccagctgg cgaaaggggg atgtgctgca 9481 aggcgattaa gttgggtaac gccagggttt tcccagtcac gacgttgtaa aacgacggcc 9541 agtgaattgt aatacgactc actata // LOCUS Exported 11345 bp ds-DNA circular SYN 10-JUL-2017 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS pNOC-Cas9-GFP-HygC SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 11345) AUTHORS . TITLE Direct Submission JOURNAL Exported Thursday, Jul 13, 2017 from SnapGene Viewer 3.3.4 http://www.snapgene.com FEATURES Location/Qualifiers source 1..11345 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 110..1059 /label=EF promoter CDS 1073..1093 /codon_start=1 /product="nuclear localization signal of SV40 large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 1094..5194 /codon_start=1 /product="Cas9 (Csn1) endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system" /label=Cas9 /note="generates RNA-guided double strand breaks in DNA" /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD ATLIHQSITGLYETRIDLSQLGGD" CDS 5225..5941 /codon_start=1 /product="enhanced GFP" /label=EGFP /note="mammalian codon-optimized" /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTFTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITHGMDELYK" CDS 5942..5962 /codon_start=1 /product="nuclear localization signal of SV40 large T antigen" /label=SV40 NLS /translation="PKKKRKV" terminator 5994..6593 /label=LDSP terminator promoter 6680..7449 /label=LDSP promoter CDS 7474..8499 /codon_start=1 /gene="aph(4)-Ia" /product="aminoglycoside phosphotransferase from E. coli" /label=HygR /note="confers resistance to hygromycin" /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK E" terminator 8500..8730 /label=35S terminator rep_origin complement(8908..9496) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" CDS complement(9667..10527) /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" promoter complement(10528..10632) /gene="bla" /label=AmpR promoter rep_origin complement(10710..11165) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind 11306..11322 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" ORIGIN 1 gggcgaattg ggcccgacgt cgcatgctcc cggccgccat ggcggccgcg ggaattcgat 61 atcactagag tatagctaca tggtagctag tagtcgtcaa aggaaataaa gacatagcgt 121 aaagtataca aaatgacttc cgcggcgcgg gagcggcagg cagcacttgc aaaacgaagc 181 ccaatgggcc agattacggt caacctcgtc attttattac ggtcgtcagc aagagcggtt 241 tagaccgtcg ccggcacggg tggctcccga tattcttggc tttcgtgggt ggaggcggcg 301 tgtgactcct catctttaag tagtggtcgc ttttgctgca tgcttttgat cacctcgtga 361 ttgatggctg gattctcagc tgtcttctga catctttggg tgcatgggtt gaccggtgtc 421 gcgggaggca gaccctgttc cgtggaggtg tgtggctgcg gatgcggcga cacggaagaa 481 gggcgtgcat cacttctctc ttagtcgatt tacgatgcgc ttctgcgcac agctcattca 541 cccacccgcc accactcctc ttgcacatgt atgcaggtgt cgggccaagt tagtcgcgcg 601 ctgagtggcc cgaaccccgc atgtggtagt ggaatgcgtg gtcggcagaa gccttgagca 661 cagcgaaaga agaaaacaag ataagcaggg gatggctgcg gtttgcgcgt gcctttgccg 721 cgcagcccgt cacaacaata cgtatccctc atgggaatta cactcacctg tttcggcgcg 781 caggtgggac atgcctcgtt cagcatgaga cgcaaggctt ctcgttgttg ggccggacgg 841 cggcgtggct tctttgctgc actgccggcg ctgcaccacc tcgccctgcc agccaccggc 901 cagggtcgtg gcatcgatgg gcatgtacgc gcctcacgca cggcccgcgc cggcattttg 961 ttcataggac tttgataaac acctctggct cattctccct ctcctcctca cccatccacc 1021 acgagcacag cacccccacc cctcaaccct cacttcgtaa cactcgagca tgcccaagaa 1081 aaagcggaag gtggacaaga agtactccat tgggctcgat atcggcacaa acagcgtcgg 1141 ctgggccgtc attacggacg agtacaaggt gccgagcaaa aaattcaaag ttctgggcaa 1201 taccgatcgc cacagcataa agaagaacct cattggcgcc ctcctgttcg actccgggga 1261 gacggccgaa gccacgcggc tcaaaagaac agcacggcgc agatataccc gcagaaagaa 1321 tcggatctgc tacctgcagg agatctttag taatgagatg gctaaggtgg atgactcttt 1381 cttccatagg ctggaggagt cctttttggt ggaggaggat aaaaagcacg agcgccaccc 1441 aatctttggc aatatcgtgg acgaggtggc gtaccatgaa aagtacccaa ccatatatca 1501 tctgaggaag aagcttgtag acagtactga taaggctgac ttgcggttga tctatctcgc 1561 gctggcgcat atgatcaaat ttcggggaca cttcctcatc gagggggacc tgaacccaga 1621 caacagcgat gtcgacaaac tctttatcca actggttcag acttacaatc agcttttcga 1681 agagaacccg atcaacgcat ccggagttga cgccaaagca atcctgagcg ctaggctgtc 1741 caaatcccgg cggctcgaaa acctcatcgc acagctccct ggggagaaga agaacggcct 1801 gtttggtaat cttatcgccc tgtcactcgg gctgaccccc aactttaaat ctaacttcga 1861 cctggccgaa gatgccaagc ttcaactgag caaagacacc tacgatgatg atctcgacaa 1921 tctgctggcc cagatcggcg accagtacgc agaccttttt ttggcggcaa agaacctgtc 1981 agacgccatt ctgctgagtg atattctgcg agtgaacacg gagatcacca aagctccgct 2041 gagcgctagt atgatcaagc gctatgatga gcaccaccaa gacttgactt tgctgaaggc 2101 ccttgtcaga cagcaactgc ctgagaagta caaggaaatt ttcttcgatc agtctaaaaa 2161 tggctacgcc ggatacattg acggcggagc aagccaggag gaattttaca aatttattaa 2221 gcccatcttg gaaaaaatgg acggcaccga ggagctgctg gtaaagctta acagagaaga 2281 tctgttgcgc aaacagcgca ctttcgacaa tggaagcatc ccccaccaga ttcacctggg 2341 cgaactgcac gctatcctca ggcggcaaga ggatttctac ccctttttga aagataacag 2401 ggaaaagatt gagaaaatcc tcacatttcg gataccctac tatgtaggcc ccctcgcccg 2461 gggaaattcc agattcgcgt ggatgactcg caaatcagaa gagaccatca ctccctggaa 2521 cttcgaggaa gtcgtggata agggggcctc tgcccagtcc ttcatcgaaa ggatgactaa 2581 ctttgataaa aatctgccta acgaaaaggt gcttcctaaa cactctctgc tgtacgagta 2641 cttcacagtt tataacgagc tcaccaaggt caaatacgtc acagaaggga tgagaaagcc 2701 agcattcctg tctggagagc agaagaaagc tatcgtggac ctcctcttca agacgaaccg 2761 gaaagttacc gtgaaacagc tcaaagaaga ctatttcaaa aagattgaat gtttcgactc 2821 tgttgaaatc agcggagtgg aggatcgctt caacgcatcc ctgggaacgt atcacgatct 2881 cctgaaaatc attaaagaca aggacttcct ggacaatgag gagaacgagg acattcttga 2941 ggacattgtc ctcaccctta cgttgtttga agatagggag atgattgaag aacgcttgaa 3001 aacttacgct catctcttcg acgacaaagt catgaaacag ctcaagaggc gccgatatac 3061 aggatggggg cggctgtcaa gaaaactgat caatgggatc cgagacaagc agagtggaaa 3121 gacaatcctg gattttctta agtccgatgg atttgccaac cggaacttca tgcagttgat 3181 ccatgatgac tctctcacct ttaaggagga catccagaaa gcacaagttt ctggccaggg 3241 ggacagtctt cacgagcaca tcgctaatct tgcaggtagc ccagctatca aaaagggaat 3301 actgcagacc gttaaggtcg tggatgaact cgtcaaagta atgggaaggc ataagcccga 3361 gaatatcgtt atcgagatgg cccgagagaa ccaaactacc cagaagggac agaagaacag 3421 tagggaaagg atgaagagga ttgaagaggg tataaaagaa ctggggtccc aaatccttaa 3481 ggaacaccca gttgaaaaca cccagcttca gaatgagaag ctctacctgt actacctgca 3541 gaacggcagg gacatgtacg tggatcagga actggacatc aatcggctct ccgactacga 3601 cgtggatcat atcgtgcccc agtcttttct caaagatgat tctattgata ataaagtgtt 3661 gacaagatcc gataaaaata gagggaagag tgataacgtc ccctcagaag aagttgtcaa 3721 gaaaatgaaa aattattggc ggcagctgct gaacgccaaa ctgatcacac aacggaagtt 3781 cgataatctg actaaggctg aacgaggtgg cctgtctgag ttggataaag ccggcttcat 3841 caaaaggcag cttgttgaga cacgccagat caccaagcac gtggcccaaa ttctcgattc 3901 acgcatgaac accaagtacg atgaaaatga caaactgatt cgagaggtga aagttattac 3961 tctgaagtct aagctggtct cagatttcag aaaggacttt cagttttata aggtgagaga 4021 gatcaacaat taccaccatg cgcatgatgc ctacctgaat gcagtggtag gcactgcact 4081 tatcaaaaaa tatcccaagc ttgaatctga atttgtttac ggagactata aagtgtacga 4141 tgttaggaaa atgatcgcaa agtctgagca ggaaataggc aaggccaccg ctaagtactt 4201 cttttacagc aatattatga attttttcaa gaccgagatt acactggcca atggagagat 4261 tcggaagcga ccacttatcg aaacaaacgg agaaacagga gaaatcgtgt gggacaaggg 4321 tagggatttc gcgacagtcc ggaaggtcct gtccatgccg caggtgaaca tcgttaaaaa 4381 gaccgaagta cagaccggag gcttctccaa ggaaagtatc ctcccgaaaa ggaacagcga 4441 caagctgatc gcacgcaaaa aagattggga ccccaagaaa tacggcggat tcgattctcc 4501 tacagtcgct tacagtgtac tggttgtggc caaagtggag aaagggaagt ctaaaaaact 4561 caaaagcgtc aaggaactgc tgggcatcac aatcatggag cgatcaagct tcgaaaaaaa 4621 ccccatcgac tttctcgagg cgaaaggata taaagaggtc aaaaaagacc tcatcattaa 4681 gcttcccaag tactctctct ttgagcttga aaacggccgg aaacgaatgc tcgctagtgc 4741 gggcgagctg cagaaaggta acgagctggc actgccctct aaatacgtta atttcttgta 4801 tctggccagc cactatgaaa agctcaaagg gtctcccgaa gataatgagc agaagcagct 4861 gttcgtggaa caacacaaac actaccttga tgagatcatc gagcaaataa gcgaattctc 4921 caaaagagtg atcctcgccg acgctaacct cgataaggtg ctttctgctt acaataagca 4981 cagggataag cccatcaggg agcaggcaga aaacattatc cacttgttta ctctgaccaa 5041 cttgggcgcg cctgcagcct tcaagtactt cgacaccacc atagacagaa agcggtacac 5101 ctctacaaag gaggtcctgg acgccacact gattcatcag tcaattacgg ggctctatga 5161 aacaagaatc gacctctctc agctcggtgg agacagcagg gctgacgggt ccggcagcgg 5221 gtccatggtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct 5281 ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg gcgatgccac 5341 ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc 5401 caccctcgtg accaccttca cctacggcgt gcagtgcttc agccgctacc ccgaccacat 5461 gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat 5521 cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac 5581 cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg 5641 gcacaagctg gagtacaact acaacagcca caacgtctat atcatggccg acaagcagaa 5701 gaacggcatc aaggtgaact tcaagatccg ccacaacatc gaggacggca gcgtgcagct 5761 cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa 5821 ccactacctg agcacccagt ccgccctgag caaagacccc aacgagaagc gcgatcacat 5881 ggtcctgctg gagttcgtga ccgccgccgg gatcactcac ggcatggacg agctgtacaa 5941 gccgaaaaag aagaggaagg tctgacaatt ggaaagatcc aagagagacg agtagagatt 6001 ttttttttgg gattgatgtt tgtcgttctt tgagttgtcg tcgagttacg ccttttgtaa 6061 gaatgttccg caggagagga ggaggatggg catgagtgag ggtgagaggg cttgcccgct 6121 ttttttttta aaaacgctga agacgtggtt gtcaaacaaa ccccccatag aaacgatttt 6181 gttacggtgc ggtccagacg tcacttgaat ggctccgcgg aaaggccagg gagggaaggg 6241 gggagggagg aaacatgaaa catgttgaac ggctcaacag ggtttggggg acaagagagg 6301 tagcgccctg atggactgct ccctcccctc ctttccctca atgtctcatt catccatgct 6361 tcccccttct ctctctcccc tccgttccat cccccgcggg cgtggtagtg gcgtgatggg 6421 atccactaaa atgtacgtgt aagaaaagcc ggtgagctta cgcttttgtg aaagtgggag 6481 tacgagtgtt gtgtgtgtgt gtagtggttt cagaccccag acagaggcga agcagaaaaa 6541 gcagacgatg aagacgacga agaaatgagc agtctatttt tatcgtggaa acagaagagg 6601 tgatatcgtc tcctgcaggc atgcaagctg atccactaga ggccatggcg gccgcactag 6661 tggatccccc gggatgcata cttggaagat ggagtggatg gaggaggagg cgagcgtagc 6721 agcaagcgtg agttatacag ccaggcacat gtcgcaatcc ttcggtctcg ggcttaaaat 6781 ccacgcacta atcacgctgg gccatgcaaa gagcaatgcc gaggcccacc acacaaaacg 6841 ctgtgtcgcg cgttgcggcc tgaagcttca tacttcttag tcgccgccaa aagggctcga 6901 gagacgagac ccgttggcat gaccgatgtt gttcgacgcg gtttgcttcg tcacagtcga 6961 cgtgattcag gaatctggag cctgcagatc atttttttca gcctgatatc gttcttttcc 7021 actgagaacc atcagaccac cttttcttcc attgtgtgaa ggagtaggag ttgccgtgct 7081 gctttgtggg agacatctgc gatggtgacc agcctcccgt cgtctggtcg acgtgacgag 7141 cctcttcact gttcttcgac ggagagacgc aagcgagacg gctctagacc ttttggacac 7201 gcattctgtg tgtgaactag tggacagtga taccacgtct gaaagctcac cactgcccat 7261 ggtgcagcta cttgtcacaa agttttgact ccgtcggtat caccattcgc gctcgtgtgc 7321 ctggttgttc cgccacgccg gcctgccccg gggcggggca atattctaaa atctcacgca 7381 aaacaccgca cttacccctc acacatattc gtgatagacc accaccaatc tcagcccgca 7441 tcaacacagg agggccccgg ggggcaataa gatatgaaaa agcctgaact caccgcgacg 7501 tctgtcgaga agtttctgat cgaaaagttc gacagcgtct ccgacctgat gcagctctcg 7561 gagggcgaag aatctcgtgc tttcagcttc gatgtaggag ggcgtggata tgtcctgcgg 7621 gtaaatagct gcgccgatgg tttctacaaa gatcgttatg tttatcggca ctttgcatcg 7681 gccgcgctcc cgattccgga agtgcttgac attggggaat tcagcgagag cctgacctat 7741 tgcatctccc gccgtgcaca gggtgtcacg ttgcaagacc tgcctgaaac cgaactgccc 7801 gctgttctgc agccggtcgc ggaggccatg gatgcgatcg ctgcggccga tcttagccag 7861 acgagcgggt tcggcccatt cggaccgcaa ggaatcggtc aatacactac atggcgtgat 7921 ttcatatgcg cgattgctga tccccatgtg tatcactggc aaactgtgat ggacgacacc 7981 gtcagtgcgt ccgtcgcgca ggctctcgat gagctgatgc tttgggccga ggactgcccc 8041 gaagtccggc acctcgtgca cgcggatttc ggctccaaca atgtcctgac ggacaatggc 8101 cgcataacag cggtcattga ctggagcgag gcgatgttcg gggattccca atacgaggtc 8161 gccaacatct tcttctggag gccgtggttg gcttgtatgg agcagcagac gcgctacttc 8221 gagcggaggc atccggagct tgcaggatcg ccgcggctcc gggcgtatat gctccgcatt 8281 ggtcttgacc aactctatca gagcttggtt gacggcaatt tcgatgatgc agcttgggcg 8341 cagggtcgat gcgacgcaat cgtccgatcc ggagccggga ctgtcgggcg tacacaaatc 8401 gcccgcagaa gcgcggccgt ctggaccgat ggctgtgtag aagtactcgc cgatagtgga 8461 aaccgacgcc ccagcactcg tccgagggca aaggaataga gtagatgccg accggagtcc 8521 gcaaaaatca ccagtctctc tctacaaatc tatctctctc tatttttctc cagaataatg 8581 tgtgagtagt tcccagataa gggaattagg gttcttatag ggtttcgctc atgtgttgag 8641 catataagaa acccttagta tgtatttgta tttgtaaaat acttctatca ataaaatttc 8701 taattcctaa aaccaaaatc cagtgacctg agcttcctcg ctcactgact cgctgcgctc 8761 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac 8821 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa 8881 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca 8941 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc 9001 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata 9061 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta 9121 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca 9181 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga 9241 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg 9301 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg 9361 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg 9421 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag 9481 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa 9541 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat 9601 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc 9661 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc 9721 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc 9781 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc 9841 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc 9901 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt 9961 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc 10021 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa 10081 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt 10141 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg 10201 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc 10261 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa 10321 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt 10381 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt 10441 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag 10501 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta 10561 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat 10621 aggggttccg cgcacatttc cccgaaaagt gccacctgat gcggtgtgaa ataccgcaca 10681 gatgcgtaag gagaaaatac cgcatcagga aattgtaagc gttaatattt tgttaaaatt 10741 cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat 10801 cccttataaa tcaaaagaat agaccgagat agggttgagt gttgttccag tttggaacaa 10861 gagtccacta ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg 10921 cgatggccca ctacgtgaac catcacccta atcaagtttt ttggggtcga ggtgccgtaa 10981 agcactaaat cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc 11041 gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag 11101 tgtagcggtc acgctgcgcg taaccaccac acccgccgcg cttaatgcgc cgctacaggg 11161 cgcgtccatt cgccattcag gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct 11221 tcgctattac gccagctggc gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg 11281 ccagggtttt cccagtcacg acgttgtaaa acgacggcca gtgaattgta atacgactca 11341 ctata // LOCUS Exported 13085 bp ds-DNA circular SYN 10-JUL-2017 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS pNOC-stacked-Cas9-GFP SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 13085) AUTHORS . TITLE Direct Submission JOURNAL Exported Friday, Jul 14, 2017 from SnapGene Viewer 3.3.4 http://www.snapgene.com FEATURES Location/Qualifiers source 1..13085 /organism="synthetic DNA construct" /mol_type="other DNA" terminator 418..1599 /label=CS terminator promoter 1619..2802 /label=Ribi CDS 2813..2833 /codon_start=1 /product="nuclear localization signal of SV40 large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 2834..6934 /codon_start=1 /product="Cas9 (Csn1) endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system" /label=Cas9 /note="generates RNA-guided double strand breaks in DNA" /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD ATLIHQSITGLYETRIDLSQLGGD" CDS 6965..7681 /codon_start=1 /product="enhanced GFP" /label=EGFP /note="mammalian codon-optimized" /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTFTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITHGMDELYK" CDS 7682..7702 /codon_start=1 /product="nuclear localization signal of SV40 large T antigen" /label=SV40 NLS /translation="PKKKRKV" terminator 7734..8333 /label=LDSP terminator promoter 8420..9189 /label=LDSP promoter CDS 9214..10239 /codon_start=1 /gene="aph(4)-Ia" /product="aminoglycoside phosphotransferase from E. coli" /label=HygR /note="confers resistance to hygromycin" /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK E" terminator 10240..10470 /label=35S terminator rep_origin complement(10648..11236) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" CDS complement(11407..12267) /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" promoter complement(12268..12372) /gene="bla" /label=AmpR promoter rep_origin complement(12450..12905) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind 13046..13062 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" promoter 13069..2 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" ORIGIN 1 gggcgaattg ggcccgacgt cgcatgctcc cggccgccat ggcggccgcg ggaattcgat 61 atcactagag tatagctaca tggtagctag tagtcgtcaa aggaaataaa gacatagcgt 121 aaagtataca aaatgacttc cgcggcgcgg gagcggcagg cagcacttgc aaaacgaagc 181 ccaatgggcc agattacggt caacctcgtc attttattac ggtcgtcagc aagagcggtt 241 tagaccgtcg ccggcacggg tggctcccga tattcttggc tttcgtgggt ggaggcggcg 301 tgtgactcct catctttaag tagtggtcgc ttttgctgca tgcttttgat cacctcgtga 361 ttgatggctg gattctcagc tgtcttctga catctttggg tgcatgggtt gaccggtcga 421 ggaggcattg tatttacagc ggagagcgaa gaatgaagtg agaggaagtt aggagggagg 481 aaaagacaaa gacgggctgc attaagagaa agtgaagagc atcagcagcg catcacagaa 541 aatgattgag ggaacaacat cttcaaatgt atgaaacaaa aaggatggaa gtggaagagc 601 aaagctgacg cccttttcgt cgtcgttgct ggtgttaaaa ttgcgcgttt cttttttttc 661 cgtctttgtc gtcttttgac ttttttatct tcttttctgt cttcttttat tcttctcttc 721 ttcttcatca ctccctcttc atttttcttc ttctttttcc tcgtctattg tcctcgggca 781 ttaccagttg tgctgctcac gtgcgcgcaa gcggtgaaag ggtgagagga tacacggaca 841 acaatcattg aaaattacgc ttaaagccgg tgcctggaaa agtccacgcg cagatggcct 901 ttgcttgcct tgctgctgct gcagctgctg ctgctgtcgc ctgtgatggt aatggttgtt 961 gttgtggtcc tgcttgcttg ctagtttgca ttgcgtttgc ggttgcctta tgaccgactc 1021 cgtgtgattg atccttaaac atattttatc attgatgtgg attgtgtgac gacggcaaaa 1081 aagaattact ttacacatga ctatcacacc tgctcccctc ccttctcctt tcaatcggtc 1141 aacttcccaa ccgctcctcg aaaatatttc agttgaatcc tctactacgt accccttact 1201 taaatttact ttctctctcc ccatcaatgc gccgttgttc tgaagttgta ttattgatgg 1261 gagatgattg gcactgcacc agacctggcc aaaagccacc acaggtggcc acatactctt 1321 gcatgggtat actctaccga ggcaaagatt aaagtgcacg tcccctgccc ctctctctcc 1381 ctacttcctt cagtctcagt ggttccgtgc ccgttcagca ctacatcgcc ctgcccgctg 1441 ttccttctca aacagccacc catcccaacc cacatacaca gaaacaacct ttcctacaca 1501 ttttcccaag tcttctcttc actattctac actaatacac caccgtgata tgccggttat 1561 ctagatgtcc tttctccttt ctcatccttt ctttcccatg gtaccaaggc ctatcgattc 1621 tgtggatgga gggagggaag gaggggggga gtgagtgagt cgagagaacg acgactgggg 1681 caaagagagg atgacgccac tgcaacaaaa gacagggaag aatagaggct gttggaaaac 1741 aggaacacag tgaagaatag aggctgcagg gtaacaggca catttatcgc agagaggcgg 1801 gcggcagggg cgatacgtaa agagacccct aaacttctcc gcgtcatccg ttcacgctac 1861 ctccttccct ccctttactc ccattgatca ccaaggcagc gcccgcagcc accagcagcc 1921 accgtcctca gtgtcacagc tagacgcgtc cccatcaggc agcagaccac cagaatatgc 1981 gctttcgctt cccatcatat ccctccctcc tccccttcta ttcctcatca tgcatcattt 2041 ctttctcctt gctgcatgcc cctcttctta ccttggctaa gatctaccgg aggaagaagt 2101 cttcaacgat agataaatgc ctgtatcgtg atggttgttt gaatggtgga agtgagccag 2161 gtattgagaa cgaggggcgc aagggtgcgt ggaagaaggc agggagaggc gaggcgaagg 2221 agaagggcat gtggtgggaa cgaagcctcc acaccacagc cacccatgct tttctctgtt 2281 cgagttcccg ggttctactc tcacacacta cacgtgcaca gaaaacattc acgcacaaca 2341 gagatacaga gggatccgtc gagatggtct acacgtccta atgtcccctg ctttcgaagc 2401 ccagagccat cgctttctgt ttttatccat tcgcaacaca tcctgttctc gcgacctctc 2461 ctcttcctgg ccaccatcgc ttctccgtgc catgccctgt acatccttcc ctccctcgtt 2521 gtctaccaca ctaatcatgc gctctgcagc agacactcgg ctactatttg tgtcgtacgg 2581 attgagggga gggaggggaa gggaggcaag aagtaaatcg gtggtcaaca acagacgcgg 2641 tctccccacc ctccctcatt cctttcccct cctataatga gcgaagaaag ccatggacac 2701 cgagtcaccc actctcctaa tccaccacca ccagggcact aacggactca cgctcaccaa 2761 actcggtaac aatatccact gcacacagat acacacgcaa cactcgagca tgcccaagaa 2821 aaagcggaag gtggacaaga agtactccat tgggctcgat atcggcacaa acagcgtcgg 2881 ctgggccgtc attacggacg agtacaaggt gccgagcaaa aaattcaaag ttctgggcaa 2941 taccgatcgc cacagcataa agaagaacct cattggcgcc ctcctgttcg actccgggga 3001 gacggccgaa gccacgcggc tcaaaagaac agcacggcgc agatataccc gcagaaagaa 3061 tcggatctgc tacctgcagg agatctttag taatgagatg gctaaggtgg atgactcttt 3121 cttccatagg ctggaggagt cctttttggt ggaggaggat aaaaagcacg agcgccaccc 3181 aatctttggc aatatcgtgg acgaggtggc gtaccatgaa aagtacccaa ccatatatca 3241 tctgaggaag aagcttgtag acagtactga taaggctgac ttgcggttga tctatctcgc 3301 gctggcgcat atgatcaaat ttcggggaca cttcctcatc gagggggacc tgaacccaga 3361 caacagcgat gtcgacaaac tctttatcca actggttcag acttacaatc agcttttcga 3421 agagaacccg atcaacgcat ccggagttga cgccaaagca atcctgagcg ctaggctgtc 3481 caaatcccgg cggctcgaaa acctcatcgc acagctccct ggggagaaga agaacggcct 3541 gtttggtaat cttatcgccc tgtcactcgg gctgaccccc aactttaaat ctaacttcga 3601 cctggccgaa gatgccaagc ttcaactgag caaagacacc tacgatgatg atctcgacaa 3661 tctgctggcc cagatcggcg accagtacgc agaccttttt ttggcggcaa agaacctgtc 3721 agacgccatt ctgctgagtg atattctgcg agtgaacacg gagatcacca aagctccgct 3781 gagcgctagt atgatcaagc gctatgatga gcaccaccaa gacttgactt tgctgaaggc 3841 ccttgtcaga cagcaactgc ctgagaagta caaggaaatt ttcttcgatc agtctaaaaa 3901 tggctacgcc ggatacattg acggcggagc aagccaggag gaattttaca aatttattaa 3961 gcccatcttg gaaaaaatgg acggcaccga ggagctgctg gtaaagctta acagagaaga 4021 tctgttgcgc aaacagcgca ctttcgacaa tggaagcatc ccccaccaga ttcacctggg 4081 cgaactgcac gctatcctca ggcggcaaga ggatttctac ccctttttga aagataacag 4141 ggaaaagatt gagaaaatcc tcacatttcg gataccctac tatgtaggcc ccctcgcccg 4201 gggaaattcc agattcgcgt ggatgactcg caaatcagaa gagaccatca ctccctggaa 4261 cttcgaggaa gtcgtggata agggggcctc tgcccagtcc ttcatcgaaa ggatgactaa 4321 ctttgataaa aatctgccta acgaaaaggt gcttcctaaa cactctctgc tgtacgagta 4381 cttcacagtt tataacgagc tcaccaaggt caaatacgtc acagaaggga tgagaaagcc 4441 agcattcctg tctggagagc agaagaaagc tatcgtggac ctcctcttca agacgaaccg 4501 gaaagttacc gtgaaacagc tcaaagaaga ctatttcaaa aagattgaat gtttcgactc 4561 tgttgaaatc agcggagtgg aggatcgctt caacgcatcc ctgggaacgt atcacgatct 4621 cctgaaaatc attaaagaca aggacttcct ggacaatgag gagaacgagg acattcttga 4681 ggacattgtc ctcaccctta cgttgtttga agatagggag atgattgaag aacgcttgaa 4741 aacttacgct catctcttcg acgacaaagt catgaaacag ctcaagaggc gccgatatac 4801 aggatggggg cggctgtcaa gaaaactgat caatgggatc cgagacaagc agagtggaaa 4861 gacaatcctg gattttctta agtccgatgg atttgccaac cggaacttca tgcagttgat 4921 ccatgatgac tctctcacct ttaaggagga catccagaaa gcacaagttt ctggccaggg 4981 ggacagtctt cacgagcaca tcgctaatct tgcaggtagc ccagctatca aaaagggaat 5041 actgcagacc gttaaggtcg tggatgaact cgtcaaagta atgggaaggc ataagcccga 5101 gaatatcgtt atcgagatgg cccgagagaa ccaaactacc cagaagggac agaagaacag 5161 tagggaaagg atgaagagga ttgaagaggg tataaaagaa ctggggtccc aaatccttaa 5221 ggaacaccca gttgaaaaca cccagcttca gaatgagaag ctctacctgt actacctgca 5281 gaacggcagg gacatgtacg tggatcagga actggacatc aatcggctct ccgactacga 5341 cgtggatcat atcgtgcccc agtcttttct caaagatgat tctattgata ataaagtgtt 5401 gacaagatcc gataaaaata gagggaagag tgataacgtc ccctcagaag aagttgtcaa 5461 gaaaatgaaa aattattggc ggcagctgct gaacgccaaa ctgatcacac aacggaagtt 5521 cgataatctg actaaggctg aacgaggtgg cctgtctgag ttggataaag ccggcttcat 5581 caaaaggcag cttgttgaga cacgccagat caccaagcac gtggcccaaa ttctcgattc 5641 acgcatgaac accaagtacg atgaaaatga caaactgatt cgagaggtga aagttattac 5701 tctgaagtct aagctggtct cagatttcag aaaggacttt cagttttata aggtgagaga 5761 gatcaacaat taccaccatg cgcatgatgc ctacctgaat gcagtggtag gcactgcact 5821 tatcaaaaaa tatcccaagc ttgaatctga atttgtttac ggagactata aagtgtacga 5881 tgttaggaaa atgatcgcaa agtctgagca ggaaataggc aaggccaccg ctaagtactt 5941 cttttacagc aatattatga attttttcaa gaccgagatt acactggcca atggagagat 6001 tcggaagcga ccacttatcg aaacaaacgg agaaacagga gaaatcgtgt gggacaaggg 6061 tagggatttc gcgacagtcc ggaaggtcct gtccatgccg caggtgaaca tcgttaaaaa 6121 gaccgaagta cagaccggag gcttctccaa ggaaagtatc ctcccgaaaa ggaacagcga 6181 caagctgatc gcacgcaaaa aagattggga ccccaagaaa tacggcggat tcgattctcc 6241 tacagtcgct tacagtgtac tggttgtggc caaagtggag aaagggaagt ctaaaaaact 6301 caaaagcgtc aaggaactgc tgggcatcac aatcatggag cgatcaagct tcgaaaaaaa 6361 ccccatcgac tttctcgagg cgaaaggata taaagaggtc aaaaaagacc tcatcattaa 6421 gcttcccaag tactctctct ttgagcttga aaacggccgg aaacgaatgc tcgctagtgc 6481 gggcgagctg cagaaaggta acgagctggc actgccctct aaatacgtta atttcttgta 6541 tctggccagc cactatgaaa agctcaaagg gtctcccgaa gataatgagc agaagcagct 6601 gttcgtggaa caacacaaac actaccttga tgagatcatc gagcaaataa gcgaattctc 6661 caaaagagtg atcctcgccg acgctaacct cgataaggtg ctttctgctt acaataagca 6721 cagggataag cccatcaggg agcaggcaga aaacattatc cacttgttta ctctgaccaa 6781 cttgggcgcg cctgcagcct tcaagtactt cgacaccacc atagacagaa agcggtacac 6841 ctctacaaag gaggtcctgg acgccacact gattcatcag tcaattacgg ggctctatga 6901 aacaagaatc gacctctctc agctcggtgg agacagcagg gctgacgggt ccggcagcgg 6961 gtccatggtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct 7021 ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg gcgatgccac 7081 ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc 7141 caccctcgtg accaccttca cctacggcgt gcagtgcttc agccgctacc ccgaccacat 7201 gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat 7261 cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac 7321 cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg 7381 gcacaagctg gagtacaact acaacagcca caacgtctat atcatggccg acaagcagaa 7441 gaacggcatc aaggtgaact tcaagatccg ccacaacatc gaggacggca gcgtgcagct 7501 cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa 7561 ccactacctg agcacccagt ccgccctgag caaagacccc aacgagaagc gcgatcacat 7621 ggtcctgctg gagttcgtga ccgccgccgg gatcactcac ggcatggacg agctgtacaa 7681 gccgaaaaag aagaggaagg tctgacaatt ggaaagatcc aagagagacg agtagagatt 7741 ttttttttgg gattgatgtt tgtcgttctt tgagttgtcg tcgagttacg ccttttgtaa 7801 gaatgttccg caggagagga ggaggatggg catgagtgag ggtgagaggg cttgcccgct 7861 ttttttttta aaaacgctga agacgtggtt gtcaaacaaa ccccccatag aaacgatttt 7921 gttacggtgc ggtccagacg tcacttgaat ggctccgcgg aaaggccagg gagggaaggg 7981 gggagggagg aaacatgaaa catgttgaac ggctcaacag ggtttggggg acaagagagg 8041 tagcgccctg atggactgct ccctcccctc ctttccctca atgtctcatt catccatgct 8101 tcccccttct ctctctcccc tccgttccat cccccgcggg cgtggtagtg gcgtgatggg 8161 atccactaaa atgtacgtgt aagaaaagcc ggtgagctta cgcttttgtg aaagtgggag 8221 tacgagtgtt gtgtgtgtgt gtagtggttt cagaccccag acagaggcga agcagaaaaa 8281 gcagacgatg aagacgacga agaaatgagc agtctatttt tatcgtggaa acagaagagg 8341 tgatatcgtc tcctgcaggc atgcaagctg atccactaga ggccatggcg gccgcactag 8401 tggatccccc gggatgcata cttggaagat ggagtggatg gaggaggagg cgagcgtagc 8461 agcaagcgtg agttatacag ccaggcacat gtcgcaatcc ttcggtctcg ggcttaaaat 8521 ccacgcacta atcacgctgg gccatgcaaa gagcaatgcc gaggcccacc acacaaaacg 8581 ctgtgtcgcg cgttgcggcc tgaagcttca tacttcttag tcgccgccaa aagggctcga 8641 gagacgagac ccgttggcat gaccgatgtt gttcgacgcg gtttgcttcg tcacagtcga 8701 cgtgattcag gaatctggag cctgcagatc atttttttca gcctgatatc gttcttttcc 8761 actgagaacc atcagaccac cttttcttcc attgtgtgaa ggagtaggag ttgccgtgct 8821 gctttgtggg agacatctgc gatggtgacc agcctcccgt cgtctggtcg acgtgacgag 8881 cctcttcact gttcttcgac ggagagacgc aagcgagacg gctctagacc ttttggacac 8941 gcattctgtg tgtgaactag tggacagtga taccacgtct gaaagctcac cactgcccat 9001 ggtgcagcta cttgtcacaa agttttgact ccgtcggtat caccattcgc gctcgtgtgc 9061 ctggttgttc cgccacgccg gcctgccccg gggcggggca atattctaaa atctcacgca 9121 aaacaccgca cttacccctc acacatattc gtgatagacc accaccaatc tcagcccgca 9181 tcaacacagg agggccccgg ggggcaataa gatatgaaaa agcctgaact caccgcgacg 9241 tctgtcgaga agtttctgat cgaaaagttc gacagcgtct ccgacctgat gcagctctcg 9301 gagggcgaag aatctcgtgc tttcagcttc gatgtaggag ggcgtggata tgtcctgcgg 9361 gtaaatagct gcgccgatgg tttctacaaa gatcgttatg tttatcggca ctttgcatcg 9421 gccgcgctcc cgattccgga agtgcttgac attggggaat tcagcgagag cctgacctat 9481 tgcatctccc gccgtgcaca gggtgtcacg ttgcaagacc tgcctgaaac cgaactgccc 9541 gctgttctgc agccggtcgc ggaggccatg gatgcgatcg ctgcggccga tcttagccag 9601 acgagcgggt tcggcccatt cggaccgcaa ggaatcggtc aatacactac atggcgtgat 9661 ttcatatgcg cgattgctga tccccatgtg tatcactggc aaactgtgat ggacgacacc 9721 gtcagtgcgt ccgtcgcgca ggctctcgat gagctgatgc tttgggccga ggactgcccc 9781 gaagtccggc acctcgtgca cgcggatttc ggctccaaca atgtcctgac ggacaatggc 9841 cgcataacag cggtcattga ctggagcgag gcgatgttcg gggattccca atacgaggtc 9901 gccaacatct tcttctggag gccgtggttg gcttgtatgg agcagcagac gcgctacttc 9961 gagcggaggc atccggagct tgcaggatcg ccgcggctcc gggcgtatat gctccgcatt 10021 ggtcttgacc aactctatca gagcttggtt gacggcaatt tcgatgatgc agcttgggcg 10081 cagggtcgat gcgacgcaat cgtccgatcc ggagccggga ctgtcgggcg tacacaaatc 10141 gcccgcagaa gcgcggccgt ctggaccgat ggctgtgtag aagtactcgc cgatagtgga 10201 aaccgacgcc ccagcactcg tccgagggca aaggaataga gtagatgccg accggagtcc 10261 gcaaaaatca ccagtctctc tctacaaatc tatctctctc tatttttctc cagaataatg 10321 tgtgagtagt tcccagataa gggaattagg gttcttatag ggtttcgctc atgtgttgag 10381 catataagaa acccttagta tgtatttgta tttgtaaaat acttctatca ataaaatttc 10441 taattcctaa aaccaaaatc cagtgacctg agcttcctcg ctcactgact cgctgcgctc 10501 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac 10561 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa 10621 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca 10681 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc 10741 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata 10801 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta 10861 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca 10921 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga 10981 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg 11041 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg 11101 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg 11161 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag 11221 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa 11281 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat 11341 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc 11401 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc 11461 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc 11521 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc 11581 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc 11641 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt 11701 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc 11761 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa 11821 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt 11881 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg 11941 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc 12001 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa 12061 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt 12121 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt 12181 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag 12241 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta 12301 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat 12361 aggggttccg cgcacatttc cccgaaaagt gccacctgat gcggtgtgaa ataccgcaca 12421 gatgcgtaag gagaaaatac cgcatcagga aattgtaagc gttaatattt tgttaaaatt 12481 cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat 12541 cccttataaa tcaaaagaat agaccgagat agggttgagt gttgttccag tttggaacaa 12601 gagtccacta ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg 12661 cgatggccca ctacgtgaac catcacccta atcaagtttt ttggggtcga ggtgccgtaa 12721 agcactaaat cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc 12781 gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag 12841 tgtagcggtc acgctgcgcg taaccaccac acccgccgcg cttaatgcgc cgctacaggg 12901 cgcgtccatt cgccattcag gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct 12961 tcgctattac gccagctggc gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg 13021 ccagggtttt cccagtcacg acgttgtaaa acgacggcca gtgaattgta atacgactca 13081 ctata // LOCUS Exported 13072 bp ds-DNA circular SYN 10-JUL-2017 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS pNOC-CRISPR-GFP SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 13072) AUTHORS . TITLE Direct Submission JOURNAL Exported Thursday, Jul 13, 2017 from SnapGene Viewer 3.3.4 http://www.snapgene.com FEATURES Location/Qualifiers source 1..13072 /organism="synthetic DNA construct" /mol_type="other DNA" terminator 418..1418 /label=CS terminator misc_feature 1425..1501 /label=HDV misc_RNA complement(1502..1577) /label=g RNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" promoter 1606..2789 /label=Ribi CDS 2800..2820 /codon_start=1 /product="nuclear localization signal of SV40 large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 2821..6921 /codon_start=1 /product="Cas9 (Csn1) endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system" /label=Cas9 /note="generates RNA-guided double strand breaks in DNA" /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD ATLIHQSITGLYETRIDLSQLGGD" CDS 6952..7668 /codon_start=1 /product="enhanced GFP" /label=EGFP /note="mammalian codon-optimized" /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTFTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITHGMDELYK" CDS 7669..7689 /codon_start=1 /product="nuclear localization signal of SV40 large T antigen" /label=SV40 NLS /translation="PKKKRKV" terminator 7721..8320 /label=LDSP terminator promoter 8407..9176 /label=LDSP promoter CDS 9201..10226 /codon_start=1 /gene="aph(4)-Ia" /product="aminoglycoside phosphotransferase from E. coli" /label=HygR /note="confers resistance to hygromycin" /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK E" terminator 10227..10457 /label=35S terminator rep_origin complement(10635..11223) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" CDS complement(11394..12254) /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" promoter complement(12255..12359) /gene="bla" /label=AmpR promoter rep_origin complement(12437..12892) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind 13033..13049 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" ORIGIN 1 gggcgaattg ggcccgacgt cgcatgctcc cggccgccat ggcggccgcg ggaattcgat 61 atcactagag tatagctaca tggtagctag tagtcgtcaa aggaaataaa gacatagcgt 121 aaagtataca aaatgacttc cgcggcgcgg gagcggcagg cagcacttgc aaaacgaagc 181 ccaatgggcc agattacggt caacctcgtc attttattac ggtcgtcagc aagagcggtt 241 tagaccgtcg ccggcacggg tggctcccga tattcttggc tttcgtgggt ggaggcggcg 301 tgtgactcct catctttaag tagtggtcgc ttttgctgca tgcttttgat cacctcgtga 361 ttgatggctg gattctcagc tgtcttctga catctttggg tgcatgggtt gaccggtgca 421 aagctgacgc ccttttcgtc gtcgttgctg gtgttaaaat tgcgcgtttc ttttttttcc 481 gtctttgtcg tcttttgact tttttatctt cttttctgtc ttcttttatt cttctcttct 541 tcttcatcac tccctcttca tttttcttct tctttttcct cgtctattgt cctcgggcat 601 taccagttgt gctgctcacg tgcgcgcaag cggtgaaagg gtgagaggat acacggacaa 661 caatcattga aaattacgct taaagccggt gcctggaaaa gtccacgcgc agatggcctt 721 tgcttgcctt gctgctgctg cagctgctgc tgctgtcgcc tgtgatggta atggttgttg 781 ttgtggtcct gcttgcttgc tagtttgcat tgcgtttgcg gttgccttat gaccgactcc 841 gtgtgattga tccttaaaca tattttatca ttgatgtgga ttgtgtgacg acggcaaaaa 901 agaattactt tacacatgac tatcacacct gctcccctcc cttctccttt caatcggtca 961 acttcccaac cgctcctcga aaatatttca gttgaatcct ctactacgta ccccttactt 1021 aaatttactt tctctctccc catcaatgcg ccgttgttct gaagttgtat tattgatggg 1081 agatgattgg cactgcacca gacctggcca aaagccacca caggtggcca catactcttg 1141 catgggtata ctctaccgag gcaaagatta aagtgcacgt cccctgcccc tctctctccc 1201 tacttccttc agtctcagtg gttccgtgcc cgttcagcac tacatcgccc tgcccgctgt 1261 tccttctcaa acagccaccc atcccaaccc acatacacag aaacaacctt tcctacacat 1321 tttcccaagt cttctcttca ctattctaca ctaatacacc accgtgatat gccggttatc 1381 tagatgtcct ttctcctttc tcatcctttc tttcccatgg tacccaggag tcccattcgc 1441 catgccgaag catgttgccc agccggcgcc agcgaggagg ctgggaccat gccggccaaa 1501 agcaccgact cggtgccact ttttcaagtt gataacggac tagccttatt ttaacttgct 1561 atttctagct ctaaaacgaa gagccaaggc ctgctcttca tcgattctgt ggatggaggg 1621 agggaaggag ggggggagtg agtgagtcga gagaacgacg actggggcaa agagaggatg 1681 acgccactgc aacaaaagac agggaagaat agaggctgtt ggaaaacagg aacacagtga 1741 agaatagagg ctgcagggta acaggcacat ttatcgcaga gaggcgggcg gcaggggcga 1801 tacgtaaaga gacccctaaa cttctccgcg tcatccgttc acgctacctc cttccctccc 1861 tttactccca ttgatcacca aggcagcgcc cgcagccacc agcagccacc gtcctcagtg 1921 tcacagctag acgcgtcccc atcaggcagc agaccaccag aatatgcgct ttcgcttccc 1981 atcatatccc tccctcctcc ccttctattc ctcatcatgc atcatttctt tctccttgct 2041 gcatgcccct cttcttacct tggctaagat ctaccggagg aagaagtctt caacgataga 2101 taaatgcctg tatcgtgatg gttgtttgaa tggtggaagt gagccaggta ttgagaacga 2161 ggggcgcaag ggtgcgtgga agaaggcagg gagaggcgag gcgaaggaga agggcatgtg 2221 gtgggaacga agcctccaca ccacagccac ccatgctttt ctctgttcga gttcccgggt 2281 tctactctca cacactacac gtgcacagaa aacattcacg cacaacagag atacagaggg 2341 atccgtcgag atggtctaca cgtcctaatg tcccctgctt tcgaagccca gagccatcgc 2401 tttctgtttt tatccattcg caacacatcc tgttctcgcg acctctcctc ttcctggcca 2461 ccatcgcttc tccgtgccat gccctgtaca tccttccctc cctcgttgtc taccacacta 2521 atcatgcgct ctgcagcaga cactcggcta ctatttgtgt cgtacggatt gaggggaggg 2581 aggggaaggg aggcaagaag taaatcggtg gtcaacaaca gacgcggtct ccccaccctc 2641 cctcattcct ttcccctcct ataatgagcg aagaaagcca tggacaccga gtcacccact 2701 ctcctaatcc accaccacca gggcactaac ggactcacgc tcaccaaact cggtaacaat 2761 atccactgca cacagataca cacgcaacac tcgagcatgc ccaagaaaaa gcggaaggtg 2821 gacaagaagt actccattgg gctcgatatc ggcacaaaca gcgtcggctg ggccgtcatt 2881 acggacgagt acaaggtgcc gagcaaaaaa ttcaaagttc tgggcaatac cgatcgccac 2941 agcataaaga agaacctcat tggcgccctc ctgttcgact ccggggagac ggccgaagcc 3001 acgcggctca aaagaacagc acggcgcaga tatacccgca gaaagaatcg gatctgctac 3061 ctgcaggaga tctttagtaa tgagatggct aaggtggatg actctttctt ccataggctg 3121 gaggagtcct ttttggtgga ggaggataaa aagcacgagc gccacccaat ctttggcaat 3181 atcgtggacg aggtggcgta ccatgaaaag tacccaacca tatatcatct gaggaagaag 3241 cttgtagaca gtactgataa ggctgacttg cggttgatct atctcgcgct ggcgcatatg 3301 atcaaatttc ggggacactt cctcatcgag ggggacctga acccagacaa cagcgatgtc 3361 gacaaactct ttatccaact ggttcagact tacaatcagc ttttcgaaga gaacccgatc 3421 aacgcatccg gagttgacgc caaagcaatc ctgagcgcta ggctgtccaa atcccggcgg 3481 ctcgaaaacc tcatcgcaca gctccctggg gagaagaaga acggcctgtt tggtaatctt 3541 atcgccctgt cactcgggct gacccccaac tttaaatcta acttcgacct ggccgaagat 3601 gccaagcttc aactgagcaa agacacctac gatgatgatc tcgacaatct gctggcccag 3661 atcggcgacc agtacgcaga cctttttttg gcggcaaaga acctgtcaga cgccattctg 3721 ctgagtgata ttctgcgagt gaacacggag atcaccaaag ctccgctgag cgctagtatg 3781 atcaagcgct atgatgagca ccaccaagac ttgactttgc tgaaggccct tgtcagacag 3841 caactgcctg agaagtacaa ggaaattttc ttcgatcagt ctaaaaatgg ctacgccgga 3901 tacattgacg gcggagcaag ccaggaggaa ttttacaaat ttattaagcc catcttggaa 3961 aaaatggacg gcaccgagga gctgctggta aagcttaaca gagaagatct gttgcgcaaa 4021 cagcgcactt tcgacaatgg aagcatcccc caccagattc acctgggcga actgcacgct 4081 atcctcaggc ggcaagagga tttctacccc tttttgaaag ataacaggga aaagattgag 4141 aaaatcctca catttcggat accctactat gtaggccccc tcgcccgggg aaattccaga 4201 ttcgcgtgga tgactcgcaa atcagaagag accatcactc cctggaactt cgaggaagtc 4261 gtggataagg gggcctctgc ccagtccttc atcgaaagga tgactaactt tgataaaaat 4321 ctgcctaacg aaaaggtgct tcctaaacac tctctgctgt acgagtactt cacagtttat 4381 aacgagctca ccaaggtcaa atacgtcaca gaagggatga gaaagccagc attcctgtct 4441 ggagagcaga agaaagctat cgtggacctc ctcttcaaga cgaaccggaa agttaccgtg 4501 aaacagctca aagaagacta tttcaaaaag attgaatgtt tcgactctgt tgaaatcagc 4561 ggagtggagg atcgcttcaa cgcatccctg ggaacgtatc acgatctcct gaaaatcatt 4621 aaagacaagg acttcctgga caatgaggag aacgaggaca ttcttgagga cattgtcctc 4681 acccttacgt tgtttgaaga tagggagatg attgaagaac gcttgaaaac ttacgctcat 4741 ctcttcgacg acaaagtcat gaaacagctc aagaggcgcc gatatacagg atgggggcgg 4801 ctgtcaagaa aactgatcaa tgggatccga gacaagcaga gtggaaagac aatcctggat 4861 tttcttaagt ccgatggatt tgccaaccgg aacttcatgc agttgatcca tgatgactct 4921 ctcaccttta aggaggacat ccagaaagca caagtttctg gccaggggga cagtcttcac 4981 gagcacatcg ctaatcttgc aggtagccca gctatcaaaa agggaatact gcagaccgtt 5041 aaggtcgtgg atgaactcgt caaagtaatg ggaaggcata agcccgagaa tatcgttatc 5101 gagatggccc gagagaacca aactacccag aagggacaga agaacagtag ggaaaggatg 5161 aagaggattg aagagggtat aaaagaactg gggtcccaaa tccttaagga acacccagtt 5221 gaaaacaccc agcttcagaa tgagaagctc tacctgtact acctgcagaa cggcagggac 5281 atgtacgtgg atcaggaact ggacatcaat cggctctccg actacgacgt ggatcatatc 5341 gtgccccagt cttttctcaa agatgattct attgataata aagtgttgac aagatccgat 5401 aaaaatagag ggaagagtga taacgtcccc tcagaagaag ttgtcaagaa aatgaaaaat 5461 tattggcggc agctgctgaa cgccaaactg atcacacaac ggaagttcga taatctgact 5521 aaggctgaac gaggtggcct gtctgagttg gataaagccg gcttcatcaa aaggcagctt 5581 gttgagacac gccagatcac caagcacgtg gcccaaattc tcgattcacg catgaacacc 5641 aagtacgatg aaaatgacaa actgattcga gaggtgaaag ttattactct gaagtctaag 5701 ctggtctcag atttcagaaa ggactttcag ttttataagg tgagagagat caacaattac 5761 caccatgcgc atgatgccta cctgaatgca gtggtaggca ctgcacttat caaaaaatat 5821 cccaagcttg aatctgaatt tgtttacgga gactataaag tgtacgatgt taggaaaatg 5881 atcgcaaagt ctgagcagga aataggcaag gccaccgcta agtacttctt ttacagcaat 5941 attatgaatt ttttcaagac cgagattaca ctggccaatg gagagattcg gaagcgacca 6001 cttatcgaaa caaacggaga aacaggagaa atcgtgtggg acaagggtag ggatttcgcg 6061 acagtccgga aggtcctgtc catgccgcag gtgaacatcg ttaaaaagac cgaagtacag 6121 accggaggct tctccaagga aagtatcctc ccgaaaagga acagcgacaa gctgatcgca 6181 cgcaaaaaag attgggaccc caagaaatac ggcggattcg attctcctac agtcgcttac 6241 agtgtactgg ttgtggccaa agtggagaaa gggaagtcta aaaaactcaa aagcgtcaag 6301 gaactgctgg gcatcacaat catggagcga tcaagcttcg aaaaaaaccc catcgacttt 6361 ctcgaggcga aaggatataa agaggtcaaa aaagacctca tcattaagct tcccaagtac 6421 tctctctttg agcttgaaaa cggccggaaa cgaatgctcg ctagtgcggg cgagctgcag 6481 aaaggtaacg agctggcact gccctctaaa tacgttaatt tcttgtatct ggccagccac 6541 tatgaaaagc tcaaagggtc tcccgaagat aatgagcaga agcagctgtt cgtggaacaa 6601 cacaaacact accttgatga gatcatcgag caaataagcg aattctccaa aagagtgatc 6661 ctcgccgacg ctaacctcga taaggtgctt tctgcttaca ataagcacag ggataagccc 6721 atcagggagc aggcagaaaa cattatccac ttgtttactc tgaccaactt gggcgcgcct 6781 gcagccttca agtacttcga caccaccata gacagaaagc ggtacacctc tacaaaggag 6841 gtcctggacg ccacactgat tcatcagtca attacggggc tctatgaaac aagaatcgac 6901 ctctctcagc tcggtggaga cagcagggct gacgggtccg gcagcgggtc catggtgagc 6961 aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta 7021 aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg 7081 accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc 7141 accttcacct acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac 7201 ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac 7261 gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc 7321 atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag 7381 tacaactaca acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag 7441 gtgaacttca agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac 7501 cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc 7561 acccagtccg ccctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag 7621 ttcgtgaccg ccgccgggat cactcacggc atggacgagc tgtacaagcc gaaaaagaag 7681 aggaaggtct gacaattgga aagatccaag agagacgagt agagattttt tttttgggat 7741 tgatgtttgt cgttctttga gttgtcgtcg agttacgcct tttgtaagaa tgttccgcag 7801 gagaggagga ggatgggcat gagtgagggt gagagggctt gcccgctttt ttttttaaaa 7861 acgctgaaga cgtggttgtc aaacaaaccc cccatagaaa cgattttgtt acggtgcggt 7921 ccagacgtca cttgaatggc tccgcggaaa ggccagggag ggaagggggg agggaggaaa 7981 catgaaacat gttgaacggc tcaacagggt ttgggggaca agagaggtag cgccctgatg 8041 gactgctccc tcccctcctt tccctcaatg tctcattcat ccatgcttcc cccttctctc 8101 tctcccctcc gttccatccc ccgcgggcgt ggtagtggcg tgatgggatc cactaaaatg 8161 tacgtgtaag aaaagccggt gagcttacgc ttttgtgaaa gtgggagtac gagtgttgtg 8221 tgtgtgtgta gtggtttcag accccagaca gaggcgaagc agaaaaagca gacgatgaag 8281 acgacgaaga aatgagcagt ctatttttat cgtggaaaca gaagaggtga tatcgtctcc 8341 tgcaggcatg caagctgatc cactagaggc catggcggcc gcactagtgg atcccccggg 8401 atgcatactt ggaagatgga gtggatggag gaggaggcga gcgtagcagc aagcgtgagt 8461 tatacagcca ggcacatgtc gcaatccttc ggtctcgggc ttaaaatcca cgcactaatc 8521 acgctgggcc atgcaaagag caatgccgag gcccaccaca caaaacgctg tgtcgcgcgt 8581 tgcggcctga agcttcatac ttcttagtcg ccgccaaaag ggctcgagag acgagacccg 8641 ttggcatgac cgatgttgtt cgacgcggtt tgcttcgtca cagtcgacgt gattcaggaa 8701 tctggagcct gcagatcatt tttttcagcc tgatatcgtt cttttccact gagaaccatc 8761 agaccacctt ttcttccatt gtgtgaagga gtaggagttg ccgtgctgct ttgtgggaga 8821 catctgcgat ggtgaccagc ctcccgtcgt ctggtcgacg tgacgagcct cttcactgtt 8881 cttcgacgga gagacgcaag cgagacggct ctagaccttt tggacacgca ttctgtgtgt 8941 gaactagtgg acagtgatac cacgtctgaa agctcaccac tgcccatggt gcagctactt 9001 gtcacaaagt tttgactccg tcggtatcac cattcgcgct cgtgtgcctg gttgttccgc 9061 cacgccggcc tgccccgggg cggggcaata ttctaaaatc tcacgcaaaa caccgcactt 9121 acccctcaca catattcgtg atagaccacc accaatctca gcccgcatca acacaggagg 9181 gccccggggg gcaataagat atgaaaaagc ctgaactcac cgcgacgtct gtcgagaagt 9241 ttctgatcga aaagttcgac agcgtctccg acctgatgca gctctcggag ggcgaagaat 9301 ctcgtgcttt cagcttcgat gtaggagggc gtggatatgt cctgcgggta aatagctgcg 9361 ccgatggttt ctacaaagat cgttatgttt atcggcactt tgcatcggcc gcgctcccga 9421 ttccggaagt gcttgacatt ggggaattca gcgagagcct gacctattgc atctcccgcc 9481 gtgcacaggg tgtcacgttg caagacctgc ctgaaaccga actgcccgct gttctgcagc 9541 cggtcgcgga ggccatggat gcgatcgctg cggccgatct tagccagacg agcgggttcg 9601 gcccattcgg accgcaagga atcggtcaat acactacatg gcgtgatttc atatgcgcga 9661 ttgctgatcc ccatgtgtat cactggcaaa ctgtgatgga cgacaccgtc agtgcgtccg 9721 tcgcgcaggc tctcgatgag ctgatgcttt gggccgagga ctgccccgaa gtccggcacc 9781 tcgtgcacgc ggatttcggc tccaacaatg tcctgacgga caatggccgc ataacagcgg 9841 tcattgactg gagcgaggcg atgttcgggg attcccaata cgaggtcgcc aacatcttct 9901 tctggaggcc gtggttggct tgtatggagc agcagacgcg ctacttcgag cggaggcatc 9961 cggagcttgc aggatcgccg cggctccggg cgtatatgct ccgcattggt cttgaccaac 10021 tctatcagag cttggttgac ggcaatttcg atgatgcagc ttgggcgcag ggtcgatgcg 10081 acgcaatcgt ccgatccgga gccgggactg tcgggcgtac acaaatcgcc cgcagaagcg 10141 cggccgtctg gaccgatggc tgtgtagaag tactcgccga tagtggaaac cgacgcccca 10201 gcactcgtcc gagggcaaag gaatagagta gatgccgacc ggagtccgca aaaatcacca 10261 gtctctctct acaaatctat ctctctctat ttttctccag aataatgtgt gagtagttcc 10321 cagataaggg aattagggtt cttatagggt ttcgctcatg tgttgagcat ataagaaacc 10381 cttagtatgt atttgtattt gtaaaatact tctatcaata aaatttctaa ttcctaaaac 10441 caaaatccag tgacctgagc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg 10501 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat 10561 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc 10621 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc 10681 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga 10741 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt 10801 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg 10861 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc 10921 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg 10981 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc 11041 ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg 11101 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc 11161 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct 11221 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt 11281 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa 11341 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa 11401 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc 11461 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct 11521 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca 11581 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt 11641 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt 11701 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc 11761 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc 11821 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt 11881 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact 11941 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc 12001 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt 12061 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg 12121 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct 12181 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa 12241 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt 12301 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc 12361 acatttcccc gaaaagtgcc acctgatgcg gtgtgaaata ccgcacagat gcgtaaggag 12421 aaaataccgc atcaggaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt 12481 tgttaaatca gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca 12541 aaagaataga ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta 12601 aagaacgtgg actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta 12661 cgtgaaccat caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg 12721 aaccctaaag ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga 12781 aaggaaggga agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg 12841 ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtccattcgc 12901 cattcaggct gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc 12961 agctggcgaa agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc 13021 agtcacgacg ttgtaaaacg acggccagtg aattgtaata cgactcacta ta // LOCUS Exported 12379 bp ds-DNA circular SYN 14-JUL-2017 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS pNOC-CRISPR-HA SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 12379) AUTHORS . TITLE Direct Submission JOURNAL Exported Friday, Jul 14, 2017 from SnapGene Viewer 3.3.4 http://www.snapgene.com FEATURES Location/Qualifiers source 1..12379 /organism="synthetic DNA construct" /mol_type="other DNA" terminator 418..1418 /label=CS terminator misc_feature 1425..1501 /label=hdv misc_RNA complement(1502..1577) /label=g RNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" promoter 1606..2789 /label=Ribi CDS 2800..2820 /codon_start=1 /product="nuclear localization signal of SV40 large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 2821..6921 /codon_start=1 /product="Cas9 (Csn1) endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system" /label=Cas9 /note="generates RNA-guided double strand breaks in DNA" /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD ATLIHQSITGLYETRIDLSQLGGD" CDS 6946..6972 /codon_start=1 /product="HA (human influenza hemagglutinin) epitope tag" /label=HA /translation="YPYDVPDYA" CDS 6976..6996 /codon_start=1 /product="nuclear localization signal of SV40 large T antigen" /label=SV40 NLS /translation="PKKKRKV" terminator 7028..7627 /label=LDSP terminator promoter 7714..8483 /label=LDSP promoter CDS 8508..9533 /codon_start=1 /gene="aph(4)-Ia" /product="aminoglycoside phosphotransferase from E. coli" /label=HygR /note="confers resistance to hygromycin" /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK E" terminator 9534..9764 /label=35S terminator rep_origin complement(9942..10530) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" CDS complement(10701..11561) /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" promoter complement(11562..11666) /gene="bla" /label=AmpR promoter rep_origin complement(11744..12199) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind 12340..12356 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" ORIGIN 1 gggcgaattg ggcccgacgt cgcatgctcc cggccgccat ggcggccgcg ggaattcgat 61 atcactagag tatagctaca tggtagctag tagtcgtcaa aggaaataaa gacatagcgt 121 aaagtataca aaatgacttc cgcggcgcgg gagcggcagg cagcacttgc aaaacgaagc 181 ccaatgggcc agattacggt caacctcgtc attttattac ggtcgtcagc aagagcggtt 241 tagaccgtcg ccggcacggg tggctcccga tattcttggc tttcgtgggt ggaggcggcg 301 tgtgactcct catctttaag tagtggtcgc ttttgctgca tgcttttgat cacctcgtga 361 ttgatggctg gattctcagc tgtcttctga catctttggg tgcatgggtt gaccggtgca 421 aagctgacgc ccttttcgtc gtcgttgctg gtgttaaaat tgcgcgtttc ttttttttcc 481 gtctttgtcg tcttttgact tttttatctt cttttctgtc ttcttttatt cttctcttct 541 tcttcatcac tccctcttca tttttcttct tctttttcct cgtctattgt cctcgggcat 601 taccagttgt gctgctcacg tgcgcgcaag cggtgaaagg gtgagaggat acacggacaa 661 caatcattga aaattacgct taaagccggt gcctggaaaa gtccacgcgc agatggcctt 721 tgcttgcctt gctgctgctg cagctgctgc tgctgtcgcc tgtgatggta atggttgttg 781 ttgtggtcct gcttgcttgc tagtttgcat tgcgtttgcg gttgccttat gaccgactcc 841 gtgtgattga tccttaaaca tattttatca ttgatgtgga ttgtgtgacg acggcaaaaa 901 agaattactt tacacatgac tatcacacct gctcccctcc cttctccttt caatcggtca 961 acttcccaac cgctcctcga aaatatttca gttgaatcct ctactacgta ccccttactt 1021 aaatttactt tctctctccc catcaatgcg ccgttgttct gaagttgtat tattgatggg 1081 agatgattgg cactgcacca gacctggcca aaagccacca caggtggcca catactcttg 1141 catgggtata ctctaccgag gcaaagatta aagtgcacgt cccctgcccc tctctctccc 1201 tacttccttc agtctcagtg gttccgtgcc cgttcagcac tacatcgccc tgcccgctgt 1261 tccttctcaa acagccaccc atcccaaccc acatacacag aaacaacctt tcctacacat 1321 tttcccaagt cttctcttca ctattctaca ctaatacacc accgtgatat gccggttatc 1381 tagatgtcct ttctcctttc tcatcctttc tttcccatgg tacccaggag tcccattcgc 1441 catgccgaag catgttgccc agccggcgcc agcgaggagg ctgggaccat gccggccaaa 1501 agcaccgact cggtgccact ttttcaagtt gataacggac tagccttatt ttaacttgct 1561 atttctagct ctaaaacgaa gagccaaggc ctgctcttca tcgattctgt ggatggaggg 1621 agggaaggag ggggggagtg agtgagtcga gagaacgacg actggggcaa agagaggatg 1681 acgccactgc aacaaaagac agggaagaat agaggctgtt ggaaaacagg aacacagtga 1741 agaatagagg ctgcagggta acaggcacat ttatcgcaga gaggcgggcg gcaggggcga 1801 tacgtaaaga gacccctaaa cttctccgcg tcatccgttc acgctacctc cttccctccc 1861 tttactccca ttgatcacca aggcagcgcc cgcagccacc agcagccacc gtcctcagtg 1921 tcacagctag acgcgtcccc atcaggcagc agaccaccag aatatgcgct ttcgcttccc 1981 atcatatccc tccctcctcc ccttctattc ctcatcatgc atcatttctt tctccttgct 2041 gcatgcccct cttcttacct tggctaagat ctaccggagg aagaagtctt caacgataga 2101 taaatgcctg tatcgtgatg gttgtttgaa tggtggaagt gagccaggta ttgagaacga 2161 ggggcgcaag ggtgcgtgga agaaggcagg gagaggcgag gcgaaggaga agggcatgtg 2221 gtgggaacga agcctccaca ccacagccac ccatgctttt ctctgttcga gttcccgggt 2281 tctactctca cacactacac gtgcacagaa aacattcacg cacaacagag atacagaggg 2341 atccgtcgag atggtctaca cgtcctaatg tcccctgctt tcgaagccca gagccatcgc 2401 tttctgtttt tatccattcg caacacatcc tgttctcgcg acctctcctc ttcctggcca 2461 ccatcgcttc tccgtgccat gccctgtaca tccttccctc cctcgttgtc taccacacta 2521 atcatgcgct ctgcagcaga cactcggcta ctatttgtgt cgtacggatt gaggggaggg 2581 aggggaaggg aggcaagaag taaatcggtg gtcaacaaca gacgcggtct ccccaccctc 2641 cctcattcct ttcccctcct ataatgagcg aagaaagcca tggacaccga gtcacccact 2701 ctcctaatcc accaccacca gggcactaac ggactcacgc tcaccaaact cggtaacaat 2761 atccactgca cacagataca cacgcaacac tcgagcatgc ccaagaaaaa gcggaaggtg 2821 gacaagaagt actccattgg gctcgatatc ggcacaaaca gcgtcggctg ggccgtcatt 2881 acggacgagt acaaggtgcc gagcaaaaaa ttcaaagttc tgggcaatac cgatcgccac 2941 agcataaaga agaacctcat tggcgccctc ctgttcgact ccggggagac ggccgaagcc 3001 acgcggctca aaagaacagc acggcgcaga tatacccgca gaaagaatcg gatctgctac 3061 ctgcaggaga tctttagtaa tgagatggct aaggtggatg actctttctt ccataggctg 3121 gaggagtcct ttttggtgga ggaggataaa aagcacgagc gccacccaat ctttggcaat 3181 atcgtggacg aggtggcgta ccatgaaaag tacccaacca tatatcatct gaggaagaag 3241 cttgtagaca gtactgataa ggctgacttg cggttgatct atctcgcgct ggcgcatatg 3301 atcaaatttc ggggacactt cctcatcgag ggggacctga acccagacaa cagcgatgtc 3361 gacaaactct ttatccaact ggttcagact tacaatcagc ttttcgaaga gaacccgatc 3421 aacgcatccg gagttgacgc caaagcaatc ctgagcgcta ggctgtccaa atcccggcgg 3481 ctcgaaaacc tcatcgcaca gctccctggg gagaagaaga acggcctgtt tggtaatctt 3541 atcgccctgt cactcgggct gacccccaac tttaaatcta acttcgacct ggccgaagat 3601 gccaagcttc aactgagcaa agacacctac gatgatgatc tcgacaatct gctggcccag 3661 atcggcgacc agtacgcaga cctttttttg gcggcaaaga acctgtcaga cgccattctg 3721 ctgagtgata ttctgcgagt gaacacggag atcaccaaag ctccgctgag cgctagtatg 3781 atcaagcgct atgatgagca ccaccaagac ttgactttgc tgaaggccct tgtcagacag 3841 caactgcctg agaagtacaa ggaaattttc ttcgatcagt ctaaaaatgg ctacgccgga 3901 tacattgacg gcggagcaag ccaggaggaa ttttacaaat ttattaagcc catcttggaa 3961 aaaatggacg gcaccgagga gctgctggta aagcttaaca gagaagatct gttgcgcaaa 4021 cagcgcactt tcgacaatgg aagcatcccc caccagattc acctgggcga actgcacgct 4081 atcctcaggc ggcaagagga tttctacccc tttttgaaag ataacaggga aaagattgag 4141 aaaatcctca catttcggat accctactat gtaggccccc tcgcccgggg aaattccaga 4201 ttcgcgtgga tgactcgcaa atcagaagag accatcactc cctggaactt cgaggaagtc 4261 gtggataagg gggcctctgc ccagtccttc atcgaaagga tgactaactt tgataaaaat 4321 ctgcctaacg aaaaggtgct tcctaaacac tctctgctgt acgagtactt cacagtttat 4381 aacgagctca ccaaggtcaa atacgtcaca gaagggatga gaaagccagc attcctgtct 4441 ggagagcaga agaaagctat cgtggacctc ctcttcaaga cgaaccggaa agttaccgtg 4501 aaacagctca aagaagacta tttcaaaaag attgaatgtt tcgactctgt tgaaatcagc 4561 ggagtggagg atcgcttcaa cgcatccctg ggaacgtatc acgatctcct gaaaatcatt 4621 aaagacaagg acttcctgga caatgaggag aacgaggaca ttcttgagga cattgtcctc 4681 acccttacgt tgtttgaaga tagggagatg attgaagaac gcttgaaaac ttacgctcat 4741 ctcttcgacg acaaagtcat gaaacagctc aagaggcgcc gatatacagg atgggggcgg 4801 ctgtcaagaa aactgatcaa tgggatccga gacaagcaga gtggaaagac aatcctggat 4861 tttcttaagt ccgatggatt tgccaaccgg aacttcatgc agttgatcca tgatgactct 4921 ctcaccttta aggaggacat ccagaaagca caagtttctg gccaggggga cagtcttcac 4981 gagcacatcg ctaatcttgc aggtagccca gctatcaaaa agggaatact gcagaccgtt 5041 aaggtcgtgg atgaactcgt caaagtaatg ggaaggcata agcccgagaa tatcgttatc 5101 gagatggccc gagagaacca aactacccag aagggacaga agaacagtag ggaaaggatg 5161 aagaggattg aagagggtat aaaagaactg gggtcccaaa tccttaagga acacccagtt 5221 gaaaacaccc agcttcagaa tgagaagctc tacctgtact acctgcagaa cggcagggac 5281 atgtacgtgg atcaggaact ggacatcaat cggctctccg actacgacgt ggatcatatc 5341 gtgccccagt cttttctcaa agatgattct attgataata aagtgttgac aagatccgat 5401 aaaaatagag ggaagagtga taacgtcccc tcagaagaag ttgtcaagaa aatgaaaaat 5461 tattggcggc agctgctgaa cgccaaactg atcacacaac ggaagttcga taatctgact 5521 aaggctgaac gaggtggcct gtctgagttg gataaagccg gcttcatcaa aaggcagctt 5581 gttgagacac gccagatcac caagcacgtg gcccaaattc tcgattcacg catgaacacc 5641 aagtacgatg aaaatgacaa actgattcga gaggtgaaag ttattactct gaagtctaag 5701 ctggtctcag atttcagaaa ggactttcag ttttataagg tgagagagat caacaattac 5761 caccatgcgc atgatgccta cctgaatgca gtggtaggca ctgcacttat caaaaaatat 5821 cccaagcttg aatctgaatt tgtttacgga gactataaag tgtacgatgt taggaaaatg 5881 atcgcaaagt ctgagcagga aataggcaag gccaccgcta agtacttctt ttacagcaat 5941 attatgaatt ttttcaagac cgagattaca ctggccaatg gagagattcg gaagcgacca 6001 cttatcgaaa caaacggaga aacaggagaa atcgtgtggg acaagggtag ggatttcgcg 6061 acagtccgga aggtcctgtc catgccgcag gtgaacatcg ttaaaaagac cgaagtacag 6121 accggaggct tctccaagga aagtatcctc ccgaaaagga acagcgacaa gctgatcgca 6181 cgcaaaaaag attgggaccc caagaaatac ggcggattcg attctcctac agtcgcttac 6241 agtgtactgg ttgtggccaa agtggagaaa gggaagtcta aaaaactcaa aagcgtcaag 6301 gaactgctgg gcatcacaat catggagcga tcaagcttcg aaaaaaaccc catcgacttt 6361 ctcgaggcga aaggatataa agaggtcaaa aaagacctca tcattaagct tcccaagtac 6421 tctctctttg agcttgaaaa cggccggaaa cgaatgctcg ctagtgcggg cgagctgcag 6481 aaaggtaacg agctggcact gccctctaaa tacgttaatt tcttgtatct ggccagccac 6541 tatgaaaagc tcaaagggtc tcccgaagat aatgagcaga agcagctgtt cgtggaacaa 6601 cacaaacact accttgatga gatcatcgag caaataagcg aattctccaa aagagtgatc 6661 ctcgccgacg ctaacctcga taaggtgctt tctgcttaca ataagcacag ggataagccc 6721 atcagggagc aggcagaaaa cattatccac ttgtttactc tgaccaactt gggcgcgcct 6781 gcagccttca agtacttcga caccaccata gacagaaagc ggtacacctc tacaaaggag 6841 gtcctggacg ccacactgat tcatcagtca attacggggc tctatgaaac aagaatcgac 6901 ctctctcagc tcggtggaga cgggtccggc agcgggtccg ttaactaccc ctatgacgtg 6961 ccggactacg ctagcccgaa aaagaagagg aaggtctgac aattggaaag atccaagaga 7021 gacgagtaga gatttttttt ttgggattga tgtttgtcgt tctttgagtt gtcgtcgagt 7081 tacgcctttt gtaagaatgt tccgcaggag aggaggagga tgggcatgag tgagggtgag 7141 agggcttgcc cgcttttttt tttaaaaacg ctgaagacgt ggttgtcaaa caaacccccc 7201 atagaaacga ttttgttacg gtgcggtcca gacgtcactt gaatggctcc gcggaaaggc 7261 cagggaggga aggggggagg gaggaaacat gaaacatgtt gaacggctca acagggtttg 7321 ggggacaaga gaggtagcgc cctgatggac tgctccctcc cctcctttcc ctcaatgtct 7381 cattcatcca tgcttccccc ttctctctct cccctccgtt ccatcccccg cgggcgtggt 7441 agtggcgtga tgggatccac taaaatgtac gtgtaagaaa agccggtgag cttacgcttt 7501 tgtgaaagtg ggagtacgag tgttgtgtgt gtgtgtagtg gtttcagacc ccagacagag 7561 gcgaagcaga aaaagcagac gatgaagacg acgaagaaat gagcagtcta tttttatcgt 7621 ggaaacagaa gaggtgatat cgtctcctgc aggcatgcaa gctgatccac tagaggccat 7681 ggcggccgca ctagtggatc ccccgggatg catacttgga agatggagtg gatggaggag 7741 gaggcgagcg tagcagcaag cgtgagttat acagccaggc acatgtcgca atccttcggt 7801 ctcgggctta aaatccacgc actaatcacg ctgggccatg caaagagcaa tgccgaggcc 7861 caccacacaa aacgctgtgt cgcgcgttgc ggcctgaagc ttcatacttc ttagtcgccg 7921 ccaaaagggc tcgagagacg agacccgttg gcatgaccga tgttgttcga cgcggtttgc 7981 ttcgtcacag tcgacgtgat tcaggaatct ggagcctgca gatcattttt ttcagcctga 8041 tatcgttctt ttccactgag aaccatcaga ccaccttttc ttccattgtg tgaaggagta 8101 ggagttgccg tgctgctttg tgggagacat ctgcgatggt gaccagcctc ccgtcgtctg 8161 gtcgacgtga cgagcctctt cactgttctt cgacggagag acgcaagcga gacggctcta 8221 gaccttttgg acacgcattc tgtgtgtgaa ctagtggaca gtgataccac gtctgaaagc 8281 tcaccactgc ccatggtgca gctacttgtc acaaagtttt gactccgtcg gtatcaccat 8341 tcgcgctcgt gtgcctggtt gttccgccac gccggcctgc cccggggcgg ggcaatattc 8401 taaaatctca cgcaaaacac cgcacttacc cctcacacat attcgtgata gaccaccacc 8461 aatctcagcc cgcatcaaca caggagggcc ccggggggca ataagatatg aaaaagcctg 8521 aactcaccgc gacgtctgtc gagaagtttc tgatcgaaaa gttcgacagc gtctccgacc 8581 tgatgcagct ctcggagggc gaagaatctc gtgctttcag cttcgatgta ggagggcgtg 8641 gatatgtcct gcgggtaaat agctgcgccg atggtttcta caaagatcgt tatgtttatc 8701 ggcactttgc atcggccgcg ctcccgattc cggaagtgct tgacattggg gaattcagcg 8761 agagcctgac ctattgcatc tcccgccgtg cacagggtgt cacgttgcaa gacctgcctg 8821 aaaccgaact gcccgctgtt ctgcagccgg tcgcggaggc catggatgcg atcgctgcgg 8881 ccgatcttag ccagacgagc gggttcggcc cattcggacc gcaaggaatc ggtcaataca 8941 ctacatggcg tgatttcata tgcgcgattg ctgatcccca tgtgtatcac tggcaaactg 9001 tgatggacga caccgtcagt gcgtccgtcg cgcaggctct cgatgagctg atgctttggg 9061 ccgaggactg ccccgaagtc cggcacctcg tgcacgcgga tttcggctcc aacaatgtcc 9121 tgacggacaa tggccgcata acagcggtca ttgactggag cgaggcgatg ttcggggatt 9181 cccaatacga ggtcgccaac atcttcttct ggaggccgtg gttggcttgt atggagcagc 9241 agacgcgcta cttcgagcgg aggcatccgg agcttgcagg atcgccgcgg ctccgggcgt 9301 atatgctccg cattggtctt gaccaactct atcagagctt ggttgacggc aatttcgatg 9361 atgcagcttg ggcgcagggt cgatgcgacg caatcgtccg atccggagcc gggactgtcg 9421 ggcgtacaca aatcgcccgc agaagcgcgg ccgtctggac cgatggctgt gtagaagtac 9481 tcgccgatag tggaaaccga cgccccagca ctcgtccgag ggcaaaggaa tagagtagat 9541 gccgaccgga gtccgcaaaa atcaccagtc tctctctaca aatctatctc tctctatttt 9601 tctccagaat aatgtgtgag tagttcccag ataagggaat tagggttctt atagggtttc 9661 gctcatgtgt tgagcatata agaaaccctt agtatgtatt tgtatttgta aaatacttct 9721 atcaataaaa tttctaattc ctaaaaccaa aatccagtga cctgagcttc ctcgctcact 9781 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta 9841 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag 9901 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc 9961 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta 10021 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg 10081 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc 10141 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac 10201 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac 10261 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg 10321 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga 10381 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt 10441 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag 10501 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct 10561 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg 10621 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat 10681 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc 10741 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg 10801 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct 10861 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca 10921 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg 10981 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg 11041 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc 11101 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag 11161 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg 11221 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag 11281 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat 11341 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg 11401 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca 11461 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca 11521 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat 11581 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag 11641 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgatgcggtg 11701 tgaaataccg cacagatgcg taaggagaaa ataccgcatc aggaaattgt aagcgttaat 11761 attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa ccaataggcc 11821 gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt gagtgttgtt 11881 ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa agggcgaaaa 11941 accgtctatc agggcgatgg cccactacgt gaaccatcac cctaatcaag ttttttgggg 12001 tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt tagagcttga 12061 cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct 12121 agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat 12181 gcgccgctac agggcgcgtc cattcgccat tcaggctgcg caactgttgg gaagggcgat 12241 cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat 12301 taagttgggt aacgccaggg ttttcccagt cacgacgttg taaaacgacg gccagtgaat 12361 tgtaatacga ctcactata // LOCUS Exported 12379 bp ds-DNA circular SYN 14-JUL-2017 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS pNOC-CRISPR-HA SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 12379) AUTHORS . TITLE Direct Submission JOURNAL Exported Friday, Jul 14, 2017 from SnapGene Viewer 3.3.4 http://www.snapgene.com FEATURES Location/Qualifiers source 1..12379 /organism="synthetic DNA construct" /mol_type="other DNA" terminator 418..1418 /label=CS terminator misc_feature 1425..1501 /label=hdv misc_RNA complement(1502..1577) /label=g RNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" promoter 1606..2789 /label=Ribi CDS 2800..2820 /codon_start=1 /product="nuclear localization signal of SV40 large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 2821..6921 /codon_start=1 /product="Cas9 (Csn1) endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system" /label=Cas9 /note="generates RNA-guided double strand breaks in DNA" /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD ATLIHQSITGLYETRIDLSQLGGD" CDS 6946..6972 /codon_start=1 /product="HA (human influenza hemagglutinin) epitope tag" /label=HA /translation="YPYDVPDYA" CDS 6976..6996 /codon_start=1 /product="nuclear localization signal of SV40 large T antigen" /label=SV40 NLS /translation="PKKKRKV" terminator 7028..7627 /label=LDSP terminator promoter 7714..8483 /label=LDSP promoter CDS 8508..9533 /codon_start=1 /gene="aph(4)-Ia" /product="aminoglycoside phosphotransferase from E. coli" /label=HygR /note="confers resistance to hygromycin" /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK E" terminator 9534..9764 /label=35S terminator rep_origin complement(9942..10530) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" CDS complement(10701..11561) /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" promoter complement(11562..11666) /gene="bla" /label=AmpR promoter rep_origin complement(11744..12199) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind 12340..12356 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" ORIGIN 1 gggcgaattg ggcccgacgt cgcatgctcc cggccgccat ggcggccgcg ggaattcgat 61 atcactagag tatagctaca tggtagctag tagtcgtcaa aggaaataaa gacatagcgt 121 aaagtataca aaatgacttc cgcggcgcgg gagcggcagg cagcacttgc aaaacgaagc 181 ccaatgggcc agattacggt caacctcgtc attttattac ggtcgtcagc aagagcggtt 241 tagaccgtcg ccggcacggg tggctcccga tattcttggc tttcgtgggt ggaggcggcg 301 tgtgactcct catctttaag tagtggtcgc ttttgctgca tgcttttgat cacctcgtga 361 ttgatggctg gattctcagc tgtcttctga catctttggg tgcatgggtt gaccggtgca 421 aagctgacgc ccttttcgtc gtcgttgctg gtgttaaaat tgcgcgtttc ttttttttcc 481 gtctttgtcg tcttttgact tttttatctt cttttctgtc ttcttttatt cttctcttct 541 tcttcatcac tccctcttca tttttcttct tctttttcct cgtctattgt cctcgggcat 601 taccagttgt gctgctcacg tgcgcgcaag cggtgaaagg gtgagaggat acacggacaa 661 caatcattga aaattacgct taaagccggt gcctggaaaa gtccacgcgc agatggcctt 721 tgcttgcctt gctgctgctg cagctgctgc tgctgtcgcc tgtgatggta atggttgttg 781 ttgtggtcct gcttgcttgc tagtttgcat tgcgtttgcg gttgccttat gaccgactcc 841 gtgtgattga tccttaaaca tattttatca ttgatgtgga ttgtgtgacg acggcaaaaa 901 agaattactt tacacatgac tatcacacct gctcccctcc cttctccttt caatcggtca 961 acttcccaac cgctcctcga aaatatttca gttgaatcct ctactacgta ccccttactt 1021 aaatttactt tctctctccc catcaatgcg ccgttgttct gaagttgtat tattgatggg 1081 agatgattgg cactgcacca gacctggcca aaagccacca caggtggcca catactcttg 1141 catgggtata ctctaccgag gcaaagatta aagtgcacgt cccctgcccc tctctctccc 1201 tacttccttc agtctcagtg gttccgtgcc cgttcagcac tacatcgccc tgcccgctgt 1261 tccttctcaa acagccaccc atcccaaccc acatacacag aaacaacctt tcctacacat 1321 tttcccaagt cttctcttca ctattctaca ctaatacacc accgtgatat gccggttatc 1381 tagatgtcct ttctcctttc tcatcctttc tttcccatgg tacccaggag tcccattcgc 1441 catgccgaag catgttgccc agccggcgcc agcgaggagg ctgggaccat gccggccaaa 1501 agcaccgact cggtgccact ttttcaagtt gataacggac tagccttatt ttaacttgct 1561 atttctagct ctaaaacgaa gagccaaggc ctgctcttca tcgattctgt ggatggaggg 1621 agggaaggag ggggggagtg agtgagtcga gagaacgacg actggggcaa agagaggatg 1681 acgccactgc aacaaaagac agggaagaat agaggctgtt ggaaaacagg aacacagtga 1741 agaatagagg ctgcagggta acaggcacat ttatcgcaga gaggcgggcg gcaggggcga 1801 tacgtaaaga gacccctaaa cttctccgcg tcatccgttc acgctacctc cttccctccc 1861 tttactccca ttgatcacca aggcagcgcc cgcagccacc agcagccacc gtcctcagtg 1921 tcacagctag acgcgtcccc atcaggcagc agaccaccag aatatgcgct ttcgcttccc 1981 atcatatccc tccctcctcc ccttctattc ctcatcatgc atcatttctt tctccttgct 2041 gcatgcccct cttcttacct tggctaagat ctaccggagg aagaagtctt caacgataga 2101 taaatgcctg tatcgtgatg gttgtttgaa tggtggaagt gagccaggta ttgagaacga 2161 ggggcgcaag ggtgcgtgga agaaggcagg gagaggcgag gcgaaggaga agggcatgtg 2221 gtgggaacga agcctccaca ccacagccac ccatgctttt ctctgttcga gttcccgggt 2281 tctactctca cacactacac gtgcacagaa aacattcacg cacaacagag atacagaggg 2341 atccgtcgag atggtctaca cgtcctaatg tcccctgctt tcgaagccca gagccatcgc 2401 tttctgtttt tatccattcg caacacatcc tgttctcgcg acctctcctc ttcctggcca 2461 ccatcgcttc tccgtgccat gccctgtaca tccttccctc cctcgttgtc taccacacta 2521 atcatgcgct ctgcagcaga cactcggcta ctatttgtgt cgtacggatt gaggggaggg 2581 aggggaaggg aggcaagaag taaatcggtg gtcaacaaca gacgcggtct ccccaccctc 2641 cctcattcct ttcccctcct ataatgagcg aagaaagcca tggacaccga gtcacccact 2701 ctcctaatcc accaccacca gggcactaac ggactcacgc tcaccaaact cggtaacaat 2761 atccactgca cacagataca cacgcaacac tcgagcatgc ccaagaaaaa gcggaaggtg 2821 gacaagaagt actccattgg gctcgatatc ggcacaaaca gcgtcggctg ggccgtcatt 2881 acggacgagt acaaggtgcc gagcaaaaaa ttcaaagttc tgggcaatac cgatcgccac 2941 agcataaaga agaacctcat tggcgccctc ctgttcgact ccggggagac ggccgaagcc 3001 acgcggctca aaagaacagc acggcgcaga tatacccgca gaaagaatcg gatctgctac 3061 ctgcaggaga tctttagtaa tgagatggct aaggtggatg actctttctt ccataggctg 3121 gaggagtcct ttttggtgga ggaggataaa aagcacgagc gccacccaat ctttggcaat 3181 atcgtggacg aggtggcgta ccatgaaaag tacccaacca tatatcatct gaggaagaag 3241 cttgtagaca gtactgataa ggctgacttg cggttgatct atctcgcgct ggcgcatatg 3301 atcaaatttc ggggacactt cctcatcgag ggggacctga acccagacaa cagcgatgtc 3361 gacaaactct ttatccaact ggttcagact tacaatcagc ttttcgaaga gaacccgatc 3421 aacgcatccg gagttgacgc caaagcaatc ctgagcgcta ggctgtccaa atcccggcgg 3481 ctcgaaaacc tcatcgcaca gctccctggg gagaagaaga acggcctgtt tggtaatctt 3541 atcgccctgt cactcgggct gacccccaac tttaaatcta acttcgacct ggccgaagat 3601 gccaagcttc aactgagcaa agacacctac gatgatgatc tcgacaatct gctggcccag 3661 atcggcgacc agtacgcaga cctttttttg gcggcaaaga acctgtcaga cgccattctg 3721 ctgagtgata ttctgcgagt gaacacggag atcaccaaag ctccgctgag cgctagtatg 3781 atcaagcgct atgatgagca ccaccaagac ttgactttgc tgaaggccct tgtcagacag 3841 caactgcctg agaagtacaa ggaaattttc ttcgatcagt ctaaaaatgg ctacgccgga 3901 tacattgacg gcggagcaag ccaggaggaa ttttacaaat ttattaagcc catcttggaa 3961 aaaatggacg gcaccgagga gctgctggta aagcttaaca gagaagatct gttgcgcaaa 4021 cagcgcactt tcgacaatgg aagcatcccc caccagattc acctgggcga actgcacgct 4081 atcctcaggc ggcaagagga tttctacccc tttttgaaag ataacaggga aaagattgag 4141 aaaatcctca catttcggat accctactat gtaggccccc tcgcccgggg aaattccaga 4201 ttcgcgtgga tgactcgcaa atcagaagag accatcactc cctggaactt cgaggaagtc 4261 gtggataagg gggcctctgc ccagtccttc atcgaaagga tgactaactt tgataaaaat 4321 ctgcctaacg aaaaggtgct tcctaaacac tctctgctgt acgagtactt cacagtttat 4381 aacgagctca ccaaggtcaa atacgtcaca gaagggatga gaaagccagc attcctgtct 4441 ggagagcaga agaaagctat cgtggacctc ctcttcaaga cgaaccggaa agttaccgtg 4501 aaacagctca aagaagacta tttcaaaaag attgaatgtt tcgactctgt tgaaatcagc 4561 ggagtggagg atcgcttcaa cgcatccctg ggaacgtatc acgatctcct gaaaatcatt 4621 aaagacaagg acttcctgga caatgaggag aacgaggaca ttcttgagga cattgtcctc 4681 acccttacgt tgtttgaaga tagggagatg attgaagaac gcttgaaaac ttacgctcat 4741 ctcttcgacg acaaagtcat gaaacagctc aagaggcgcc gatatacagg atgggggcgg 4801 ctgtcaagaa aactgatcaa tgggatccga gacaagcaga gtggaaagac aatcctggat 4861 tttcttaagt ccgatggatt tgccaaccgg aacttcatgc agttgatcca tgatgactct 4921 ctcaccttta aggaggacat ccagaaagca caagtttctg gccaggggga cagtcttcac 4981 gagcacatcg ctaatcttgc aggtagccca gctatcaaaa agggaatact gcagaccgtt 5041 aaggtcgtgg atgaactcgt caaagtaatg ggaaggcata agcccgagaa tatcgttatc 5101 gagatggccc gagagaacca aactacccag aagggacaga agaacagtag ggaaaggatg 5161 aagaggattg aagagggtat aaaagaactg gggtcccaaa tccttaagga acacccagtt 5221 gaaaacaccc agcttcagaa tgagaagctc tacctgtact acctgcagaa cggcagggac 5281 atgtacgtgg atcaggaact ggacatcaat cggctctccg actacgacgt ggatcatatc 5341 gtgccccagt cttttctcaa agatgattct attgataata aagtgttgac aagatccgat 5401 aaaaatagag ggaagagtga taacgtcccc tcagaagaag ttgtcaagaa aatgaaaaat 5461 tattggcggc agctgctgaa cgccaaactg atcacacaac ggaagttcga taatctgact 5521 aaggctgaac gaggtggcct gtctgagttg gataaagccg gcttcatcaa aaggcagctt 5581 gttgagacac gccagatcac caagcacgtg gcccaaattc tcgattcacg catgaacacc 5641 aagtacgatg aaaatgacaa actgattcga gaggtgaaag ttattactct gaagtctaag 5701 ctggtctcag atttcagaaa ggactttcag ttttataagg tgagagagat caacaattac 5761 caccatgcgc atgatgccta cctgaatgca gtggtaggca ctgcacttat caaaaaatat 5821 cccaagcttg aatctgaatt tgtttacgga gactataaag tgtacgatgt taggaaaatg 5881 atcgcaaagt ctgagcagga aataggcaag gccaccgcta agtacttctt ttacagcaat 5941 attatgaatt ttttcaagac cgagattaca ctggccaatg gagagattcg gaagcgacca 6001 cttatcgaaa caaacggaga aacaggagaa atcgtgtggg acaagggtag ggatttcgcg 6061 acagtccgga aggtcctgtc catgccgcag gtgaacatcg ttaaaaagac cgaagtacag 6121 accggaggct tctccaagga aagtatcctc ccgaaaagga acagcgacaa gctgatcgca 6181 cgcaaaaaag attgggaccc caagaaatac ggcggattcg attctcctac agtcgcttac 6241 agtgtactgg ttgtggccaa agtggagaaa gggaagtcta aaaaactcaa aagcgtcaag 6301 gaactgctgg gcatcacaat catggagcga tcaagcttcg aaaaaaaccc catcgacttt 6361 ctcgaggcga aaggatataa agaggtcaaa aaagacctca tcattaagct tcccaagtac 6421 tctctctttg agcttgaaaa cggccggaaa cgaatgctcg ctagtgcggg cgagctgcag 6481 aaaggtaacg agctggcact gccctctaaa tacgttaatt tcttgtatct ggccagccac 6541 tatgaaaagc tcaaagggtc tcccgaagat aatgagcaga agcagctgtt cgtggaacaa 6601 cacaaacact accttgatga gatcatcgag caaataagcg aattctccaa aagagtgatc 6661 ctcgccgacg ctaacctcga taaggtgctt tctgcttaca ataagcacag ggataagccc 6721 atcagggagc aggcagaaaa cattatccac ttgtttactc tgaccaactt gggcgcgcct 6781 gcagccttca agtacttcga caccaccata gacagaaagc ggtacacctc tacaaaggag 6841 gtcctggacg ccacactgat tcatcagtca attacggggc tctatgaaac aagaatcgac 6901 ctctctcagc tcggtggaga cgggtccggc agcgggtccg ttaactaccc ctatgacgtg 6961 ccggactacg ctagcccgaa aaagaagagg aaggtctgac aattggaaag atccaagaga 7021 gacgagtaga gatttttttt ttgggattga tgtttgtcgt tctttgagtt gtcgtcgagt 7081 tacgcctttt gtaagaatgt tccgcaggag aggaggagga tgggcatgag tgagggtgag 7141 agggcttgcc cgcttttttt tttaaaaacg ctgaagacgt ggttgtcaaa caaacccccc 7201 atagaaacga ttttgttacg gtgcggtcca gacgtcactt gaatggctcc gcggaaaggc 7261 cagggaggga aggggggagg gaggaaacat gaaacatgtt gaacggctca acagggtttg 7321 ggggacaaga gaggtagcgc cctgatggac tgctccctcc cctcctttcc ctcaatgtct 7381 cattcatcca tgcttccccc ttctctctct cccctccgtt ccatcccccg cgggcgtggt 7441 agtggcgtga tgggatccac taaaatgtac gtgtaagaaa agccggtgag cttacgcttt 7501 tgtgaaagtg ggagtacgag tgttgtgtgt gtgtgtagtg gtttcagacc ccagacagag 7561 gcgaagcaga aaaagcagac gatgaagacg acgaagaaat gagcagtcta tttttatcgt 7621 ggaaacagaa gaggtgatat cgtctcctgc aggcatgcaa gctgatccac tagaggccat 7681 ggcggccgca ctagtggatc ccccgggatg catacttgga agatggagtg gatggaggag 7741 gaggcgagcg tagcagcaag cgtgagttat acagccaggc acatgtcgca atccttcggt 7801 ctcgggctta aaatccacgc actaatcacg ctgggccatg caaagagcaa tgccgaggcc 7861 caccacacaa aacgctgtgt cgcgcgttgc ggcctgaagc ttcatacttc ttagtcgccg 7921 ccaaaagggc tcgagagacg agacccgttg gcatgaccga tgttgttcga cgcggtttgc 7981 ttcgtcacag tcgacgtgat tcaggaatct ggagcctgca gatcattttt ttcagcctga 8041 tatcgttctt ttccactgag aaccatcaga ccaccttttc ttccattgtg tgaaggagta 8101 ggagttgccg tgctgctttg tgggagacat ctgcgatggt gaccagcctc ccgtcgtctg 8161 gtcgacgtga cgagcctctt cactgttctt cgacggagag acgcaagcga gacggctcta 8221 gaccttttgg acacgcattc tgtgtgtgaa ctagtggaca gtgataccac gtctgaaagc 8281 tcaccactgc ccatggtgca gctacttgtc acaaagtttt gactccgtcg gtatcaccat 8341 tcgcgctcgt gtgcctggtt gttccgccac gccggcctgc cccggggcgg ggcaatattc 8401 taaaatctca cgcaaaacac cgcacttacc cctcacacat attcgtgata gaccaccacc 8461 aatctcagcc cgcatcaaca caggagggcc ccggggggca ataagatatg aaaaagcctg 8521 aactcaccgc gacgtctgtc gagaagtttc tgatcgaaaa gttcgacagc gtctccgacc 8581 tgatgcagct ctcggagggc gaagaatctc gtgctttcag cttcgatgta ggagggcgtg 8641 gatatgtcct gcgggtaaat agctgcgccg atggtttcta caaagatcgt tatgtttatc 8701 ggcactttgc atcggccgcg ctcccgattc cggaagtgct tgacattggg gaattcagcg 8761 agagcctgac ctattgcatc tcccgccgtg cacagggtgt cacgttgcaa gacctgcctg 8821 aaaccgaact gcccgctgtt ctgcagccgg tcgcggaggc catggatgcg atcgctgcgg 8881 ccgatcttag ccagacgagc gggttcggcc cattcggacc gcaaggaatc ggtcaataca 8941 ctacatggcg tgatttcata tgcgcgattg ctgatcccca tgtgtatcac tggcaaactg 9001 tgatggacga caccgtcagt gcgtccgtcg cgcaggctct cgatgagctg atgctttggg 9061 ccgaggactg ccccgaagtc cggcacctcg tgcacgcgga tttcggctcc aacaatgtcc 9121 tgacggacaa tggccgcata acagcggtca ttgactggag cgaggcgatg ttcggggatt 9181 cccaatacga ggtcgccaac atcttcttct ggaggccgtg gttggcttgt atggagcagc 9241 agacgcgcta cttcgagcgg aggcatccgg agcttgcagg atcgccgcgg ctccgggcgt 9301 atatgctccg cattggtctt gaccaactct atcagagctt ggttgacggc aatttcgatg 9361 atgcagcttg ggcgcagggt cgatgcgacg caatcgtccg atccggagcc gggactgtcg 9421 ggcgtacaca aatcgcccgc agaagcgcgg ccgtctggac cgatggctgt gtagaagtac 9481 tcgccgatag tggaaaccga cgccccagca ctcgtccgag ggcaaaggaa tagagtagat 9541 gccgaccgga gtccgcaaaa atcaccagtc tctctctaca aatctatctc tctctatttt 9601 tctccagaat aatgtgtgag tagttcccag ataagggaat tagggttctt atagggtttc 9661 gctcatgtgt tgagcatata agaaaccctt agtatgtatt tgtatttgta aaatacttct 9721 atcaataaaa tttctaattc ctaaaaccaa aatccagtga cctgagcttc ctcgctcact 9781 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta 9841 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag 9901 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc 9961 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta 10021 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg 10081 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc 10141 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac 10201 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac 10261 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg 10321 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga 10381 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt 10441 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag 10501 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct 10561 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg 10621 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat 10681 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc 10741 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg 10801 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct 10861 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca 10921 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg 10981 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg 11041 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc 11101 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag 11161 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg 11221 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag 11281 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat 11341 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg 11401 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca 11461 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca 11521 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat 11581 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag 11641 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgatgcggtg 11701 tgaaataccg cacagatgcg taaggagaaa ataccgcatc aggaaattgt aagcgttaat 11761 attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa ccaataggcc 11821 gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt gagtgttgtt 11881 ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa agggcgaaaa 11941 accgtctatc agggcgatgg cccactacgt gaaccatcac cctaatcaag ttttttgggg 12001 tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt tagagcttga 12061 cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct 12121 agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat 12181 gcgccgctac agggcgcgtc cattcgccat tcaggctgcg caactgttgg gaagggcgat 12241 cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat 12301 taagttgggt aacgccaggg ttttcccagt cacgacgttg taaaacgacg gccagtgaat 12361 tgtaatacga ctcactata // LOCUS Exported 12919 bp ds-DNA circular SYN 10-JUL-2017 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS pNOC-CRISPR SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 12919) AUTHORS . TITLE Direct Submission JOURNAL Exported Friday, Jul 14, 2017 from SnapGene Viewer 3.3.4 http://www.snapgene.com FEATURES Location/Qualifiers source 1..12919 /organism="synthetic DNA construct" /mol_type="other DNA" terminator 418..1418 /label=CS terminator misc_feature 1425..1501 /label=HDV misc_RNA complement(1502..1577) /label=g RNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" promoter 1606..2789 /label=Ribi CDS 2800..2820 /codon_start=1 /product="nuclear localization signal of SV40 large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 2821..6921 /codon_start=1 /product="Cas9 (Csn1) endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system" /label=Cas9 /note="generates RNA-guided double strand breaks in DNA" /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD ATLIHQSITGLYETRIDLSQLGGD" CDS 6958..7473 /codon_start=1 /label=Nlux /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW RLCERILAG" CDS 7474..7500 /codon_start=1 /product="HA (human influenza hemagglutinin) epitope tag" /label=HA /translation="YPYDVPDYA" CDS 7516..7536 /codon_start=1 /product="nuclear localization signal of SV40 large T antigen" /label=SV40 NLS /translation="PKKKRKV" terminator 7568..8167 /label=LDSP terminator promoter 8254..9023 /label=LDSP promoter CDS 9048..10073 /codon_start=1 /gene="aph(4)-Ia" /product="aminoglycoside phosphotransferase from E. coli" /label=HygR /note="confers resistance to hygromycin" /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK E" terminator 10074..10304 /label=35S terminator rep_origin complement(10482..11070) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" CDS complement(11241..12101) /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" promoter complement(12102..12206) /gene="bla" /label=AmpR promoter rep_origin complement(12284..12739) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind 12880..12896 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" ORIGIN 1 gggcgaattg ggcccgacgt cgcatgctcc cggccgccat ggcggccgcg ggaattcgat 61 atcactagag tatagctaca tggtagctag tagtcgtcaa aggaaataaa gacatagcgt 121 aaagtataca aaatgacttc cgcggcgcgg gagcggcagg cagcacttgc aaaacgaagc 181 ccaatgggcc agattacggt caacctcgtc attttattac ggtcgtcagc aagagcggtt 241 tagaccgtcg ccggcacggg tggctcccga tattcttggc tttcgtgggt ggaggcggcg 301 tgtgactcct catctttaag tagtggtcgc ttttgctgca tgcttttgat cacctcgtga 361 ttgatggctg gattctcagc tgtcttctga catctttggg tgcatgggtt gaccggtgca 421 aagctgacgc ccttttcgtc gtcgttgctg gtgttaaaat tgcgcgtttc ttttttttcc 481 gtctttgtcg tcttttgact tttttatctt cttttctgtc ttcttttatt cttctcttct 541 tcttcatcac tccctcttca tttttcttct tctttttcct cgtctattgt cctcgggcat 601 taccagttgt gctgctcacg tgcgcgcaag cggtgaaagg gtgagaggat acacggacaa 661 caatcattga aaattacgct taaagccggt gcctggaaaa gtccacgcgc agatggcctt 721 tgcttgcctt gctgctgctg cagctgctgc tgctgtcgcc tgtgatggta atggttgttg 781 ttgtggtcct gcttgcttgc tagtttgcat tgcgtttgcg gttgccttat gaccgactcc 841 gtgtgattga tccttaaaca tattttatca ttgatgtgga ttgtgtgacg acggcaaaaa 901 agaattactt tacacatgac tatcacacct gctcccctcc cttctccttt caatcggtca 961 acttcccaac cgctcctcga aaatatttca gttgaatcct ctactacgta ccccttactt 1021 aaatttactt tctctctccc catcaatgcg ccgttgttct gaagttgtat tattgatggg 1081 agatgattgg cactgcacca gacctggcca aaagccacca caggtggcca catactcttg 1141 catgggtata ctctaccgag gcaaagatta aagtgcacgt cccctgcccc tctctctccc 1201 tacttccttc agtctcagtg gttccgtgcc cgttcagcac tacatcgccc tgcccgctgt 1261 tccttctcaa acagccaccc atcccaaccc acatacacag aaacaacctt tcctacacat 1321 tttcccaagt cttctcttca ctattctaca ctaatacacc accgtgatat gccggttatc 1381 tagatgtcct ttctcctttc tcatcctttc tttcccatgg tacccaggag tcccattcgc 1441 catgccgaag catgttgccc agccggcgcc agcgaggagg ctgggaccat gccggccaaa 1501 agcaccgact cggtgccact ttttcaagtt gataacggac tagccttatt ttaacttgct 1561 atttctagct ctaaaacgaa gagccaaggc ctgctcttca tcgattctgt ggatggaggg 1621 agggaaggag ggggggagtg agtgagtcga gagaacgacg actggggcaa agagaggatg 1681 acgccactgc aacaaaagac agggaagaat agaggctgtt ggaaaacagg aacacagtga 1741 agaatagagg ctgcagggta acaggcacat ttatcgcaga gaggcgggcg gcaggggcga 1801 tacgtaaaga gacccctaaa cttctccgcg tcatccgttc acgctacctc cttccctccc 1861 tttactccca ttgatcacca aggcagcgcc cgcagccacc agcagccacc gtcctcagtg 1921 tcacagctag acgcgtcccc atcaggcagc agaccaccag aatatgcgct ttcgcttccc 1981 atcatatccc tccctcctcc ccttctattc ctcatcatgc atcatttctt tctccttgct 2041 gcatgcccct cttcttacct tggctaagat ctaccggagg aagaagtctt caacgataga 2101 taaatgcctg tatcgtgatg gttgtttgaa tggtggaagt gagccaggta ttgagaacga 2161 ggggcgcaag ggtgcgtgga agaaggcagg gagaggcgag gcgaaggaga agggcatgtg 2221 gtgggaacga agcctccaca ccacagccac ccatgctttt ctctgttcga gttcccgggt 2281 tctactctca cacactacac gtgcacagaa aacattcacg cacaacagag atacagaggg 2341 atccgtcgag atggtctaca cgtcctaatg tcccctgctt tcgaagccca gagccatcgc 2401 tttctgtttt tatccattcg caacacatcc tgttctcgcg acctctcctc ttcctggcca 2461 ccatcgcttc tccgtgccat gccctgtaca tccttccctc cctcgttgtc taccacacta 2521 atcatgcgct ctgcagcaga cactcggcta ctatttgtgt cgtacggatt gaggggaggg 2581 aggggaaggg aggcaagaag taaatcggtg gtcaacaaca gacgcggtct ccccaccctc 2641 cctcattcct ttcccctcct ataatgagcg aagaaagcca tggacaccga gtcacccact 2701 ctcctaatcc accaccacca gggcactaac ggactcacgc tcaccaaact cggtaacaat 2761 atccactgca cacagataca cacgcaacac tcgagcatgc ccaagaaaaa gcggaaggtg 2821 gacaagaagt actccattgg gctcgatatc ggcacaaaca gcgtcggctg ggccgtcatt 2881 acggacgagt acaaggtgcc gagcaaaaaa ttcaaagttc tgggcaatac cgatcgccac 2941 agcataaaga agaacctcat tggcgccctc ctgttcgact ccggggagac ggccgaagcc 3001 acgcggctca aaagaacagc acggcgcaga tatacccgca gaaagaatcg gatctgctac 3061 ctgcaggaga tctttagtaa tgagatggct aaggtggatg actctttctt ccataggctg 3121 gaggagtcct ttttggtgga ggaggataaa aagcacgagc gccacccaat ctttggcaat 3181 atcgtggacg aggtggcgta ccatgaaaag tacccaacca tatatcatct gaggaagaag 3241 cttgtagaca gtactgataa ggctgacttg cggttgatct atctcgcgct ggcgcatatg 3301 atcaaatttc ggggacactt cctcatcgag ggggacctga acccagacaa cagcgatgtc 3361 gacaaactct ttatccaact ggttcagact tacaatcagc ttttcgaaga gaacccgatc 3421 aacgcatccg gagttgacgc caaagcaatc ctgagcgcta ggctgtccaa atcccggcgg 3481 ctcgaaaacc tcatcgcaca gctccctggg gagaagaaga acggcctgtt tggtaatctt 3541 atcgccctgt cactcgggct gacccccaac tttaaatcta acttcgacct ggccgaagat 3601 gccaagcttc aactgagcaa agacacctac gatgatgatc tcgacaatct gctggcccag 3661 atcggcgacc agtacgcaga cctttttttg gcggcaaaga acctgtcaga cgccattctg 3721 ctgagtgata ttctgcgagt gaacacggag atcaccaaag ctccgctgag cgctagtatg 3781 atcaagcgct atgatgagca ccaccaagac ttgactttgc tgaaggccct tgtcagacag 3841 caactgcctg agaagtacaa ggaaattttc ttcgatcagt ctaaaaatgg ctacgccgga 3901 tacattgacg gcggagcaag ccaggaggaa ttttacaaat ttattaagcc catcttggaa 3961 aaaatggacg gcaccgagga gctgctggta aagcttaaca gagaagatct gttgcgcaaa 4021 cagcgcactt tcgacaatgg aagcatcccc caccagattc acctgggcga actgcacgct 4081 atcctcaggc ggcaagagga tttctacccc tttttgaaag ataacaggga aaagattgag 4141 aaaatcctca catttcggat accctactat gtaggccccc tcgcccgggg aaattccaga 4201 ttcgcgtgga tgactcgcaa atcagaagag accatcactc cctggaactt cgaggaagtc 4261 gtggataagg gggcctctgc ccagtccttc atcgaaagga tgactaactt tgataaaaat 4321 ctgcctaacg aaaaggtgct tcctaaacac tctctgctgt acgagtactt cacagtttat 4381 aacgagctca ccaaggtcaa atacgtcaca gaagggatga gaaagccagc attcctgtct 4441 ggagagcaga agaaagctat cgtggacctc ctcttcaaga cgaaccggaa agttaccgtg 4501 aaacagctca aagaagacta tttcaaaaag attgaatgtt tcgactctgt tgaaatcagc 4561 ggagtggagg atcgcttcaa cgcatccctg ggaacgtatc acgatctcct gaaaatcatt 4621 aaagacaagg acttcctgga caatgaggag aacgaggaca ttcttgagga cattgtcctc 4681 acccttacgt tgtttgaaga tagggagatg attgaagaac gcttgaaaac ttacgctcat 4741 ctcttcgacg acaaagtcat gaaacagctc aagaggcgcc gatatacagg atgggggcgg 4801 ctgtcaagaa aactgatcaa tgggatccga gacaagcaga gtggaaagac aatcctggat 4861 tttcttaagt ccgatggatt tgccaaccgg aacttcatgc agttgatcca tgatgactct 4921 ctcaccttta aggaggacat ccagaaagca caagtttctg gccaggggga cagtcttcac 4981 gagcacatcg ctaatcttgc aggtagccca gctatcaaaa agggaatact gcagaccgtt 5041 aaggtcgtgg atgaactcgt caaagtaatg ggaaggcata agcccgagaa tatcgttatc 5101 gagatggccc gagagaacca aactacccag aagggacaga agaacagtag ggaaaggatg 5161 aagaggattg aagagggtat aaaagaactg gggtcccaaa tccttaagga acacccagtt 5221 gaaaacaccc agcttcagaa tgagaagctc tacctgtact acctgcagaa cggcagggac 5281 atgtacgtgg atcaggaact ggacatcaat cggctctccg actacgacgt ggatcatatc 5341 gtgccccagt cttttctcaa agatgattct attgataata aagtgttgac aagatccgat 5401 aaaaatagag ggaagagtga taacgtcccc tcagaagaag ttgtcaagaa aatgaaaaat 5461 tattggcggc agctgctgaa cgccaaactg atcacacaac ggaagttcga taatctgact 5521 aaggctgaac gaggtggcct gtctgagttg gataaagccg gcttcatcaa aaggcagctt 5581 gttgagacac gccagatcac caagcacgtg gcccaaattc tcgattcacg catgaacacc 5641 aagtacgatg aaaatgacaa actgattcga gaggtgaaag ttattactct gaagtctaag 5701 ctggtctcag atttcagaaa ggactttcag ttttataagg tgagagagat caacaattac 5761 caccatgcgc atgatgccta cctgaatgca gtggtaggca ctgcacttat caaaaaatat 5821 cccaagcttg aatctgaatt tgtttacgga gactataaag tgtacgatgt taggaaaatg 5881 atcgcaaagt ctgagcagga aataggcaag gccaccgcta agtacttctt ttacagcaat 5941 attatgaatt ttttcaagac cgagattaca ctggccaatg gagagattcg gaagcgacca 6001 cttatcgaaa caaacggaga aacaggagaa atcgtgtggg acaagggtag ggatttcgcg 6061 acagtccgga aggtcctgtc catgccgcag gtgaacatcg ttaaaaagac cgaagtacag 6121 accggaggct tctccaagga aagtatcctc ccgaaaagga acagcgacaa gctgatcgca 6181 cgcaaaaaag attgggaccc caagaaatac ggcggattcg attctcctac agtcgcttac 6241 agtgtactgg ttgtggccaa agtggagaaa gggaagtcta aaaaactcaa aagcgtcaag 6301 gaactgctgg gcatcacaat catggagcga tcaagcttcg aaaaaaaccc catcgacttt 6361 ctcgaggcga aaggatataa agaggtcaaa aaagacctca tcattaagct tcccaagtac 6421 tctctctttg agcttgaaaa cggccggaaa cgaatgctcg ctagtgcggg cgagctgcag 6481 aaaggtaacg agctggcact gccctctaaa tacgttaatt tcttgtatct ggccagccac 6541 tatgaaaagc tcaaagggtc tcccgaagat aatgagcaga agcagctgtt cgtggaacaa 6601 cacaaacact accttgatga gatcatcgag caaataagcg aattctccaa aagagtgatc 6661 ctcgccgacg ctaacctcga taaggtgctt tctgcttaca ataagcacag ggataagccc 6721 atcagggagc aggcagaaaa cattatccac ttgtttactc tgaccaactt gggcgcgcct 6781 gcagccttca agtacttcga caccaccata gacagaaagc ggtacacctc tacaaaggag 6841 gtcctggacg ccacactgat tcatcagtca attacggggc tctatgaaac aagaatcgac 6901 ctctctcagc tcggtggaga cagcagggct gacgggtccg gcagcgggtc cgttaacatg 6961 gtgtttactc tcgaggactt cgtgggcgac tggcggcaga cagctggcta taacctcgac 7021 caggtgctgg agcagggcgg tgtgtcctcg cttttccaga acctgggcgt ctccgtgacc 7081 ccaatccagc gcatcgtcct gagcggcgag aacggcctga agatcgacat ccacgtcatc 7141 attccctacg agggcctgag cggggaccaa atgggccaga tcgagaagat cttcaaggtt 7201 gtgtaccccg tggacgatca ccacttcaag gtgatactgc actacgggac gctggtcatc 7261 gacggtgtga cgccgaacat gattgactac ttcggccgtc cttacgaggg cattgccgtg 7321 ttcgacggca agaagatcac ggtgaccggc accctgtgga acggcaataa gatcattgac 7381 gagcggctga tcaacccgga tggctcgctg ttgttccgcg tgaccatcaa cggggtaacc 7441 ggctggcgcc tgtgcgagcg catcctggcc ggatacccct acgacgtgcc cgactacgcg 7501 tccatggtgg ctagcccgaa aaagaagagg aaggtctgac aattggaaag atccaagaga 7561 gacgagtaga gatttttttt ttgggattga tgtttgtcgt tctttgagtt gtcgtcgagt 7621 tacgcctttt gtaagaatgt tccgcaggag aggaggagga tgggcatgag tgagggtgag 7681 agggcttgcc cgcttttttt tttaaaaacg ctgaagacgt ggttgtcaaa caaacccccc 7741 atagaaacga ttttgttacg gtgcggtcca gacgtcactt gaatggctcc gcggaaaggc 7801 cagggaggga aggggggagg gaggaaacat gaaacatgtt gaacggctca acagggtttg 7861 ggggacaaga gaggtagcgc cctgatggac tgctccctcc cctcctttcc ctcaatgtct 7921 cattcatcca tgcttccccc ttctctctct cccctccgtt ccatcccccg cgggcgtggt 7981 agtggcgtga tgggatccac taaaatgtac gtgtaagaaa agccggtgag cttacgcttt 8041 tgtgaaagtg ggagtacgag tgttgtgtgt gtgtgtagtg gtttcagacc ccagacagag 8101 gcgaagcaga aaaagcagac gatgaagacg acgaagaaat gagcagtcta tttttatcgt 8161 ggaaacagaa gaggtgatat cgtctcctgc aggcatgcaa gctgatccac tagaggccat 8221 ggcggccgca ctagtggatc ccccgggatg catacttgga agatggagtg gatggaggag 8281 gaggcgagcg tagcagcaag cgtgagttat acagccaggc acatgtcgca atccttcggt 8341 ctcgggctta aaatccacgc actaatcacg ctgggccatg caaagagcaa tgccgaggcc 8401 caccacacaa aacgctgtgt cgcgcgttgc ggcctgaagc ttcatacttc ttagtcgccg 8461 ccaaaagggc tcgagagacg agacccgttg gcatgaccga tgttgttcga cgcggtttgc 8521 ttcgtcacag tcgacgtgat tcaggaatct ggagcctgca gatcattttt ttcagcctga 8581 tatcgttctt ttccactgag aaccatcaga ccaccttttc ttccattgtg tgaaggagta 8641 ggagttgccg tgctgctttg tgggagacat ctgcgatggt gaccagcctc ccgtcgtctg 8701 gtcgacgtga cgagcctctt cactgttctt cgacggagag acgcaagcga gacggctcta 8761 gaccttttgg acacgcattc tgtgtgtgaa ctagtggaca gtgataccac gtctgaaagc 8821 tcaccactgc ccatggtgca gctacttgtc acaaagtttt gactccgtcg gtatcaccat 8881 tcgcgctcgt gtgcctggtt gttccgccac gccggcctgc cccggggcgg ggcaatattc 8941 taaaatctca cgcaaaacac cgcacttacc cctcacacat attcgtgata gaccaccacc 9001 aatctcagcc cgcatcaaca caggagggcc ccggggggca ataagatatg aaaaagcctg 9061 aactcaccgc gacgtctgtc gagaagtttc tgatcgaaaa gttcgacagc gtctccgacc 9121 tgatgcagct ctcggagggc gaagaatctc gtgctttcag cttcgatgta ggagggcgtg 9181 gatatgtcct gcgggtaaat agctgcgccg atggtttcta caaagatcgt tatgtttatc 9241 ggcactttgc atcggccgcg ctcccgattc cggaagtgct tgacattggg gaattcagcg 9301 agagcctgac ctattgcatc tcccgccgtg cacagggtgt cacgttgcaa gacctgcctg 9361 aaaccgaact gcccgctgtt ctgcagccgg tcgcggaggc catggatgcg atcgctgcgg 9421 ccgatcttag ccagacgagc gggttcggcc cattcggacc gcaaggaatc ggtcaataca 9481 ctacatggcg tgatttcata tgcgcgattg ctgatcccca tgtgtatcac tggcaaactg 9541 tgatggacga caccgtcagt gcgtccgtcg cgcaggctct cgatgagctg atgctttggg 9601 ccgaggactg ccccgaagtc cggcacctcg tgcacgcgga tttcggctcc aacaatgtcc 9661 tgacggacaa tggccgcata acagcggtca ttgactggag cgaggcgatg ttcggggatt 9721 cccaatacga ggtcgccaac atcttcttct ggaggccgtg gttggcttgt atggagcagc 9781 agacgcgcta cttcgagcgg aggcatccgg agcttgcagg atcgccgcgg ctccgggcgt 9841 atatgctccg cattggtctt gaccaactct atcagagctt ggttgacggc aatttcgatg 9901 atgcagcttg ggcgcagggt cgatgcgacg caatcgtccg atccggagcc gggactgtcg 9961 ggcgtacaca aatcgcccgc agaagcgcgg ccgtctggac cgatggctgt gtagaagtac 10021 tcgccgatag tggaaaccga cgccccagca ctcgtccgag ggcaaaggaa tagagtagat 10081 gccgaccgga gtccgcaaaa atcaccagtc tctctctaca aatctatctc tctctatttt 10141 tctccagaat aatgtgtgag tagttcccag ataagggaat tagggttctt atagggtttc 10201 gctcatgtgt tgagcatata agaaaccctt agtatgtatt tgtatttgta aaatacttct 10261 atcaataaaa tttctaattc ctaaaaccaa aatccagtga cctgagcttc ctcgctcact 10321 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta 10381 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag 10441 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc 10501 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta 10561 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg 10621 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc 10681 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac 10741 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac 10801 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg 10861 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga 10921 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt 10981 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag 11041 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct 11101 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg 11161 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat 11221 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc 11281 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg 11341 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct 11401 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca 11461 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg 11521 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg 11581 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc 11641 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag 11701 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg 11761 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag 11821 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat 11881 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg 11941 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca 12001 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca 12061 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat 12121 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag 12181 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgatgcggtg 12241 tgaaataccg cacagatgcg taaggagaaa ataccgcatc aggaaattgt aagcgttaat 12301 attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa ccaataggcc 12361 gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt gagtgttgtt 12421 ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa agggcgaaaa 12481 accgtctatc agggcgatgg cccactacgt gaaccatcac cctaatcaag ttttttgggg 12541 tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt tagagcttga 12601 cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct 12661 agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat 12721 gcgccgctac agggcgcgtc cattcgccat tcaggctgcg caactgttgg gaagggcgat 12781 cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat 12841 taagttgggt aacgccaggg ttttcccagt cacgacgttg taaaacgacg gccagtgaat 12901 tgtaatacga ctcactata // LOCUS Exported 13473 bp ds-DNA circular SYN 10-JUL-2017 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS pNOC-ARS-CRISPR SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 13473) AUTHORS . TITLE Direct Submission JOURNAL Exported Thursday, Jul 13, 2017 from SnapGene Viewer 3.3.4 http://www.snapgene.com FEATURES Location/Qualifiers source 1..13473 /organism="synthetic DNA construct" /mol_type="other DNA" misc_feature 439..942 /label=CEN/ARS /note="S. cerevisiae CEN6 centromere fused to an autonomously replicating sequence" terminator 972..1972 /label=CS terminator misc_feature 1979..2055 /label=HDV misc_RNA complement(2056..2131) /label=g RNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" promoter 2160..3343 /label=Ribi CDS 3354..3374 /codon_start=1 /product="nuclear localization signal of SV40 large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 3375..7475 /codon_start=1 /product="Cas9 (Csn1) endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system" /label=Cas9 /note="generates RNA-guided double strand breaks in DNA" /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD ATLIHQSITGLYETRIDLSQLGGD" CDS 7512..8027 /codon_start=1 /label=Nlux /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW RLCERILAG" CDS 8028..8054 /codon_start=1 /product="HA (human influenza hemagglutinin) epitope tag" /label=HA /translation="YPYDVPDYA" CDS 8070..8090 /codon_start=1 /product="nuclear localization signal of SV40 large T antigen" /label=SV40 NLS /translation="PKKKRKV" terminator 8122..8721 /label=LDSP terminator promoter 8808..9577 /label=LDSP promoter CDS 9602..10627 /codon_start=1 /gene="aph(4)-Ia" /product="aminoglycoside phosphotransferase from E. coli" /label=HygR /note="confers resistance to hygromycin" /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK E" terminator 10628..10858 /label=35S terminator rep_origin complement(11036..11624) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" CDS complement(11795..12655) /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" promoter complement(12656..12760) /gene="bla" /label=AmpR promoter rep_origin complement(12838..13293) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind 13434..13450 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" ORIGIN 1 gggcgaattg ggcccgacgt cgcatgctcc cggccgccat ggcggccgcg ggaattcgat 61 atcactagag tatagctaca tggtagctag tagtcgtcaa aggaaataaa gacatagcgt 121 aaagtataca aaatgacttc cgcggcgcgg gagcggcagg cagcacttgc aaaacgaagc 181 ccaatgggcc agattacggt caacctcgtc attttattac ggtcgtcagc aagagcggtt 241 tagaccgtcg ccggcacggg tggctcccga tattcttggc tttcgtgggt ggaggcggcg 301 tgtgactcct catctttaag tagtggtcgc ttttgctgca tgcttttgat cacctcgtga 361 ttgatggctg gattctcagc tgtcttctga catctttggg tgcatgggtt gaccggtggc 421 cggccggttt cttagacgga tcgcttgcct gtaacttaca cgcgcctcgt atcttttaat 481 gatggaataa tttgggaatt tactctgtgt ttatttattt ttatgttttg tatttggatt 541 ttagaaagta aataaagaag gtagaagagt tacggaatga agaaaaaaaa ataaacaaag 601 gtttaaaaaa tttcaacaaa aagcgtactt tacatatata tttattagac aagaaaagca 661 gattaaatag atatacattc gattaacgat aagtaaaatg taaaatcaca ggattttcgt 721 gtgtggtctt ctacacagac aagatgaaac aattcggcat taatacctga gagcaggaag 781 agcaagataa aaggtagtat ttgttggcga tccccctaga gtcttttaca tcttcggaaa 841 acaaaaacta ttttttcttt aatttctttt tttactttct atttttaatt tatatattta 901 tattaaaaaa tttaaattat aattattttt atagcacgtg atgaaaagga cccaggtgtt 961 taaacaccgg tgcaaagctg acgccctttt cgtcgtcgtt gctggtgtta aaattgcgcg 1021 tttctttttt ttccgtcttt gtcgtctttt gactttttta tcttcttttc tgtcttcttt 1081 tattcttctc ttcttcttca tcactccctc ttcatttttc ttcttctttt tcctcgtcta 1141 ttgtcctcgg gcattaccag ttgtgctgct cacgtgcgcg caagcggtga aagggtgaga 1201 ggatacacgg acaacaatca ttgaaaatta cgcttaaagc cggtgcctgg aaaagtccac 1261 gcgcagatgg cctttgcttg ccttgctgct gctgcagctg ctgctgctgt cgcctgtgat 1321 ggtaatggtt gttgttgtgg tcctgcttgc ttgctagttt gcattgcgtt tgcggttgcc 1381 ttatgaccga ctccgtgtga ttgatcctta aacatatttt atcattgatg tggattgtgt 1441 gacgacggca aaaaagaatt actttacaca tgactatcac acctgctccc ctcccttctc 1501 ctttcaatcg gtcaacttcc caaccgctcc tcgaaaatat ttcagttgaa tcctctacta 1561 cgtacccctt acttaaattt actttctctc tccccatcaa tgcgccgttg ttctgaagtt 1621 gtattattga tgggagatga ttggcactgc accagacctg gccaaaagcc accacaggtg 1681 gccacatact cttgcatggg tatactctac cgaggcaaag attaaagtgc acgtcccctg 1741 cccctctctc tccctacttc cttcagtctc agtggttccg tgcccgttca gcactacatc 1801 gccctgcccg ctgttccttc tcaaacagcc acccatccca acccacatac acagaaacaa 1861 cctttcctac acattttccc aagtcttctc ttcactattc tacactaata caccaccgtg 1921 atatgccggt tatctagatg tcctttctcc tttctcatcc tttctttccc atggtaccca 1981 ggagtcccat tcgccatgcc gaagcatgtt gcccagccgg cgccagcgag gaggctggga 2041 ccatgccggc caaaagcacc gactcggtgc cactttttca agttgataac ggactagcct 2101 tattttaact tgctatttct agctctaaaa cgaagagcca aggcctgctc ttcatcgatt 2161 ctgtggatgg agggagggaa ggaggggggg agtgagtgag tcgagagaac gacgactggg 2221 gcaaagagag gatgacgcca ctgcaacaaa agacagggaa gaatagaggc tgttggaaaa 2281 caggaacaca gtgaagaata gaggctgcag ggtaacaggc acatttatcg cagagaggcg 2341 ggcggcaggg gcgatacgta aagagacccc taaacttctc cgcgtcatcc gttcacgcta 2401 cctccttccc tccctttact cccattgatc accaaggcag cgcccgcagc caccagcagc 2461 caccgtcctc agtgtcacag ctagacgcgt ccccatcagg cagcagacca ccagaatatg 2521 cgctttcgct tcccatcata tccctccctc ctccccttct attcctcatc atgcatcatt 2581 tctttctcct tgctgcatgc ccctcttctt accttggcta agatctaccg gaggaagaag 2641 tcttcaacga tagataaatg cctgtatcgt gatggttgtt tgaatggtgg aagtgagcca 2701 ggtattgaga acgaggggcg caagggtgcg tggaagaagg cagggagagg cgaggcgaag 2761 gagaagggca tgtggtggga acgaagcctc cacaccacag ccacccatgc ttttctctgt 2821 tcgagttccc gggttctact ctcacacact acacgtgcac agaaaacatt cacgcacaac 2881 agagatacag agggatccgt cgagatggtc tacacgtcct aatgtcccct gctttcgaag 2941 cccagagcca tcgctttctg tttttatcca ttcgcaacac atcctgttct cgcgacctct 3001 cctcttcctg gccaccatcg cttctccgtg ccatgccctg tacatccttc cctccctcgt 3061 tgtctaccac actaatcatg cgctctgcag cagacactcg gctactattt gtgtcgtacg 3121 gattgagggg agggagggga agggaggcaa gaagtaaatc ggtggtcaac aacagacgcg 3181 gtctccccac cctccctcat tcctttcccc tcctataatg agcgaagaaa gccatggaca 3241 ccgagtcacc cactctccta atccaccacc accagggcac taacggactc acgctcacca 3301 aactcggtaa caatatccac tgcacacaga tacacacgca acactcgagc atgcccaaga 3361 aaaagcggaa ggtggacaag aagtactcca ttgggctcga tatcggcaca aacagcgtcg 3421 gctgggccgt cattacggac gagtacaagg tgccgagcaa aaaattcaaa gttctgggca 3481 ataccgatcg ccacagcata aagaagaacc tcattggcgc cctcctgttc gactccgggg 3541 agacggccga agccacgcgg ctcaaaagaa cagcacggcg cagatatacc cgcagaaaga 3601 atcggatctg ctacctgcag gagatcttta gtaatgagat ggctaaggtg gatgactctt 3661 tcttccatag gctggaggag tcctttttgg tggaggagga taaaaagcac gagcgccacc 3721 caatctttgg caatatcgtg gacgaggtgg cgtaccatga aaagtaccca accatatatc 3781 atctgaggaa gaagcttgta gacagtactg ataaggctga cttgcggttg atctatctcg 3841 cgctggcgca tatgatcaaa tttcggggac acttcctcat cgagggggac ctgaacccag 3901 acaacagcga tgtcgacaaa ctctttatcc aactggttca gacttacaat cagcttttcg 3961 aagagaaccc gatcaacgca tccggagttg acgccaaagc aatcctgagc gctaggctgt 4021 ccaaatcccg gcggctcgaa aacctcatcg cacagctccc tggggagaag aagaacggcc 4081 tgtttggtaa tcttatcgcc ctgtcactcg ggctgacccc caactttaaa tctaacttcg 4141 acctggccga agatgccaag cttcaactga gcaaagacac ctacgatgat gatctcgaca 4201 atctgctggc ccagatcggc gaccagtacg cagacctttt tttggcggca aagaacctgt 4261 cagacgccat tctgctgagt gatattctgc gagtgaacac ggagatcacc aaagctccgc 4321 tgagcgctag tatgatcaag cgctatgatg agcaccacca agacttgact ttgctgaagg 4381 cccttgtcag acagcaactg cctgagaagt acaaggaaat tttcttcgat cagtctaaaa 4441 atggctacgc cggatacatt gacggcggag caagccagga ggaattttac aaatttatta 4501 agcccatctt ggaaaaaatg gacggcaccg aggagctgct ggtaaagctt aacagagaag 4561 atctgttgcg caaacagcgc actttcgaca atggaagcat cccccaccag attcacctgg 4621 gcgaactgca cgctatcctc aggcggcaag aggatttcta cccctttttg aaagataaca 4681 gggaaaagat tgagaaaatc ctcacatttc ggatacccta ctatgtaggc cccctcgccc 4741 ggggaaattc cagattcgcg tggatgactc gcaaatcaga agagaccatc actccctgga 4801 acttcgagga agtcgtggat aagggggcct ctgcccagtc cttcatcgaa aggatgacta 4861 actttgataa aaatctgcct aacgaaaagg tgcttcctaa acactctctg ctgtacgagt 4921 acttcacagt ttataacgag ctcaccaagg tcaaatacgt cacagaaggg atgagaaagc 4981 cagcattcct gtctggagag cagaagaaag ctatcgtgga cctcctcttc aagacgaacc 5041 ggaaagttac cgtgaaacag ctcaaagaag actatttcaa aaagattgaa tgtttcgact 5101 ctgttgaaat cagcggagtg gaggatcgct tcaacgcatc cctgggaacg tatcacgatc 5161 tcctgaaaat cattaaagac aaggacttcc tggacaatga ggagaacgag gacattcttg 5221 aggacattgt cctcaccctt acgttgtttg aagataggga gatgattgaa gaacgcttga 5281 aaacttacgc tcatctcttc gacgacaaag tcatgaaaca gctcaagagg cgccgatata 5341 caggatgggg gcggctgtca agaaaactga tcaatgggat ccgagacaag cagagtggaa 5401 agacaatcct ggattttctt aagtccgatg gatttgccaa ccggaacttc atgcagttga 5461 tccatgatga ctctctcacc tttaaggagg acatccagaa agcacaagtt tctggccagg 5521 gggacagtct tcacgagcac atcgctaatc ttgcaggtag cccagctatc aaaaagggaa 5581 tactgcagac cgttaaggtc gtggatgaac tcgtcaaagt aatgggaagg cataagcccg 5641 agaatatcgt tatcgagatg gcccgagaga accaaactac ccagaaggga cagaagaaca 5701 gtagggaaag gatgaagagg attgaagagg gtataaaaga actggggtcc caaatcctta 5761 aggaacaccc agttgaaaac acccagcttc agaatgagaa gctctacctg tactacctgc 5821 agaacggcag ggacatgtac gtggatcagg aactggacat caatcggctc tccgactacg 5881 acgtggatca tatcgtgccc cagtcttttc tcaaagatga ttctattgat aataaagtgt 5941 tgacaagatc cgataaaaat agagggaaga gtgataacgt cccctcagaa gaagttgtca 6001 agaaaatgaa aaattattgg cggcagctgc tgaacgccaa actgatcaca caacggaagt 6061 tcgataatct gactaaggct gaacgaggtg gcctgtctga gttggataaa gccggcttca 6121 tcaaaaggca gcttgttgag acacgccaga tcaccaagca cgtggcccaa attctcgatt 6181 cacgcatgaa caccaagtac gatgaaaatg acaaactgat tcgagaggtg aaagttatta 6241 ctctgaagtc taagctggtc tcagatttca gaaaggactt tcagttttat aaggtgagag 6301 agatcaacaa ttaccaccat gcgcatgatg cctacctgaa tgcagtggta ggcactgcac 6361 ttatcaaaaa atatcccaag cttgaatctg aatttgttta cggagactat aaagtgtacg 6421 atgttaggaa aatgatcgca aagtctgagc aggaaatagg caaggccacc gctaagtact 6481 tcttttacag caatattatg aattttttca agaccgagat tacactggcc aatggagaga 6541 ttcggaagcg accacttatc gaaacaaacg gagaaacagg agaaatcgtg tgggacaagg 6601 gtagggattt cgcgacagtc cggaaggtcc tgtccatgcc gcaggtgaac atcgttaaaa 6661 agaccgaagt acagaccgga ggcttctcca aggaaagtat cctcccgaaa aggaacagcg 6721 acaagctgat cgcacgcaaa aaagattggg accccaagaa atacggcgga ttcgattctc 6781 ctacagtcgc ttacagtgta ctggttgtgg ccaaagtgga gaaagggaag tctaaaaaac 6841 tcaaaagcgt caaggaactg ctgggcatca caatcatgga gcgatcaagc ttcgaaaaaa 6901 accccatcga ctttctcgag gcgaaaggat ataaagaggt caaaaaagac ctcatcatta 6961 agcttcccaa gtactctctc tttgagcttg aaaacggccg gaaacgaatg ctcgctagtg 7021 cgggcgagct gcagaaaggt aacgagctgg cactgccctc taaatacgtt aatttcttgt 7081 atctggccag ccactatgaa aagctcaaag ggtctcccga agataatgag cagaagcagc 7141 tgttcgtgga acaacacaaa cactaccttg atgagatcat cgagcaaata agcgaattct 7201 ccaaaagagt gatcctcgcc gacgctaacc tcgataaggt gctttctgct tacaataagc 7261 acagggataa gcccatcagg gagcaggcag aaaacattat ccacttgttt actctgacca 7321 acttgggcgc gcctgcagcc ttcaagtact tcgacaccac catagacaga aagcggtaca 7381 cctctacaaa ggaggtcctg gacgccacac tgattcatca gtcaattacg gggctctatg 7441 aaacaagaat cgacctctct cagctcggtg gagacagcag ggctgacggg tccggcagcg 7501 ggtccgttaa catggtgttt actctcgagg acttcgtggg cgactggcgg cagacagctg 7561 gctataacct cgaccaggtg ctggagcagg gcggtgtgtc ctcgcttttc cagaacctgg 7621 gcgtctccgt gaccccaatc cagcgcatcg tcctgagcgg cgagaacggc ctgaagatcg 7681 acatccacgt catcattccc tacgagggcc tgagcgggga ccaaatgggc cagatcgaga 7741 agatcttcaa ggttgtgtac cccgtggacg atcaccactt caaggtgata ctgcactacg 7801 ggacgctggt catcgacggt gtgacgccga acatgattga ctacttcggc cgtccttacg 7861 agggcattgc cgtgttcgac ggcaagaaga tcacggtgac cggcaccctg tggaacggca 7921 ataagatcat tgacgagcgg ctgatcaacc cggatggctc gctgttgttc cgcgtgacca 7981 tcaacggggt aaccggctgg cgcctgtgcg agcgcatcct ggccggatac ccctacgacg 8041 tgcccgacta cgcgtccatg gtggctagcc cgaaaaagaa gaggaaggtc tgacaattgg 8101 aaagatccaa gagagacgag tagagatttt ttttttggga ttgatgtttg tcgttctttg 8161 agttgtcgtc gagttacgcc ttttgtaaga atgttccgca ggagaggagg aggatgggca 8221 tgagtgaggg tgagagggct tgcccgcttt tttttttaaa aacgctgaag acgtggttgt 8281 caaacaaacc ccccatagaa acgattttgt tacggtgcgg tccagacgtc acttgaatgg 8341 ctccgcggaa aggccaggga gggaaggggg gagggaggaa acatgaaaca tgttgaacgg 8401 ctcaacaggg tttgggggac aagagaggta gcgccctgat ggactgctcc ctcccctcct 8461 ttccctcaat gtctcattca tccatgcttc ccccttctct ctctcccctc cgttccatcc 8521 cccgcgggcg tggtagtggc gtgatgggat ccactaaaat gtacgtgtaa gaaaagccgg 8581 tgagcttacg cttttgtgaa agtgggagta cgagtgttgt gtgtgtgtgt agtggtttca 8641 gaccccagac agaggcgaag cagaaaaagc agacgatgaa gacgacgaag aaatgagcag 8701 tctattttta tcgtggaaac agaagaggtg atatcgtctc ctgcaggcat gcaagctgat 8761 ccactagagg ccatggcggc cgcactagtg gatcccccgg gatgcatact tggaagatgg 8821 agtggatgga ggaggaggcg agcgtagcag caagcgtgag ttatacagcc aggcacatgt 8881 cgcaatcctt cggtctcggg cttaaaatcc acgcactaat cacgctgggc catgcaaaga 8941 gcaatgccga ggcccaccac acaaaacgct gtgtcgcgcg ttgcggcctg aagcttcata 9001 cttcttagtc gccgccaaaa gggctcgaga gacgagaccc gttggcatga ccgatgttgt 9061 tcgacgcggt ttgcttcgtc acagtcgacg tgattcagga atctggagcc tgcagatcat 9121 ttttttcagc ctgatatcgt tcttttccac tgagaaccat cagaccacct tttcttccat 9181 tgtgtgaagg agtaggagtt gccgtgctgc tttgtgggag acatctgcga tggtgaccag 9241 cctcccgtcg tctggtcgac gtgacgagcc tcttcactgt tcttcgacgg agagacgcaa 9301 gcgagacggc tctagacctt ttggacacgc attctgtgtg tgaactagtg gacagtgata 9361 ccacgtctga aagctcacca ctgcccatgg tgcagctact tgtcacaaag ttttgactcc 9421 gtcggtatca ccattcgcgc tcgtgtgcct ggttgttccg ccacgccggc ctgccccggg 9481 gcggggcaat attctaaaat ctcacgcaaa acaccgcact tacccctcac acatattcgt 9541 gatagaccac caccaatctc agcccgcatc aacacaggag ggccccgggg ggcaataaga 9601 tatgaaaaag cctgaactca ccgcgacgtc tgtcgagaag tttctgatcg aaaagttcga 9661 cagcgtctcc gacctgatgc agctctcgga gggcgaagaa tctcgtgctt tcagcttcga 9721 tgtaggaggg cgtggatatg tcctgcgggt aaatagctgc gccgatggtt tctacaaaga 9781 tcgttatgtt tatcggcact ttgcatcggc cgcgctcccg attccggaag tgcttgacat 9841 tggggaattc agcgagagcc tgacctattg catctcccgc cgtgcacagg gtgtcacgtt 9901 gcaagacctg cctgaaaccg aactgcccgc tgttctgcag ccggtcgcgg aggccatgga 9961 tgcgatcgct gcggccgatc ttagccagac gagcgggttc ggcccattcg gaccgcaagg 10021 aatcggtcaa tacactacat ggcgtgattt catatgcgcg attgctgatc cccatgtgta 10081 tcactggcaa actgtgatgg acgacaccgt cagtgcgtcc gtcgcgcagg ctctcgatga 10141 gctgatgctt tgggccgagg actgccccga agtccggcac ctcgtgcacg cggatttcgg 10201 ctccaacaat gtcctgacgg acaatggccg cataacagcg gtcattgact ggagcgaggc 10261 gatgttcggg gattcccaat acgaggtcgc caacatcttc ttctggaggc cgtggttggc 10321 ttgtatggag cagcagacgc gctacttcga gcggaggcat ccggagcttg caggatcgcc 10381 gcggctccgg gcgtatatgc tccgcattgg tcttgaccaa ctctatcaga gcttggttga 10441 cggcaatttc gatgatgcag cttgggcgca gggtcgatgc gacgcaatcg tccgatccgg 10501 agccgggact gtcgggcgta cacaaatcgc ccgcagaagc gcggccgtct ggaccgatgg 10561 ctgtgtagaa gtactcgccg atagtggaaa ccgacgcccc agcactcgtc cgagggcaaa 10621 ggaatagagt agatgccgac cggagtccgc aaaaatcacc agtctctctc tacaaatcta 10681 tctctctcta tttttctcca gaataatgtg tgagtagttc ccagataagg gaattagggt 10741 tcttataggg tttcgctcat gtgttgagca tataagaaac ccttagtatg tatttgtatt 10801 tgtaaaatac ttctatcaat aaaatttcta attcctaaaa ccaaaatcca gtgacctgag 10861 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc 10921 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt 10981 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc 11041 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa 11101 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc 11161 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg 11221 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc 11281 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc 11341 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca 11401 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact 11461 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg 11521 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt 11581 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct 11641 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga 11701 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa 11761 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac 11821 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga 11881 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc 11941 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca 12001 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta 12061 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg 12121 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc 12181 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg 12241 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt 12301 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt 12361 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata 12421 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc 12481 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac 12541 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa 12601 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct 12661 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat 12721 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc 12781 cacctgatgc ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg catcaggaaa 12841 ttgtaagcgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt 12901 ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag 12961 ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg 13021 tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcaccctaat 13081 caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc 13141 gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga 13201 aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac 13261 ccgccgcgct taatgcgccg ctacagggcg cgtccattcg ccattcaggc tgcgcaactg 13321 ttgggaaggg cgatcggtgc gggcctcttc gctattacgc cagctggcga aagggggatg 13381 tgctgcaagg cgattaagtt gggtaacgcc agggttttcc cagtcacgac gttgtaaaac 13441 gacggccagt gaattgtaat acgactcact ata // LOCUS Exported 13514 bp ds-DNA circular SYN 13-JUL-2017 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS pNOC-ARS-CRISPR-sgNR1 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 13514) AUTHORS . TITLE Direct Submission JOURNAL Exported Thursday, Jul 13, 2017 from SnapGene Viewer 3.3.4 http://www.snapgene.com FEATURES Location/Qualifiers source 1..13514 /organism="synthetic DNA construct" /mol_type="other DNA" misc_feature 439..942 /label=CEN/ARS /note="S. cerevisiae CEN6 centromere fused to an autonomously replicating sequence" terminator 972..1972 /label=CS terminator misc_feature 1979..2055 /label=HDV misc_RNA complement(2056..2131) /label=g RNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" misc_feature 2132..2151 /label=sgNR1 misc_feature 2152..2194 /label=Hammerhead promoter 2201..3384 /label=Ribi CDS 3395..3415 /codon_start=1 /product="nuclear localization signal of SV40 large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 3416..7516 /codon_start=1 /product="Cas9 (Csn1) endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system" /label=Cas9 /note="generates RNA-guided double strand breaks in DNA" /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD ATLIHQSITGLYETRIDLSQLGGD" CDS 7553..8068 /codon_start=1 /label=Nlux /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW RLCERILAG" CDS 8069..8095 /codon_start=1 /product="HA (human influenza hemagglutinin) epitope tag" /label=HA /translation="YPYDVPDYA" CDS 8111..8131 /codon_start=1 /product="nuclear localization signal of SV40 large T antigen" /label=SV40 NLS /translation="PKKKRKV" terminator 8163..8762 /label=LDSP terminator promoter 8849..9618 /label=LDSP promoter CDS 9643..10668 /codon_start=1 /gene="aph(4)-Ia" /product="aminoglycoside phosphotransferase from E. coli" /label=HygR /note="confers resistance to hygromycin" /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK E" terminator 10669..10899 /label=35S terminator rep_origin complement(11077..11665) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" CDS complement(11836..12696) /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" promoter complement(12697..12801) /gene="bla" /label=AmpR promoter rep_origin complement(12879..13334) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind 13475..13491 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" ORIGIN 1 gggcgaattg ggcccgacgt cgcatgctcc cggccgccat ggcggccgcg ggaattcgat 61 atcactagag tatagctaca tggtagctag tagtcgtcaa aggaaataaa gacatagcgt 121 aaagtataca aaatgacttc cgcggcgcgg gagcggcagg cagcacttgc aaaacgaagc 181 ccaatgggcc agattacggt caacctcgtc attttattac ggtcgtcagc aagagcggtt 241 tagaccgtcg ccggcacggg tggctcccga tattcttggc tttcgtgggt ggaggcggcg 301 tgtgactcct catctttaag tagtggtcgc ttttgctgca tgcttttgat cacctcgtga 361 ttgatggctg gattctcagc tgtcttctga catctttggg tgcatgggtt gaccggtggc 421 cggccggttt cttagacgga tcgcttgcct gtaacttaca cgcgcctcgt atcttttaat 481 gatggaataa tttgggaatt tactctgtgt ttatttattt ttatgttttg tatttggatt 541 ttagaaagta aataaagaag gtagaagagt tacggaatga agaaaaaaaa ataaacaaag 601 gtttaaaaaa tttcaacaaa aagcgtactt tacatatata tttattagac aagaaaagca 661 gattaaatag atatacattc gattaacgat aagtaaaatg taaaatcaca ggattttcgt 721 gtgtggtctt ctacacagac aagatgaaac aattcggcat taatacctga gagcaggaag 781 agcaagataa aaggtagtat ttgttggcga tccccctaga gtcttttaca tcttcggaaa 841 acaaaaacta ttttttcttt aatttctttt tttactttct atttttaatt tatatattta 901 tattaaaaaa tttaaattat aattattttt atagcacgtg atgaaaagga cccaggtgtt 961 taaacaccgg tgcaaagctg acgccctttt cgtcgtcgtt gctggtgtta aaattgcgcg 1021 tttctttttt ttccgtcttt gtcgtctttt gactttttta tcttcttttc tgtcttcttt 1081 tattcttctc ttcttcttca tcactccctc ttcatttttc ttcttctttt tcctcgtcta 1141 ttgtcctcgg gcattaccag ttgtgctgct cacgtgcgcg caagcggtga aagggtgaga 1201 ggatacacgg acaacaatca ttgaaaatta cgcttaaagc cggtgcctgg aaaagtccac 1261 gcgcagatgg cctttgcttg ccttgctgct gctgcagctg ctgctgctgt cgcctgtgat 1321 ggtaatggtt gttgttgtgg tcctgcttgc ttgctagttt gcattgcgtt tgcggttgcc 1381 ttatgaccga ctccgtgtga ttgatcctta aacatatttt atcattgatg tggattgtgt 1441 gacgacggca aaaaagaatt actttacaca tgactatcac acctgctccc ctcccttctc 1501 ctttcaatcg gtcaacttcc caaccgctcc tcgaaaatat ttcagttgaa tcctctacta 1561 cgtacccctt acttaaattt actttctctc tccccatcaa tgcgccgttg ttctgaagtt 1621 gtattattga tgggagatga ttggcactgc accagacctg gccaaaagcc accacaggtg 1681 gccacatact cttgcatggg tatactctac cgaggcaaag attaaagtgc acgtcccctg 1741 cccctctctc tccctacttc cttcagtctc agtggttccg tgcccgttca gcactacatc 1801 gccctgcccg ctgttccttc tcaaacagcc acccatccca acccacatac acagaaacaa 1861 cctttcctac acattttccc aagtcttctc ttcactattc tacactaata caccaccgtg 1921 atatgccggt tatctagatg tcctttctcc tttctcatcc tttctttccc atggtaccca 1981 ggagtcccat tcgccatgcc gaagcatgtt gcccagccgg cgccagcgag gaggctggga 2041 ccatgccggc caaaagcacc gactcggtgc cactttttca agttgataac ggactagcct 2101 tattttaact tgctatttct agctctaaaa ctttaacgcc gagccgcaca cgacgagctt 2161 actcgtttcg tcctcacgga ctcatcaggt gtgcatcgat tctgtggatg gagggaggga 2221 aggagggggg gagtgagtga gtcgagagaa cgacgactgg ggcaaagaga ggatgacgcc 2281 actgcaacaa aagacaggga agaatagagg ctgttggaaa acaggaacac agtgaagaat 2341 agaggctgca gggtaacagg cacatttatc gcagagaggc gggcggcagg ggcgatacgt 2401 aaagagaccc ctaaacttct ccgcgtcatc cgttcacgct acctccttcc ctccctttac 2461 tcccattgat caccaaggca gcgcccgcag ccaccagcag ccaccgtcct cagtgtcaca 2521 gctagacgcg tccccatcag gcagcagacc accagaatat gcgctttcgc ttcccatcat 2581 atccctccct cctccccttc tattcctcat catgcatcat ttctttctcc ttgctgcatg 2641 cccctcttct taccttggct aagatctacc ggaggaagaa gtcttcaacg atagataaat 2701 gcctgtatcg tgatggttgt ttgaatggtg gaagtgagcc aggtattgag aacgaggggc 2761 gcaagggtgc gtggaagaag gcagggagag gcgaggcgaa ggagaagggc atgtggtggg 2821 aacgaagcct ccacaccaca gccacccatg cttttctctg ttcgagttcc cgggttctac 2881 tctcacacac tacacgtgca cagaaaacat tcacgcacaa cagagataca gagggatccg 2941 tcgagatggt ctacacgtcc taatgtcccc tgctttcgaa gcccagagcc atcgctttct 3001 gtttttatcc attcgcaaca catcctgttc tcgcgacctc tcctcttcct ggccaccatc 3061 gcttctccgt gccatgccct gtacatcctt ccctccctcg ttgtctacca cactaatcat 3121 gcgctctgca gcagacactc ggctactatt tgtgtcgtac ggattgaggg gagggagggg 3181 aagggaggca agaagtaaat cggtggtcaa caacagacgc ggtctcccca ccctccctca 3241 ttcctttccc ctcctataat gagcgaagaa agccatggac accgagtcac ccactctcct 3301 aatccaccac caccagggca ctaacggact cacgctcacc aaactcggta acaatatcca 3361 ctgcacacag atacacacgc aacactcgag catgcccaag aaaaagcgga aggtggacaa 3421 gaagtactcc attgggctcg atatcggcac aaacagcgtc ggctgggccg tcattacgga 3481 cgagtacaag gtgccgagca aaaaattcaa agttctgggc aataccgatc gccacagcat 3541 aaagaagaac ctcattggcg ccctcctgtt cgactccggg gagacggccg aagccacgcg 3601 gctcaaaaga acagcacggc gcagatatac ccgcagaaag aatcggatct gctacctgca 3661 ggagatcttt agtaatgaga tggctaaggt ggatgactct ttcttccata ggctggagga 3721 gtcctttttg gtggaggagg ataaaaagca cgagcgccac ccaatctttg gcaatatcgt 3781 ggacgaggtg gcgtaccatg aaaagtaccc aaccatatat catctgagga agaagcttgt 3841 agacagtact gataaggctg acttgcggtt gatctatctc gcgctggcgc atatgatcaa 3901 atttcgggga cacttcctca tcgaggggga cctgaaccca gacaacagcg atgtcgacaa 3961 actctttatc caactggttc agacttacaa tcagcttttc gaagagaacc cgatcaacgc 4021 atccggagtt gacgccaaag caatcctgag cgctaggctg tccaaatccc ggcggctcga 4081 aaacctcatc gcacagctcc ctggggagaa gaagaacggc ctgtttggta atcttatcgc 4141 cctgtcactc gggctgaccc ccaactttaa atctaacttc gacctggccg aagatgccaa 4201 gcttcaactg agcaaagaca cctacgatga tgatctcgac aatctgctgg cccagatcgg 4261 cgaccagtac gcagaccttt ttttggcggc aaagaacctg tcagacgcca ttctgctgag 4321 tgatattctg cgagtgaaca cggagatcac caaagctccg ctgagcgcta gtatgatcaa 4381 gcgctatgat gagcaccacc aagacttgac tttgctgaag gcccttgtca gacagcaact 4441 gcctgagaag tacaaggaaa ttttcttcga tcagtctaaa aatggctacg ccggatacat 4501 tgacggcgga gcaagccagg aggaatttta caaatttatt aagcccatct tggaaaaaat 4561 ggacggcacc gaggagctgc tggtaaagct taacagagaa gatctgttgc gcaaacagcg 4621 cactttcgac aatggaagca tcccccacca gattcacctg ggcgaactgc acgctatcct 4681 caggcggcaa gaggatttct accccttttt gaaagataac agggaaaaga ttgagaaaat 4741 cctcacattt cggataccct actatgtagg ccccctcgcc cggggaaatt ccagattcgc 4801 gtggatgact cgcaaatcag aagagaccat cactccctgg aacttcgagg aagtcgtgga 4861 taagggggcc tctgcccagt ccttcatcga aaggatgact aactttgata aaaatctgcc 4921 taacgaaaag gtgcttccta aacactctct gctgtacgag tacttcacag tttataacga 4981 gctcaccaag gtcaaatacg tcacagaagg gatgagaaag ccagcattcc tgtctggaga 5041 gcagaagaaa gctatcgtgg acctcctctt caagacgaac cggaaagtta ccgtgaaaca 5101 gctcaaagaa gactatttca aaaagattga atgtttcgac tctgttgaaa tcagcggagt 5161 ggaggatcgc ttcaacgcat ccctgggaac gtatcacgat ctcctgaaaa tcattaaaga 5221 caaggacttc ctggacaatg aggagaacga ggacattctt gaggacattg tcctcaccct 5281 tacgttgttt gaagataggg agatgattga agaacgcttg aaaacttacg ctcatctctt 5341 cgacgacaaa gtcatgaaac agctcaagag gcgccgatat acaggatggg ggcggctgtc 5401 aagaaaactg atcaatggga tccgagacaa gcagagtgga aagacaatcc tggattttct 5461 taagtccgat ggatttgcca accggaactt catgcagttg atccatgatg actctctcac 5521 ctttaaggag gacatccaga aagcacaagt ttctggccag ggggacagtc ttcacgagca 5581 catcgctaat cttgcaggta gcccagctat caaaaaggga atactgcaga ccgttaaggt 5641 cgtggatgaa ctcgtcaaag taatgggaag gcataagccc gagaatatcg ttatcgagat 5701 ggcccgagag aaccaaacta cccagaaggg acagaagaac agtagggaaa ggatgaagag 5761 gattgaagag ggtataaaag aactggggtc ccaaatcctt aaggaacacc cagttgaaaa 5821 cacccagctt cagaatgaga agctctacct gtactacctg cagaacggca gggacatgta 5881 cgtggatcag gaactggaca tcaatcggct ctccgactac gacgtggatc atatcgtgcc 5941 ccagtctttt ctcaaagatg attctattga taataaagtg ttgacaagat ccgataaaaa 6001 tagagggaag agtgataacg tcccctcaga agaagttgtc aagaaaatga aaaattattg 6061 gcggcagctg ctgaacgcca aactgatcac acaacggaag ttcgataatc tgactaaggc 6121 tgaacgaggt ggcctgtctg agttggataa agccggcttc atcaaaaggc agcttgttga 6181 gacacgccag atcaccaagc acgtggccca aattctcgat tcacgcatga acaccaagta 6241 cgatgaaaat gacaaactga ttcgagaggt gaaagttatt actctgaagt ctaagctggt 6301 ctcagatttc agaaaggact ttcagtttta taaggtgaga gagatcaaca attaccacca 6361 tgcgcatgat gcctacctga atgcagtggt aggcactgca cttatcaaaa aatatcccaa 6421 gcttgaatct gaatttgttt acggagacta taaagtgtac gatgttagga aaatgatcgc 6481 aaagtctgag caggaaatag gcaaggccac cgctaagtac ttcttttaca gcaatattat 6541 gaattttttc aagaccgaga ttacactggc caatggagag attcggaagc gaccacttat 6601 cgaaacaaac ggagaaacag gagaaatcgt gtgggacaag ggtagggatt tcgcgacagt 6661 ccggaaggtc ctgtccatgc cgcaggtgaa catcgttaaa aagaccgaag tacagaccgg 6721 aggcttctcc aaggaaagta tcctcccgaa aaggaacagc gacaagctga tcgcacgcaa 6781 aaaagattgg gaccccaaga aatacggcgg attcgattct cctacagtcg cttacagtgt 6841 actggttgtg gccaaagtgg agaaagggaa gtctaaaaaa ctcaaaagcg tcaaggaact 6901 gctgggcatc acaatcatgg agcgatcaag cttcgaaaaa aaccccatcg actttctcga 6961 ggcgaaagga tataaagagg tcaaaaaaga cctcatcatt aagcttccca agtactctct 7021 ctttgagctt gaaaacggcc ggaaacgaat gctcgctagt gcgggcgagc tgcagaaagg 7081 taacgagctg gcactgccct ctaaatacgt taatttcttg tatctggcca gccactatga 7141 aaagctcaaa gggtctcccg aagataatga gcagaagcag ctgttcgtgg aacaacacaa 7201 acactacctt gatgagatca tcgagcaaat aagcgaattc tccaaaagag tgatcctcgc 7261 cgacgctaac ctcgataagg tgctttctgc ttacaataag cacagggata agcccatcag 7321 ggagcaggca gaaaacatta tccacttgtt tactctgacc aacttgggcg cgcctgcagc 7381 cttcaagtac ttcgacacca ccatagacag aaagcggtac acctctacaa aggaggtcct 7441 ggacgccaca ctgattcatc agtcaattac ggggctctat gaaacaagaa tcgacctctc 7501 tcagctcggt ggagacagca gggctgacgg gtccggcagc gggtccgtta acatggtgtt 7561 tactctcgag gacttcgtgg gcgactggcg gcagacagct ggctataacc tcgaccaggt 7621 gctggagcag ggcggtgtgt cctcgctttt ccagaacctg ggcgtctccg tgaccccaat 7681 ccagcgcatc gtcctgagcg gcgagaacgg cctgaagatc gacatccacg tcatcattcc 7741 ctacgagggc ctgagcgggg accaaatggg ccagatcgag aagatcttca aggttgtgta 7801 ccccgtggac gatcaccact tcaaggtgat actgcactac gggacgctgg tcatcgacgg 7861 tgtgacgccg aacatgattg actacttcgg ccgtccttac gagggcattg ccgtgttcga 7921 cggcaagaag atcacggtga ccggcaccct gtggaacggc aataagatca ttgacgagcg 7981 gctgatcaac ccggatggct cgctgttgtt ccgcgtgacc atcaacgggg taaccggctg 8041 gcgcctgtgc gagcgcatcc tggccggata cccctacgac gtgcccgact acgcgtccat 8101 ggtggctagc ccgaaaaaga agaggaaggt ctgacaattg gaaagatcca agagagacga 8161 gtagagattt tttttttggg attgatgttt gtcgttcttt gagttgtcgt cgagttacgc 8221 cttttgtaag aatgttccgc aggagaggag gaggatgggc atgagtgagg gtgagagggc 8281 ttgcccgctt ttttttttaa aaacgctgaa gacgtggttg tcaaacaaac cccccataga 8341 aacgattttg ttacggtgcg gtccagacgt cacttgaatg gctccgcgga aaggccaggg 8401 agggaagggg ggagggagga aacatgaaac atgttgaacg gctcaacagg gtttggggga 8461 caagagaggt agcgccctga tggactgctc cctcccctcc tttccctcaa tgtctcattc 8521 atccatgctt cccccttctc tctctcccct ccgttccatc ccccgcgggc gtggtagtgg 8581 cgtgatggga tccactaaaa tgtacgtgta agaaaagccg gtgagcttac gcttttgtga 8641 aagtgggagt acgagtgttg tgtgtgtgtg tagtggtttc agaccccaga cagaggcgaa 8701 gcagaaaaag cagacgatga agacgacgaa gaaatgagca gtctattttt atcgtggaaa 8761 cagaagaggt gatatcgtct cctgcaggca tgcaagctga tccactagag gccatggcgg 8821 ccgcactagt ggatcccccg ggatgcatac ttggaagatg gagtggatgg aggaggaggc 8881 gagcgtagca gcaagcgtga gttatacagc caggcacatg tcgcaatcct tcggtctcgg 8941 gcttaaaatc cacgcactaa tcacgctggg ccatgcaaag agcaatgccg aggcccacca 9001 cacaaaacgc tgtgtcgcgc gttgcggcct gaagcttcat acttcttagt cgccgccaaa 9061 agggctcgag agacgagacc cgttggcatg accgatgttg ttcgacgcgg tttgcttcgt 9121 cacagtcgac gtgattcagg aatctggagc ctgcagatca tttttttcag cctgatatcg 9181 ttcttttcca ctgagaacca tcagaccacc ttttcttcca ttgtgtgaag gagtaggagt 9241 tgccgtgctg ctttgtggga gacatctgcg atggtgacca gcctcccgtc gtctggtcga 9301 cgtgacgagc ctcttcactg ttcttcgacg gagagacgca agcgagacgg ctctagacct 9361 tttggacacg cattctgtgt gtgaactagt ggacagtgat accacgtctg aaagctcacc 9421 actgcccatg gtgcagctac ttgtcacaaa gttttgactc cgtcggtatc accattcgcg 9481 ctcgtgtgcc tggttgttcc gccacgccgg cctgccccgg ggcggggcaa tattctaaaa 9541 tctcacgcaa aacaccgcac ttacccctca cacatattcg tgatagacca ccaccaatct 9601 cagcccgcat caacacagga gggccccggg gggcaataag atatgaaaaa gcctgaactc 9661 accgcgacgt ctgtcgagaa gtttctgatc gaaaagttcg acagcgtctc cgacctgatg 9721 cagctctcgg agggcgaaga atctcgtgct ttcagcttcg atgtaggagg gcgtggatat 9781 gtcctgcggg taaatagctg cgccgatggt ttctacaaag atcgttatgt ttatcggcac 9841 tttgcatcgg ccgcgctccc gattccggaa gtgcttgaca ttggggaatt cagcgagagc 9901 ctgacctatt gcatctcccg ccgtgcacag ggtgtcacgt tgcaagacct gcctgaaacc 9961 gaactgcccg ctgttctgca gccggtcgcg gaggccatgg atgcgatcgc tgcggccgat 10021 cttagccaga cgagcgggtt cggcccattc ggaccgcaag gaatcggtca atacactaca 10081 tggcgtgatt tcatatgcgc gattgctgat ccccatgtgt atcactggca aactgtgatg 10141 gacgacaccg tcagtgcgtc cgtcgcgcag gctctcgatg agctgatgct ttgggccgag 10201 gactgccccg aagtccggca cctcgtgcac gcggatttcg gctccaacaa tgtcctgacg 10261 gacaatggcc gcataacagc ggtcattgac tggagcgagg cgatgttcgg ggattcccaa 10321 tacgaggtcg ccaacatctt cttctggagg ccgtggttgg cttgtatgga gcagcagacg 10381 cgctacttcg agcggaggca tccggagctt gcaggatcgc cgcggctccg ggcgtatatg 10441 ctccgcattg gtcttgacca actctatcag agcttggttg acggcaattt cgatgatgca 10501 gcttgggcgc agggtcgatg cgacgcaatc gtccgatccg gagccgggac tgtcgggcgt 10561 acacaaatcg cccgcagaag cgcggccgtc tggaccgatg gctgtgtaga agtactcgcc 10621 gatagtggaa accgacgccc cagcactcgt ccgagggcaa aggaatagag tagatgccga 10681 ccggagtccg caaaaatcac cagtctctct ctacaaatct atctctctct atttttctcc 10741 agaataatgt gtgagtagtt cccagataag ggaattaggg ttcttatagg gtttcgctca 10801 tgtgttgagc atataagaaa cccttagtat gtatttgtat ttgtaaaata cttctatcaa 10861 taaaatttct aattcctaaa accaaaatcc agtgacctga gcttcctcgc tcactgactc 10921 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg 10981 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa 11041 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga 11101 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag 11161 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct 11221 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg 11281 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc 11341 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt 11401 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta 11461 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac 11521 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc 11581 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat 11641 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc 11701 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt 11761 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta 11821 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct 11881 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg 11941 cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga 12001 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt 12061 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt 12121 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt 12181 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat 12241 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc 12301 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc 12361 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat 12421 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag 12481 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt 12541 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc 12601 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa 12661 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg 12721 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa 12781 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgatg cggtgtgaaa 12841 taccgcacag atgcgtaagg agaaaatacc gcatcaggaa attgtaagcg ttaatatttt 12901 gttaaaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat aggccgaaat 12961 cggcaaaatc ccttataaat caaaagaata gaccgagata gggttgagtg ttgttccagt 13021 ttggaacaag agtccactat taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt 13081 ctatcagggc gatggcccac tacgtgaacc atcaccctaa tcaagttttt tggggtcgag 13141 gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg 13201 aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc 13261 gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc 13321 gctacagggc gcgtccattc gccattcagg ctgcgcaact gttgggaagg gcgatcggtg 13381 cgggcctctt cgctattacg ccagctggcg aaagggggat gtgctgcaag gcgattaagt 13441 tgggtaacgc cagggttttc ccagtcacga cgttgtaaaa cgacggccag tgaattgtaa 13501 tacgactcac tata // LOCUS Exported 13514 bp ds-DNA circular SYN 10-JUL-2017 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS pNOC-ARS-CRISPR-sgNR2 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 13514) AUTHORS . TITLE Direct Submission JOURNAL Exported Wednesday, Jul 12, 2017 from SnapGene Viewer 3.3.4 http://www.snapgene.com FEATURES Location/Qualifiers source 1..13514 /organism="synthetic DNA construct" /mol_type="other DNA" misc_feature 439..942 /label=CEN/ARS /note="S. cerevisiae CEN6 centromere fused to an autonomously replicating sequence" terminator 972..1972 /label=CS terminator misc_feature 1979..2055 /label=HDV misc_RNA complement(2056..2131) /label=g RNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" misc_feature 2132..2151 /label=sgNR2 misc_feature 2152..2194 /label=Hammerhead promoter 2201..3384 /label=Ribi CDS 3395..3415 /codon_start=1 /product="nuclear localization signal of SV40 large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 3416..7516 /codon_start=1 /product="Cas9 (Csn1) endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system" /label=Cas9 /note="generates RNA-guided double strand breaks in DNA" /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD ATLIHQSITGLYETRIDLSQLGGD" CDS 7553..8068 /codon_start=1 /label=Nlux /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW RLCERILAG" CDS 8069..8095 /codon_start=1 /product="HA (human influenza hemagglutinin) epitope tag" /label=HA /translation="YPYDVPDYA" CDS 8111..8131 /codon_start=1 /product="nuclear localization signal of SV40 large T antigen" /label=SV40 NLS /translation="PKKKRKV" terminator 8163..8762 /label=LDSP terminator promoter 8849..9618 /label=LDSP promoter CDS 9643..10668 /codon_start=1 /gene="aph(4)-Ia" /product="aminoglycoside phosphotransferase from E. coli" /label=HygR /note="confers resistance to hygromycin" /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK E" terminator 10669..10899 /label=35S terminator rep_origin complement(11077..11665) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" CDS complement(11836..12696) /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" promoter complement(12697..12801) /gene="bla" /label=AmpR promoter rep_origin complement(12879..13334) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind 13475..13491 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" ORIGIN 1 gggcgaattg ggcccgacgt cgcatgctcc cggccgccat ggcggccgcg ggaattcgat 61 atcactagag tatagctaca tggtagctag tagtcgtcaa aggaaataaa gacatagcgt 121 aaagtataca aaatgacttc cgcggcgcgg gagcggcagg cagcacttgc aaaacgaagc 181 ccaatgggcc agattacggt caacctcgtc attttattac ggtcgtcagc aagagcggtt 241 tagaccgtcg ccggcacggg tggctcccga tattcttggc tttcgtgggt ggaggcggcg 301 tgtgactcct catctttaag tagtggtcgc ttttgctgca tgcttttgat cacctcgtga 361 ttgatggctg gattctcagc tgtcttctga catctttggg tgcatgggtt gaccggtggc 421 cggccggttt cttagacgga tcgcttgcct gtaacttaca cgcgcctcgt atcttttaat 481 gatggaataa tttgggaatt tactctgtgt ttatttattt ttatgttttg tatttggatt 541 ttagaaagta aataaagaag gtagaagagt tacggaatga agaaaaaaaa ataaacaaag 601 gtttaaaaaa tttcaacaaa aagcgtactt tacatatata tttattagac aagaaaagca 661 gattaaatag atatacattc gattaacgat aagtaaaatg taaaatcaca ggattttcgt 721 gtgtggtctt ctacacagac aagatgaaac aattcggcat taatacctga gagcaggaag 781 agcaagataa aaggtagtat ttgttggcga tccccctaga gtcttttaca tcttcggaaa 841 acaaaaacta ttttttcttt aatttctttt tttactttct atttttaatt tatatattta 901 tattaaaaaa tttaaattat aattattttt atagcacgtg atgaaaagga cccaggtgtt 961 taaacaccgg tgcaaagctg acgccctttt cgtcgtcgtt gctggtgtta aaattgcgcg 1021 tttctttttt ttccgtcttt gtcgtctttt gactttttta tcttcttttc tgtcttcttt 1081 tattcttctc ttcttcttca tcactccctc ttcatttttc ttcttctttt tcctcgtcta 1141 ttgtcctcgg gcattaccag ttgtgctgct cacgtgcgcg caagcggtga aagggtgaga 1201 ggatacacgg acaacaatca ttgaaaatta cgcttaaagc cggtgcctgg aaaagtccac 1261 gcgcagatgg cctttgcttg ccttgctgct gctgcagctg ctgctgctgt cgcctgtgat 1321 ggtaatggtt gttgttgtgg tcctgcttgc ttgctagttt gcattgcgtt tgcggttgcc 1381 ttatgaccga ctccgtgtga ttgatcctta aacatatttt atcattgatg tggattgtgt 1441 gacgacggca aaaaagaatt actttacaca tgactatcac acctgctccc ctcccttctc 1501 ctttcaatcg gtcaacttcc caaccgctcc tcgaaaatat ttcagttgaa tcctctacta 1561 cgtacccctt acttaaattt actttctctc tccccatcaa tgcgccgttg ttctgaagtt 1621 gtattattga tgggagatga ttggcactgc accagacctg gccaaaagcc accacaggtg 1681 gccacatact cttgcatggg tatactctac cgaggcaaag attaaagtgc acgtcccctg 1741 cccctctctc tccctacttc cttcagtctc agtggttccg tgcccgttca gcactacatc 1801 gccctgcccg ctgttccttc tcaaacagcc acccatccca acccacatac acagaaacaa 1861 cctttcctac acattttccc aagtcttctc ttcactattc tacactaata caccaccgtg 1921 atatgccggt tatctagatg tcctttctcc tttctcatcc tttctttccc atggtaccca 1981 ggagtcccat tcgccatgcc gaagcatgtt gcccagccgg cgccagcgag gaggctggga 2041 ccatgccggc caaaagcacc gactcggtgc cactttttca agttgataac ggactagcct 2101 tattttaact tgctatttct agctctaaaa cccagtacag acgcgcgaga cgacgagctt 2161 actcgtttcg tcctcacgga ctcatcaggt ctcgatcgat tctgtggatg gagggaggga 2221 aggagggggg gagtgagtga gtcgagagaa cgacgactgg ggcaaagaga ggatgacgcc 2281 actgcaacaa aagacaggga agaatagagg ctgttggaaa acaggaacac agtgaagaat 2341 agaggctgca gggtaacagg cacatttatc gcagagaggc gggcggcagg ggcgatacgt 2401 aaagagaccc ctaaacttct ccgcgtcatc cgttcacgct acctccttcc ctccctttac 2461 tcccattgat caccaaggca gcgcccgcag ccaccagcag ccaccgtcct cagtgtcaca 2521 gctagacgcg tccccatcag gcagcagacc accagaatat gcgctttcgc ttcccatcat 2581 atccctccct cctccccttc tattcctcat catgcatcat ttctttctcc ttgctgcatg 2641 cccctcttct taccttggct aagatctacc ggaggaagaa gtcttcaacg atagataaat 2701 gcctgtatcg tgatggttgt ttgaatggtg gaagtgagcc aggtattgag aacgaggggc 2761 gcaagggtgc gtggaagaag gcagggagag gcgaggcgaa ggagaagggc atgtggtggg 2821 aacgaagcct ccacaccaca gccacccatg cttttctctg ttcgagttcc cgggttctac 2881 tctcacacac tacacgtgca cagaaaacat tcacgcacaa cagagataca gagggatccg 2941 tcgagatggt ctacacgtcc taatgtcccc tgctttcgaa gcccagagcc atcgctttct 3001 gtttttatcc attcgcaaca catcctgttc tcgcgacctc tcctcttcct ggccaccatc 3061 gcttctccgt gccatgccct gtacatcctt ccctccctcg ttgtctacca cactaatcat 3121 gcgctctgca gcagacactc ggctactatt tgtgtcgtac ggattgaggg gagggagggg 3181 aagggaggca agaagtaaat cggtggtcaa caacagacgc ggtctcccca ccctccctca 3241 ttcctttccc ctcctataat gagcgaagaa agccatggac accgagtcac ccactctcct 3301 aatccaccac caccagggca ctaacggact cacgctcacc aaactcggta acaatatcca 3361 ctgcacacag atacacacgc aacactcgag catgcccaag aaaaagcgga aggtggacaa 3421 gaagtactcc attgggctcg atatcggcac aaacagcgtc ggctgggccg tcattacgga 3481 cgagtacaag gtgccgagca aaaaattcaa agttctgggc aataccgatc gccacagcat 3541 aaagaagaac ctcattggcg ccctcctgtt cgactccggg gagacggccg aagccacgcg 3601 gctcaaaaga acagcacggc gcagatatac ccgcagaaag aatcggatct gctacctgca 3661 ggagatcttt agtaatgaga tggctaaggt ggatgactct ttcttccata ggctggagga 3721 gtcctttttg gtggaggagg ataaaaagca cgagcgccac ccaatctttg gcaatatcgt 3781 ggacgaggtg gcgtaccatg aaaagtaccc aaccatatat catctgagga agaagcttgt 3841 agacagtact gataaggctg acttgcggtt gatctatctc gcgctggcgc atatgatcaa 3901 atttcgggga cacttcctca tcgaggggga cctgaaccca gacaacagcg atgtcgacaa 3961 actctttatc caactggttc agacttacaa tcagcttttc gaagagaacc cgatcaacgc 4021 atccggagtt gacgccaaag caatcctgag cgctaggctg tccaaatccc ggcggctcga 4081 aaacctcatc gcacagctcc ctggggagaa gaagaacggc ctgtttggta atcttatcgc 4141 cctgtcactc gggctgaccc ccaactttaa atctaacttc gacctggccg aagatgccaa 4201 gcttcaactg agcaaagaca cctacgatga tgatctcgac aatctgctgg cccagatcgg 4261 cgaccagtac gcagaccttt ttttggcggc aaagaacctg tcagacgcca ttctgctgag 4321 tgatattctg cgagtgaaca cggagatcac caaagctccg ctgagcgcta gtatgatcaa 4381 gcgctatgat gagcaccacc aagacttgac tttgctgaag gcccttgtca gacagcaact 4441 gcctgagaag tacaaggaaa ttttcttcga tcagtctaaa aatggctacg ccggatacat 4501 tgacggcgga gcaagccagg aggaatttta caaatttatt aagcccatct tggaaaaaat 4561 ggacggcacc gaggagctgc tggtaaagct taacagagaa gatctgttgc gcaaacagcg 4621 cactttcgac aatggaagca tcccccacca gattcacctg ggcgaactgc acgctatcct 4681 caggcggcaa gaggatttct accccttttt gaaagataac agggaaaaga ttgagaaaat 4741 cctcacattt cggataccct actatgtagg ccccctcgcc cggggaaatt ccagattcgc 4801 gtggatgact cgcaaatcag aagagaccat cactccctgg aacttcgagg aagtcgtgga 4861 taagggggcc tctgcccagt ccttcatcga aaggatgact aactttgata aaaatctgcc 4921 taacgaaaag gtgcttccta aacactctct gctgtacgag tacttcacag tttataacga 4981 gctcaccaag gtcaaatacg tcacagaagg gatgagaaag ccagcattcc tgtctggaga 5041 gcagaagaaa gctatcgtgg acctcctctt caagacgaac cggaaagtta ccgtgaaaca 5101 gctcaaagaa gactatttca aaaagattga atgtttcgac tctgttgaaa tcagcggagt 5161 ggaggatcgc ttcaacgcat ccctgggaac gtatcacgat ctcctgaaaa tcattaaaga 5221 caaggacttc ctggacaatg aggagaacga ggacattctt gaggacattg tcctcaccct 5281 tacgttgttt gaagataggg agatgattga agaacgcttg aaaacttacg ctcatctctt 5341 cgacgacaaa gtcatgaaac agctcaagag gcgccgatat acaggatggg ggcggctgtc 5401 aagaaaactg atcaatggga tccgagacaa gcagagtgga aagacaatcc tggattttct 5461 taagtccgat ggatttgcca accggaactt catgcagttg atccatgatg actctctcac 5521 ctttaaggag gacatccaga aagcacaagt ttctggccag ggggacagtc ttcacgagca 5581 catcgctaat cttgcaggta gcccagctat caaaaaggga atactgcaga ccgttaaggt 5641 cgtggatgaa ctcgtcaaag taatgggaag gcataagccc gagaatatcg ttatcgagat 5701 ggcccgagag aaccaaacta cccagaaggg acagaagaac agtagggaaa ggatgaagag 5761 gattgaagag ggtataaaag aactggggtc ccaaatcctt aaggaacacc cagttgaaaa 5821 cacccagctt cagaatgaga agctctacct gtactacctg cagaacggca gggacatgta 5881 cgtggatcag gaactggaca tcaatcggct ctccgactac gacgtggatc atatcgtgcc 5941 ccagtctttt ctcaaagatg attctattga taataaagtg ttgacaagat ccgataaaaa 6001 tagagggaag agtgataacg tcccctcaga agaagttgtc aagaaaatga aaaattattg 6061 gcggcagctg ctgaacgcca aactgatcac acaacggaag ttcgataatc tgactaaggc 6121 tgaacgaggt ggcctgtctg agttggataa agccggcttc atcaaaaggc agcttgttga 6181 gacacgccag atcaccaagc acgtggccca aattctcgat tcacgcatga acaccaagta 6241 cgatgaaaat gacaaactga ttcgagaggt gaaagttatt actctgaagt ctaagctggt 6301 ctcagatttc agaaaggact ttcagtttta taaggtgaga gagatcaaca attaccacca 6361 tgcgcatgat gcctacctga atgcagtggt aggcactgca cttatcaaaa aatatcccaa 6421 gcttgaatct gaatttgttt acggagacta taaagtgtac gatgttagga aaatgatcgc 6481 aaagtctgag caggaaatag gcaaggccac cgctaagtac ttcttttaca gcaatattat 6541 gaattttttc aagaccgaga ttacactggc caatggagag attcggaagc gaccacttat 6601 cgaaacaaac ggagaaacag gagaaatcgt gtgggacaag ggtagggatt tcgcgacagt 6661 ccggaaggtc ctgtccatgc cgcaggtgaa catcgttaaa aagaccgaag tacagaccgg 6721 aggcttctcc aaggaaagta tcctcccgaa aaggaacagc gacaagctga tcgcacgcaa 6781 aaaagattgg gaccccaaga aatacggcgg attcgattct cctacagtcg cttacagtgt 6841 actggttgtg gccaaagtgg agaaagggaa gtctaaaaaa ctcaaaagcg tcaaggaact 6901 gctgggcatc acaatcatgg agcgatcaag cttcgaaaaa aaccccatcg actttctcga 6961 ggcgaaagga tataaagagg tcaaaaaaga cctcatcatt aagcttccca agtactctct 7021 ctttgagctt gaaaacggcc ggaaacgaat gctcgctagt gcgggcgagc tgcagaaagg 7081 taacgagctg gcactgccct ctaaatacgt taatttcttg tatctggcca gccactatga 7141 aaagctcaaa gggtctcccg aagataatga gcagaagcag ctgttcgtgg aacaacacaa 7201 acactacctt gatgagatca tcgagcaaat aagcgaattc tccaaaagag tgatcctcgc 7261 cgacgctaac ctcgataagg tgctttctgc ttacaataag cacagggata agcccatcag 7321 ggagcaggca gaaaacatta tccacttgtt tactctgacc aacttgggcg cgcctgcagc 7381 cttcaagtac ttcgacacca ccatagacag aaagcggtac acctctacaa aggaggtcct 7441 ggacgccaca ctgattcatc agtcaattac ggggctctat gaaacaagaa tcgacctctc 7501 tcagctcggt ggagacagca gggctgacgg gtccggcagc gggtccgtta acatggtgtt 7561 tactctcgag gacttcgtgg gcgactggcg gcagacagct ggctataacc tcgaccaggt 7621 gctggagcag ggcggtgtgt cctcgctttt ccagaacctg ggcgtctccg tgaccccaat 7681 ccagcgcatc gtcctgagcg gcgagaacgg cctgaagatc gacatccacg tcatcattcc 7741 ctacgagggc ctgagcgggg accaaatggg ccagatcgag aagatcttca aggttgtgta 7801 ccccgtggac gatcaccact tcaaggtgat actgcactac gggacgctgg tcatcgacgg 7861 tgtgacgccg aacatgattg actacttcgg ccgtccttac gagggcattg ccgtgttcga 7921 cggcaagaag atcacggtga ccggcaccct gtggaacggc aataagatca ttgacgagcg 7981 gctgatcaac ccggatggct cgctgttgtt ccgcgtgacc atcaacgggg taaccggctg 8041 gcgcctgtgc gagcgcatcc tggccggata cccctacgac gtgcccgact acgcgtccat 8101 ggtggctagc ccgaaaaaga agaggaaggt ctgacaattg gaaagatcca agagagacga 8161 gtagagattt tttttttggg attgatgttt gtcgttcttt gagttgtcgt cgagttacgc 8221 cttttgtaag aatgttccgc aggagaggag gaggatgggc atgagtgagg gtgagagggc 8281 ttgcccgctt ttttttttaa aaacgctgaa gacgtggttg tcaaacaaac cccccataga 8341 aacgattttg ttacggtgcg gtccagacgt cacttgaatg gctccgcgga aaggccaggg 8401 agggaagggg ggagggagga aacatgaaac atgttgaacg gctcaacagg gtttggggga 8461 caagagaggt agcgccctga tggactgctc cctcccctcc tttccctcaa tgtctcattc 8521 atccatgctt cccccttctc tctctcccct ccgttccatc ccccgcgggc gtggtagtgg 8581 cgtgatggga tccactaaaa tgtacgtgta agaaaagccg gtgagcttac gcttttgtga 8641 aagtgggagt acgagtgttg tgtgtgtgtg tagtggtttc agaccccaga cagaggcgaa 8701 gcagaaaaag cagacgatga agacgacgaa gaaatgagca gtctattttt atcgtggaaa 8761 cagaagaggt gatatcgtct cctgcaggca tgcaagctga tccactagag gccatggcgg 8821 ccgcactagt ggatcccccg ggatgcatac ttggaagatg gagtggatgg aggaggaggc 8881 gagcgtagca gcaagcgtga gttatacagc caggcacatg tcgcaatcct tcggtctcgg 8941 gcttaaaatc cacgcactaa tcacgctggg ccatgcaaag agcaatgccg aggcccacca 9001 cacaaaacgc tgtgtcgcgc gttgcggcct gaagcttcat acttcttagt cgccgccaaa 9061 agggctcgag agacgagacc cgttggcatg accgatgttg ttcgacgcgg tttgcttcgt 9121 cacagtcgac gtgattcagg aatctggagc ctgcagatca tttttttcag cctgatatcg 9181 ttcttttcca ctgagaacca tcagaccacc ttttcttcca ttgtgtgaag gagtaggagt 9241 tgccgtgctg ctttgtggga gacatctgcg atggtgacca gcctcccgtc gtctggtcga 9301 cgtgacgagc ctcttcactg ttcttcgacg gagagacgca agcgagacgg ctctagacct 9361 tttggacacg cattctgtgt gtgaactagt ggacagtgat accacgtctg aaagctcacc 9421 actgcccatg gtgcagctac ttgtcacaaa gttttgactc cgtcggtatc accattcgcg 9481 ctcgtgtgcc tggttgttcc gccacgccgg cctgccccgg ggcggggcaa tattctaaaa 9541 tctcacgcaa aacaccgcac ttacccctca cacatattcg tgatagacca ccaccaatct 9601 cagcccgcat caacacagga gggccccggg gggcaataag atatgaaaaa gcctgaactc 9661 accgcgacgt ctgtcgagaa gtttctgatc gaaaagttcg acagcgtctc cgacctgatg 9721 cagctctcgg agggcgaaga atctcgtgct ttcagcttcg atgtaggagg gcgtggatat 9781 gtcctgcggg taaatagctg cgccgatggt ttctacaaag atcgttatgt ttatcggcac 9841 tttgcatcgg ccgcgctccc gattccggaa gtgcttgaca ttggggaatt cagcgagagc 9901 ctgacctatt gcatctcccg ccgtgcacag ggtgtcacgt tgcaagacct gcctgaaacc 9961 gaactgcccg ctgttctgca gccggtcgcg gaggccatgg atgcgatcgc tgcggccgat 10021 cttagccaga cgagcgggtt cggcccattc ggaccgcaag gaatcggtca atacactaca 10081 tggcgtgatt tcatatgcgc gattgctgat ccccatgtgt atcactggca aactgtgatg 10141 gacgacaccg tcagtgcgtc cgtcgcgcag gctctcgatg agctgatgct ttgggccgag 10201 gactgccccg aagtccggca cctcgtgcac gcggatttcg gctccaacaa tgtcctgacg 10261 gacaatggcc gcataacagc ggtcattgac tggagcgagg cgatgttcgg ggattcccaa 10321 tacgaggtcg ccaacatctt cttctggagg ccgtggttgg cttgtatgga gcagcagacg 10381 cgctacttcg agcggaggca tccggagctt gcaggatcgc cgcggctccg ggcgtatatg 10441 ctccgcattg gtcttgacca actctatcag agcttggttg acggcaattt cgatgatgca 10501 gcttgggcgc agggtcgatg cgacgcaatc gtccgatccg gagccgggac tgtcgggcgt 10561 acacaaatcg cccgcagaag cgcggccgtc tggaccgatg gctgtgtaga agtactcgcc 10621 gatagtggaa accgacgccc cagcactcgt ccgagggcaa aggaatagag tagatgccga 10681 ccggagtccg caaaaatcac cagtctctct ctacaaatct atctctctct atttttctcc 10741 agaataatgt gtgagtagtt cccagataag ggaattaggg ttcttatagg gtttcgctca 10801 tgtgttgagc atataagaaa cccttagtat gtatttgtat ttgtaaaata cttctatcaa 10861 taaaatttct aattcctaaa accaaaatcc agtgacctga gcttcctcgc tcactgactc 10921 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg 10981 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa 11041 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga 11101 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag 11161 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct 11221 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg 11281 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc 11341 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt 11401 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta 11461 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac 11521 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc 11581 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat 11641 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc 11701 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt 11761 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta 11821 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct 11881 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg 11941 cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga 12001 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt 12061 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt 12121 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt 12181 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat 12241 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc 12301 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc 12361 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat 12421 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag 12481 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt 12541 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc 12601 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa 12661 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg 12721 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa 12781 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgatg cggtgtgaaa 12841 taccgcacag atgcgtaagg agaaaatacc gcatcaggaa attgtaagcg ttaatatttt 12901 gttaaaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat aggccgaaat 12961 cggcaaaatc ccttataaat caaaagaata gaccgagata gggttgagtg ttgttccagt 13021 ttggaacaag agtccactat taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt 13081 ctatcagggc gatggcccac tacgtgaacc atcaccctaa tcaagttttt tggggtcgag 13141 gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg 13201 aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc 13261 gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc 13321 gctacagggc gcgtccattc gccattcagg ctgcgcaact gttgggaagg gcgatcggtg 13381 cgggcctctt cgctattacg ccagctggcg aaagggggat gtgctgcaag gcgattaagt 13441 tgggtaacgc cagggttttc ccagtcacga cgttgtaaaa cgacggccag tgaattgtaa 13501 tacgactcac tata // LOCUS Exported 10166 bp ds-DNA circular SYN 14-JAN-2017 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS pNOC-ARS-CRISPR-compact SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 10166) AUTHORS . TITLE Direct Submission JOURNAL Exported Friday, Jul 14, 2017 from SnapGene Viewer 3.3.4 http://www.snapgene.com FEATURES Location/Qualifiers source 1..10166 /organism="synthetic DNA construct" /mol_type="other DNA" misc_feature 17..520 /label=CEN/ARS /note="S. cerevisiae CEN6 centromere fused to an autonomously replicating sequence" promoter 557..661 /gene="bla" /label=AmpR promoter CDS 662..1522 /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" rep_origin 1693..2281 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" terminator 2417..3016 /label=LDSP terminator CDS complement(3048..3068) /codon_start=1 /product="nuclear localization signal of SV40 large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS complement(3084..3110) /codon_start=1 /product="HA (human influenza hemagglutinin) epitope tag" /label=HA /translation="YPYDVPDYA" CDS complement(3111..3626) /codon_start=1 /label=Nlux /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW RLCERILAG" CDS complement(3651..7763) /codon_start=1 /label=Cas9 /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD ATLIHQSITGLYETRIDLSQLGGDSRAD" CDS complement(7764..7784) /codon_start=1 /product="nuclear localization signal of SV40 large T antigen" /label=SV40 NLS /translation="PKKKRKV" promoter 7795..8978 /label=Ribi misc_feature 9007..9086 /label=sgRNA misc_feature 9087..9154 /label=HDV terminator 9166..10139 /label=CS terminator ORIGIN 1 accggtttct tagacggatc gcttgcctgt aacttacacg cgcctcgtat cttttaatga 61 tggaataatt tgggaattta ctctgtgttt atttattttt atgttttgta tttggatttt 121 agaaagtaaa taaagaaggt agaagagtta cggaatgaag aaaaaaaaat aaacaaaggt 181 ttaaaaaatt tcaacaaaaa gcgtacttta catatatatt tattagacaa gaaaagcaga 241 ttaaatagat atacattcga ttaacgataa gtaaaatgta aaatcacagg attttcgtgt 301 gtggtcttct acacagacaa gatgaaacaa ttcggcatta atacctgaga gcaggaagta 361 caagataaaa ggtagtattt gttggcgatc cccctagagt cttttacatc ttcggaaaac 421 aaaaactatt ttttctttaa tttctttttt tactttctat ttttaattta tatatttata 481 ttaaaaaatt taaattataa ttatttttat agcacgtgat gaaaaggacc caggtggcac 541 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat 601 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag 661 tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc 721 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc 781 acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc 841 cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc 901 ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt 961 ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt 1021 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat 1081 cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct 1141 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat 1201 gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc 1261 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg 1321 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc 1381 tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta 1441 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc 1501 ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga 1561 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat 1621 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat 1681 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa 1741 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa 1801 ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt 1861 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt 1921 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata 1981 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt 2041 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac 2101 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga 2161 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg 2221 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa 2281 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat 2341 gtttaaacac tagtgcggcc gccatggcct ctagtggatc agcttgcatg cctgcaggag 2401 acgatatcac ctcttctgtt tccacgataa aaatagactg ctcatttctt cgtcgtcttc 2461 atcgtctgct ttttctgctt cgcctctgtc tggggtctga aaccactaca cacacacaca 2521 acactcgtac tcccactttc acaaaagcgt aagctcaccg gcttttctta cacgtacatt 2581 ttagtggatc ccatcacgcc actaccacgc ccgcggggga tggaacggag gggagagaga 2641 gaagggggaa gcatggatga atgagacatt gagggaaagg aggggaggga gcagtccatc 2701 agggcgctac ctctcttgtc ccccaaaccc tgttgagccg ttcaacatgt ttcatgtttc 2761 ctccctcccc ccttccctcc ctggcctttc cgcggagcca ttcaagtgac gtctggaccg 2821 caccgtaaca aaatcgtttc tatggggggt ttgtttgaca accacgtctt cagcgttttt 2881 aaaaaaaaaa gcgggcaagc cctctcaccc tcactcatgc ccatcctcct cctctcctgc 2941 ggaacattct tacaaaaggc gtaactcgac gacaactcaa agaacgacaa acatcaatcc 3001 caaaaaaaaa atctctactc gtctctcttg gatctttcca attgtcagac cttcctcttc 3061 tttttcgggc tagccaccat ggacgcgtag tcgggcacgt cgtaggggta tccggccagg 3121 atgcgctcgc acaggcgcca gccggttacc ccgttgatgg tcacgcggaa caacagcgag 3181 ccatccgggt tgatcagccg ctcgtcaatg atcttattgc cgttccacag ggtgccggtc 3241 accgtgatct tcttgccgtc gaacacggca atgccctcgt aaggacggcc gaagtagtca 3301 atcatgttcg gcgtcacacc gtcgatgacc agcgtcccgt agtgcagtat caccttgaag 3361 tggtgatcgt ccacggggta cacaaccttg aagatcttct cgatctggcc catttggtcc 3421 ccgctcaggc cctcgtaggg aatgatgacg tggatgtcga tcttcaggcc gttctcgccg 3481 ctcaggacga tgcgctggat tggggtcacg gagacgccca ggttctggaa aagcgaggac 3541 acaccgccct gctccagcac ctggtcgagg ttatagccag ctgtctgccg ccagtcgccc 3601 acgaagtcct cgagagtaaa caccatgtta acggacccgc tgccggaccc gtcagccctg 3661 ctgtctccac cgagctgaga gaggtcgatt cttgtttcat agagccccgt aattgactga 3721 tgaatcagtg tggcgtccag gacctccttt gtagaggtgt accgctttct gtctatggtg 3781 gtgtcgaagt acttgaaggc tgcaggcgcg cccaagttgg tcagagtaaa caagtggata 3841 atgttttctg cctgctccct gatgggctta tccctgtgct tattgtaagc agaaagcacc 3901 ttatcgaggt tagcgtcggc gaggatcact cttttggaga attcgcttat ttgctcgatg 3961 atctcatcaa ggtagtgttt gtgttgttcc acgaacagct gcttctgctc attatcttcg 4021 ggagaccctt tgagcttttc atagtggctg gccagataca agaaattaac gtatttagag 4081 ggcagtgcca gctcgttacc tttctgcagc tcgcccgcac tagcgagcat tcgtttccgg 4141 ccgttttcaa gctcaaagag agagtacttg ggaagcttaa tgatgaggtc ttttttgacc 4201 tctttatatc ctttcgcctc gagaaagtcg atggggtttt tttcgaagct tgatcgctcc 4261 atgattgtga tgcccagcag ttccttgacg cttttgagtt ttttagactt ccctttctcc 4321 actttggcca caaccagtac actgtaagcg actgtaggag aatcgaatcc gccgtatttc 4381 ttggggtccc aatctttttt gcgtgcgatc agcttgtcgc tgttcctttt cgggaggata 4441 ctttccttgg agaagcctcc ggtctgtact tcggtctttt taacgatgtt cacctgcggc 4501 atggacagga ccttccggac tgtcgcgaaa tccctaccct tgtcccacac gatttctcct 4561 gtttctccgt ttgtttcgat aagtggtcgc ttccgaatct ctccattggc cagtgtaatc 4621 tcggtcttga aaaaattcat aatattgctg taaaagaagt acttagcggt ggccttgcct 4681 atttcctgct cagactttgc gatcattttc ctaacatcgt acactttata gtctccgtaa 4741 acaaattcag attcaagctt gggatatttt ttgataagtg cagtgcctac cactgcattc 4801 aggtaggcat catgcgcatg gtggtaattg ttgatctctc tcaccttata aaactgaaag 4861 tcctttctga aatctgagac cagcttagac ttcagagtaa taactttcac ctctcgaatc 4921 agtttgtcat tttcatcgta cttggtgttc atgcgtgaat cgagaatttg ggccacgtgc 4981 ttggtgatct ggcgtgtctc aacaagctgc cttttgatga agccggcttt atccaactca 5041 gacaggccac ctcgttcagc cttagtcaga ttatcgaact tccgttgtgt gatcagtttg 5101 gcgttcagca gctgccgcca ataatttttc attttcttga caacttcttc tgaggggacg 5161 ttatcactct tccctctatt tttatcggat cttgtcaaca ctttattatc aatagaatca 5221 tctttgagaa aagactgggg cacgatatga tccacgtcgt agtcggagag ccgattgatg 5281 tccagttcct gatccacgta catgtccctg ccgttctgca ggtagtacag gtagagcttc 5341 tcattctgaa gctgggtgtt ttcaactggg tgttccttaa ggatttggga ccccagttct 5401 tttataccct cttcaatcct cttcatcctt tccctactgt tcttctgtcc cttctgggta 5461 gtttggttct ctcgggccat ctcgataacg atattctcgg gcttatgcct tcccattact 5521 ttgacgagtt catccacgac cttaacggtc tgcagtattc cctttttgat agctgggcta 5581 cctgcaagat tagcgatgtg ctcgtgaaga ctgtccccct ggccagaaac ttgtgctttc 5641 tggatgtcct ccttaaaggt gagagagtca tcatggatca actgcatgaa gttccggttg 5701 gcaaatccat cggacttaag aaaatccagg attgtctttc cactctgctt gtctcggatc 5761 ccattgatca gttttcttga cagccgcccc catcctgtat atcggcgcct cttgagctgt 5821 ttcatgactt tgtcgtcgaa gagatgagcg taagttttca agcgttcttc aatcatctcc 5881 ctatcttcaa acaacgtaag ggtgaggaca atgtcctcaa gaatgtcctc gttctcctca 5941 ttgtccagga agtccttgtc tttaatgatt ttcaggagat cgtgatacgt tcccagggat 6001 gcgttgaagc gatcctccac tccgctgatt tcaacagagt cgaaacattc aatctttttg 6061 aaatagtctt ctttgagctg tttcacggta actttccggt tcgtcttgaa gaggaggtcc 6121 acgatagctt tcttctgctc tccagacagg aatgctggct ttctcatccc ttctgtgacg 6181 tatttgacct tggtgagctc gttataaact gtgaagtact cgtacagcag agagtgttta 6241 ggaagcacct tttcgttagg cagattttta tcaaagttag tcatcctttc gatgaaggac 6301 tgggcagagg cccccttatc cacgacttcc tcgaagttcc agggagtgat ggtctcttct 6361 gatttgcgag tcatccacgc gaatctggaa tttccccggg cgagggggcc tacatagtag 6421 ggtatccgaa atgtgaggat tttctcaatc ttttccctgt tatctttcaa aaaggggtag 6481 aaatcctctt gccgcctgag gatagcgtgc agttcgccca ggtgaatctg gtgggggatg 6541 cttccattgt cgaaagtgcg ctgtttgcgc aacagatctt ctctgttaag ctttaccagc 6601 agctcctcgg tgccgtccat tttttccaag atgggcttaa taaatttgta aaattcctcc 6661 tggcttgctc cgccgtcaat gtatccggcg tagccatttt tagactgatc gaagaaaatt 6721 tccttgtact tctcaggcag ttgctgtctg acaagggcct tcagcaaagt caagtcttgg 6781 tggtgctcat catagcgctt gatcatacta gcgctcagcg gagctttggt gatctccgtg 6841 ttcactcgca gaatatcact cagcagaatg gcgtctgaca ggttctttgc cgccaaaaaa 6901 aggtctgcgt actggtcgcc gatctgggcc agcagattgt cgagatcatc atcgtaggtg 6961 tctttgctca gttgaagctt ggcatcttcg gccaggtcga agttagattt aaagttgggg 7021 gtcagcccga gtgacagggc gataagatta ccaaacaggc cgttcttctt ctccccaggg 7081 agctgtgcga tgaggttttc gagccgccgg gatttggaca gcctagcgct caggattgct 7141 ttggcgtcaa ctccggatgc gttgatcggg ttctcttcga aaagctgatt gtaagtctga 7201 accagttgga taaagagttt gtcgacatcg ctgttgtctg ggttcaggtc cccctcgatg 7261 aggaagtgtc cccgaaattt gatcatatgc gccagcgcga gatagatcaa ccgcaagtca 7321 gccttatcag tactgtctac aagcttcttc ctcagatgat atatggttgg gtacttttca 7381 tggtacgcca cctcgtccac gatattgcca aagattgggt ggcgctcgtg ctttttatcc 7441 tcctccacca aaaaggactc ctccagccta tggaagaaag agtcatccac cttagccatc 7501 tcattactaa agatctcctg caggtagcag atccgattct ttctgcgggt atatctgcgc 7561 cgtgctgttc ttttgagccg cgtggcttcg gccgtctccc cggagtcgaa caggagggcg 7621 ccaatgaggt tcttctttat gctgtggcga tcggtattgc ccagaacttt gaattttttg 7681 ctcggcacct tgtactcgtc cgtaatgacg gcccagccga cgctgtttgt gccgatatcg 7741 agcccaatgg agtacttctt gtccaccttc cgctttttct tgggcatgct cgagggttgc 7801 gtgtgtatct gtgtgcagtg gatattgtta ccgagtttgg tgagcgtgag tccgttagtg 7861 ccctggtggt ggtggattag gagagtgggt gactcggtgt ccatggcttt cttcgctcat 7921 tataggaggg gaaaggaatg agggagggtg gggagaccgc gtctgttgtt gaccaccgat 7981 ttacttcttg cctcccttcc cctccctccc ctcaatccgt acgacacaaa tagtagccga 8041 gtgtctgctg cagagcgcat gattagtgtg gtagacaacg agggagggaa ggatgtacag 8101 ggcatggcac ggagaagcga tggtggccag gaagaggaga ggtcgcgaga acaggatgtg 8161 ttgcgaatgg ataaaaacag aaagcgatgg ctctgggctt cgaaagcagg ggacattagg 8221 acgtgtagac catctcgacg gatccctctg tatctctgtt gtgcgtgaat gttttctgtg 8281 cacgtgtagt gtgtgagagt agaacccggg aactcgaaca gagaaaagca tgggtggctg 8341 tggtgtggag gcttcgttcc caccacatgc ccttctcctt cgcctcgcct ctccctgcct 8401 tcttccacgc acccttgcgc ccctcgttct caatacctgg ctcacttcca ccattcaaac 8461 aaccatcacg atacaggcat ttatctatcg ttgaagactt cttcctccgg tagatcttag 8521 ccaaggtaag aagaggggca tgcagcaagg agaaagaaat gatgcatgat gaggaataga 8581 aggggaggag ggagggatat gatgggaagc gaaagcgcat attctggtgg tctgctgcct 8641 gatggggacg cgtctagctg tgacactgag gacggtggct gctggtggct gcgggcgctg 8701 ccttggtgat caatgggagt aaagggaggg aaggaggtag cgtgaacgga tgacgcggag 8761 aagtttaggg gtctctttac gtatcgcccc tgccgcccgc ctctctgcga taaatgtgcc 8821 tgttaccctg cagcctctat tcttcactgt gttcctgttt tccaacagcc tctattcttc 8881 cctgtctttt gttgcagtgg cgtcatcctc tctttgcccc agtcgtcgtt ctctcgactc 8941 actcactccc cccctccttc cctccctcca tccacagaat cgatgaagag caggccttgg 9001 ctcttcgttt tagagctaga aatagcaagt taaaataagg ctagtccgtt atcaacttga 9061 aaaagtggca ccgagtcggt gcttttggcc ggcatggtcc cagcctcctc gctggcgccg 9121 gctgggcaac atgcttcggc atggcgaatg ggactcctgg gtaccatggg aaagaaagga 9181 tgagaaagga gaaaggacat ctagataacc ggcatatcac ggtggtgtat tagtgtagaa 9241 tagtgaagag aagacttggg aaaatgtgta ggaaaggttg tttctgtgta tgtgggttgg 9301 gatgggtggc tgtttgagaa ggaacagcgg gcagggcgat gtagtgctga acgggcacgg 9361 aaccactgag actgaaggaa gtagggagag agaggggcag gggacgtgca ctttaatctt 9421 tgcctcggta gagtataccc atgcaagagt atgtggccac ctgtggtggc ttttggccag 9481 gtctggtgca gtgccaatca tctcccatca ataatacaac ttcagaacaa cggcgcattg 9541 atggggagag agaaagtaaa tttaagtaag gggtacgtag tagaggattc aactgaaata 9601 ttttcgagga gcggttggga agttgaccga ttgaaaggag aagggagggg agcaggtgtg 9661 atagtcatgt gtaaagtaat tcttttttgc cgtcgtcaca caatccacat caatgataaa 9721 atatgtttaa ggatcaatca cacggagtcg gtcataaggc aaccgcaaac gcaatgcaaa 9781 ctagcaagca agcaggacca caacaacaac cattaccatc acaggcgaca gcagcagcag 9841 ctgcagcagc agcaaggcaa gcaaaggcca tctgcgcgtg gacttttcca ggcaccggct 9901 ttaagcgtaa ttttcaatga ttgttgtccg tgtatcctct caccctttca ccgcttgcgc 9961 gcacgtgagc agcacaactg gtaatgcccg aggacaatag acgaggaaaa agaagaagaa 10021 aaatgaagag ggagtgatga agaagaagag aagaataaaa gaagacagaa aagaagataa 10081 aaaagtcaaa agacgacaaa gacggaaaaa aaagaaacgc gcaattttaa caccagcaac 10141 gacgacgaaa agggcgtcag ctttgc // LOCUS Exported 10397 bp ds-DNA circular SYN 15-JAN-2017 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS pNOC-ARS-CRISPR-35S SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 10397) AUTHORS . TITLE Direct Submission JOURNAL Exported Friday, Jul 14, 2017 from SnapGene Viewer 3.3.4 http://www.snapgene.com FEATURES Location/Qualifiers source 1..10397 /organism="synthetic DNA construct" /mol_type="other DNA" misc_feature 17..520 /label=CEN/ARS /note="S. cerevisiae CEN6 centromere fused to an autonomously replicating sequence" promoter 557..661 /gene="bla" /label=AmpR promoter CDS 662..1522 /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" rep_origin 1693..2281 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" terminator 2349..2579 /label=35S terminator terminator 2648..3247 /label=LDSP terminator CDS complement(3279..3299) /codon_start=1 /product="nuclear localization signal of SV40 large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS complement(3315..3341) /codon_start=1 /product="HA (human influenza hemagglutinin) epitope tag" /label=HA /translation="YPYDVPDYA" CDS complement(3342..3857) /codon_start=1 /label=Nlux /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW RLCERILAG" CDS complement(3882..7994) /codon_start=1 /label=Cas9 /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD ATLIHQSITGLYETRIDLSQLGGDSRAD" CDS complement(7995..8015) /codon_start=1 /product="nuclear localization signal of SV40 large T antigen" /label=SV40 NLS /translation="PKKKRKV" promoter 8026..9209 /label=Ribi misc_feature 9238..9317 /label=sgRNA misc_feature 9318..9385 /label=HDV terminator 9397..10370 /label=CS terminator ORIGIN 1 accggtttct tagacggatc gcttgcctgt aacttacacg cgcctcgtat cttttaatga 61 tggaataatt tgggaattta ctctgtgttt atttattttt atgttttgta tttggatttt 121 agaaagtaaa taaagaaggt agaagagtta cggaatgaag aaaaaaaaat aaacaaaggt 181 ttaaaaaatt tcaacaaaaa gcgtacttta catatatatt tattagacaa gaaaagcaga 241 ttaaatagat atacattcga ttaacgataa gtaaaatgta aaatcacagg attttcgtgt 301 gtggtcttct acacagacaa gatgaaacaa ttcggcatta atacctgaga gcaggaagta 361 caagataaaa ggtagtattt gttggcgatc cccctagagt cttttacatc ttcggaaaac 421 aaaaactatt ttttctttaa tttctttttt tactttctat ttttaattta tatatttata 481 ttaaaaaatt taaattataa ttatttttat agcacgtgat gaaaaggacc caggtggcac 541 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat 601 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag 661 tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc 721 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc 781 acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc 841 cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc 901 ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt 961 ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt 1021 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat 1081 cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct 1141 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat 1201 gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc 1261 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg 1321 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc 1381 tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta 1441 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc 1501 ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga 1561 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat 1621 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat 1681 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa 1741 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa 1801 ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt 1861 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt 1921 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata 1981 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt 2041 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac 2101 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga 2161 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg 2221 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa 2281 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat 2341 gtttaaacag tagatgccga ccggagtccg caaaaatcac cagtctctct ctacaaatct 2401 atctctctct atttttctcc agaataatgt gtgagtagtt cccagataag ggaattaggg 2461 ttcttatagg gtttcgctca tgtgttgagc atataagaaa cccttagtat gtatttgtat 2521 ttgtaaaata cttctatcaa taaaatttct aattcctaaa accaaaatcc agtgacctga 2581 ctagtgcggc cgccatggcc tctagtggat cagcttgcat gcctgcagga gacgatatca 2641 cctcttctgt ttccacgata aaaatagact gctcatttct tcgtcgtctt catcgtctgc 2701 tttttctgct tcgcctctgt ctggggtctg aaaccactac acacacacac aacactcgta 2761 ctcccacttt cacaaaagcg taagctcacc ggcttttctt acacgtacat tttagtggat 2821 cccatcacgc cactaccacg cccgcggggg atggaacgga ggggagagag agaaggggga 2881 agcatggatg aatgagacat tgagggaaag gaggggaggg agcagtccat cagggcgcta 2941 cctctcttgt cccccaaacc ctgttgagcc gttcaacatg tttcatgttt cctccctccc 3001 cccttccctc cctggccttt ccgcggagcc attcaagtga cgtctggacc gcaccgtaac 3061 aaaatcgttt ctatgggggg tttgtttgac aaccacgtct tcagcgtttt taaaaaaaaa 3121 agcgggcaag ccctctcacc ctcactcatg cccatcctcc tcctctcctg cggaacattc 3181 ttacaaaagg cgtaactcga cgacaactca aagaacgaca aacatcaatc ccaaaaaaaa 3241 aatctctact cgtctctctt ggatctttcc aattgtcaga ccttcctctt ctttttcggg 3301 ctagccacca tggacgcgta gtcgggcacg tcgtaggggt atccggccag gatgcgctcg 3361 cacaggcgcc agccggttac cccgttgatg gtcacgcgga acaacagcga gccatccggg 3421 ttgatcagcc gctcgtcaat gatcttattg ccgttccaca gggtgccggt caccgtgatc 3481 ttcttgccgt cgaacacggc aatgccctcg taaggacggc cgaagtagtc aatcatgttc 3541 ggcgtcacac cgtcgatgac cagcgtcccg tagtgcagta tcaccttgaa gtggtgatcg 3601 tccacggggt acacaacctt gaagatcttc tcgatctggc ccatttggtc cccgctcagg 3661 ccctcgtagg gaatgatgac gtggatgtcg atcttcaggc cgttctcgcc gctcaggacg 3721 atgcgctgga ttggggtcac ggagacgccc aggttctgga aaagcgagga cacaccgccc 3781 tgctccagca cctggtcgag gttatagcca gctgtctgcc gccagtcgcc cacgaagtcc 3841 tcgagagtaa acaccatgtt aacggacccg ctgccggacc cgtcagccct gctgtctcca 3901 ccgagctgag agaggtcgat tcttgtttca tagagccccg taattgactg atgaatcagt 3961 gtggcgtcca ggacctcctt tgtagaggtg taccgctttc tgtctatggt ggtgtcgaag 4021 tacttgaagg ctgcaggcgc gcccaagttg gtcagagtaa acaagtggat aatgttttct 4081 gcctgctccc tgatgggctt atccctgtgc ttattgtaag cagaaagcac cttatcgagg 4141 ttagcgtcgg cgaggatcac tcttttggag aattcgctta tttgctcgat gatctcatca 4201 aggtagtgtt tgtgttgttc cacgaacagc tgcttctgct cattatcttc gggagaccct 4261 ttgagctttt catagtggct ggccagatac aagaaattaa cgtatttaga gggcagtgcc 4321 agctcgttac ctttctgcag ctcgcccgca ctagcgagca ttcgtttccg gccgttttca 4381 agctcaaaga gagagtactt gggaagctta atgatgaggt cttttttgac ctctttatat 4441 cctttcgcct cgagaaagtc gatggggttt ttttcgaagc ttgatcgctc catgattgtg 4501 atgcccagca gttccttgac gcttttgagt tttttagact tccctttctc cactttggcc 4561 acaaccagta cactgtaagc gactgtagga gaatcgaatc cgccgtattt cttggggtcc 4621 caatcttttt tgcgtgcgat cagcttgtcg ctgttccttt tcgggaggat actttccttg 4681 gagaagcctc cggtctgtac ttcggtcttt ttaacgatgt tcacctgcgg catggacagg 4741 accttccgga ctgtcgcgaa atccctaccc ttgtcccaca cgatttctcc tgtttctccg 4801 tttgtttcga taagtggtcg cttccgaatc tctccattgg ccagtgtaat ctcggtcttg 4861 aaaaaattca taatattgct gtaaaagaag tacttagcgg tggccttgcc tatttcctgc 4921 tcagactttg cgatcatttt cctaacatcg tacactttat agtctccgta aacaaattca 4981 gattcaagct tgggatattt tttgataagt gcagtgccta ccactgcatt caggtaggca 5041 tcatgcgcat ggtggtaatt gttgatctct ctcaccttat aaaactgaaa gtcctttctg 5101 aaatctgaga ccagcttaga cttcagagta ataactttca cctctcgaat cagtttgtca 5161 ttttcatcgt acttggtgtt catgcgtgaa tcgagaattt gggccacgtg cttggtgatc 5221 tggcgtgtct caacaagctg ccttttgatg aagccggctt tatccaactc agacaggcca 5281 cctcgttcag ccttagtcag attatcgaac ttccgttgtg tgatcagttt ggcgttcagc 5341 agctgccgcc aataattttt cattttcttg acaacttctt ctgaggggac gttatcactc 5401 ttccctctat ttttatcgga tcttgtcaac actttattat caatagaatc atctttgaga 5461 aaagactggg gcacgatatg atccacgtcg tagtcggaga gccgattgat gtccagttcc 5521 tgatccacgt acatgtccct gccgttctgc aggtagtaca ggtagagctt ctcattctga 5581 agctgggtgt tttcaactgg gtgttcctta aggatttggg accccagttc ttttataccc 5641 tcttcaatcc tcttcatcct ttccctactg ttcttctgtc ccttctgggt agtttggttc 5701 tctcgggcca tctcgataac gatattctcg ggcttatgcc ttcccattac tttgacgagt 5761 tcatccacga ccttaacggt ctgcagtatt ccctttttga tagctgggct acctgcaaga 5821 ttagcgatgt gctcgtgaag actgtccccc tggccagaaa cttgtgcttt ctggatgtcc 5881 tccttaaagg tgagagagtc atcatggatc aactgcatga agttccggtt ggcaaatcca 5941 tcggacttaa gaaaatccag gattgtcttt ccactctgct tgtctcggat cccattgatc 6001 agttttcttg acagccgccc ccatcctgta tatcggcgcc tcttgagctg tttcatgact 6061 ttgtcgtcga agagatgagc gtaagttttc aagcgttctt caatcatctc cctatcttca 6121 aacaacgtaa gggtgaggac aatgtcctca agaatgtcct cgttctcctc attgtccagg 6181 aagtccttgt ctttaatgat tttcaggaga tcgtgatacg ttcccaggga tgcgttgaag 6241 cgatcctcca ctccgctgat ttcaacagag tcgaaacatt caatcttttt gaaatagtct 6301 tctttgagct gtttcacggt aactttccgg ttcgtcttga agaggaggtc cacgatagct 6361 ttcttctgct ctccagacag gaatgctggc tttctcatcc cttctgtgac gtatttgacc 6421 ttggtgagct cgttataaac tgtgaagtac tcgtacagca gagagtgttt aggaagcacc 6481 ttttcgttag gcagattttt atcaaagtta gtcatccttt cgatgaagga ctgggcagag 6541 gcccccttat ccacgacttc ctcgaagttc cagggagtga tggtctcttc tgatttgcga 6601 gtcatccacg cgaatctgga atttccccgg gcgagggggc ctacatagta gggtatccga 6661 aatgtgagga ttttctcaat cttttccctg ttatctttca aaaaggggta gaaatcctct 6721 tgccgcctga ggatagcgtg cagttcgccc aggtgaatct ggtgggggat gcttccattg 6781 tcgaaagtgc gctgtttgcg caacagatct tctctgttaa gctttaccag cagctcctcg 6841 gtgccgtcca ttttttccaa gatgggctta ataaatttgt aaaattcctc ctggcttgct 6901 ccgccgtcaa tgtatccggc gtagccattt ttagactgat cgaagaaaat ttccttgtac 6961 ttctcaggca gttgctgtct gacaagggcc ttcagcaaag tcaagtcttg gtggtgctca 7021 tcatagcgct tgatcatact agcgctcagc ggagctttgg tgatctccgt gttcactcgc 7081 agaatatcac tcagcagaat ggcgtctgac aggttctttg ccgccaaaaa aaggtctgcg 7141 tactggtcgc cgatctgggc cagcagattg tcgagatcat catcgtaggt gtctttgctc 7201 agttgaagct tggcatcttc ggccaggtcg aagttagatt taaagttggg ggtcagcccg 7261 agtgacaggg cgataagatt accaaacagg ccgttcttct tctccccagg gagctgtgcg 7321 atgaggtttt cgagccgccg ggatttggac agcctagcgc tcaggattgc tttggcgtca 7381 actccggatg cgttgatcgg gttctcttcg aaaagctgat tgtaagtctg aaccagttgg 7441 ataaagagtt tgtcgacatc gctgttgtct gggttcaggt ccccctcgat gaggaagtgt 7501 ccccgaaatt tgatcatatg cgccagcgcg agatagatca accgcaagtc agccttatca 7561 gtactgtcta caagcttctt cctcagatga tatatggttg ggtacttttc atggtacgcc 7621 acctcgtcca cgatattgcc aaagattggg tggcgctcgt gctttttatc ctcctccacc 7681 aaaaaggact cctccagcct atggaagaaa gagtcatcca ccttagccat ctcattacta 7741 aagatctcct gcaggtagca gatccgattc tttctgcggg tatatctgcg ccgtgctgtt 7801 cttttgagcc gcgtggcttc ggccgtctcc ccggagtcga acaggagggc gccaatgagg 7861 ttcttcttta tgctgtggcg atcggtattg cccagaactt tgaatttttt gctcggcacc 7921 ttgtactcgt ccgtaatgac ggcccagccg acgctgtttg tgccgatatc gagcccaatg 7981 gagtacttct tgtccacctt ccgctttttc ttgggcatgc tcgagggttg cgtgtgtatc 8041 tgtgtgcagt ggatattgtt accgagtttg gtgagcgtga gtccgttagt gccctggtgg 8101 tggtggatta ggagagtggg tgactcggtg tccatggctt tcttcgctca ttataggagg 8161 ggaaaggaat gagggagggt ggggagaccg cgtctgttgt tgaccaccga tttacttctt 8221 gcctcccttc ccctccctcc cctcaatccg tacgacacaa atagtagccg agtgtctgct 8281 gcagagcgca tgattagtgt ggtagacaac gagggaggga aggatgtaca gggcatggca 8341 cggagaagcg atggtggcca ggaagaggag aggtcgcgag aacaggatgt gttgcgaatg 8401 gataaaaaca gaaagcgatg gctctgggct tcgaaagcag gggacattag gacgtgtaga 8461 ccatctcgac ggatccctct gtatctctgt tgtgcgtgaa tgttttctgt gcacgtgtag 8521 tgtgtgagag tagaacccgg gaactcgaac agagaaaagc atgggtggct gtggtgtgga 8581 ggcttcgttc ccaccacatg cccttctcct tcgcctcgcc tctccctgcc ttcttccacg 8641 cacccttgcg cccctcgttc tcaatacctg gctcacttcc accattcaaa caaccatcac 8701 gatacaggca tttatctatc gttgaagact tcttcctccg gtagatctta gccaaggtaa 8761 gaagaggggc atgcagcaag gagaaagaaa tgatgcatga tgaggaatag aaggggagga 8821 gggagggata tgatgggaag cgaaagcgca tattctggtg gtctgctgcc tgatggggac 8881 gcgtctagct gtgacactga ggacggtggc tgctggtggc tgcgggcgct gccttggtga 8941 tcaatgggag taaagggagg gaaggaggta gcgtgaacgg atgacgcgga gaagtttagg 9001 ggtctcttta cgtatcgccc ctgccgcccg cctctctgcg ataaatgtgc ctgttaccct 9061 gcagcctcta ttcttcactg tgttcctgtt ttccaacagc ctctattctt ccctgtcttt 9121 tgttgcagtg gcgtcatcct ctctttgccc cagtcgtcgt tctctcgact cactcactcc 9181 ccccctcctt ccctccctcc atccacagaa tcgatgaaga gcaggccttg gctcttcgtt 9241 ttagagctag aaatagcaag ttaaaataag gctagtccgt tatcaacttg aaaaagtggc 9301 accgagtcgg tgcttttggc cggcatggtc ccagcctcct cgctggcgcc ggctgggcaa 9361 catgcttcgg catggcgaat gggactcctg ggtaccatgg gaaagaaagg atgagaaagg 9421 agaaaggaca tctagataac cggcatatca cggtggtgta ttagtgtaga atagtgaaga 9481 gaagacttgg gaaaatgtgt aggaaaggtt gtttctgtgt atgtgggttg ggatgggtgg 9541 ctgtttgaga aggaacagcg ggcagggcga tgtagtgctg aacgggcacg gaaccactga 9601 gactgaagga agtagggaga gagaggggca ggggacgtgc actttaatct ttgcctcggt 9661 agagtatacc catgcaagag tatgtggcca cctgtggtgg cttttggcca ggtctggtgc 9721 agtgccaatc atctcccatc aataatacaa cttcagaaca acggcgcatt gatggggaga 9781 gagaaagtaa atttaagtaa ggggtacgta gtagaggatt caactgaaat attttcgagg 9841 agcggttggg aagttgaccg attgaaagga gaagggaggg gagcaggtgt gatagtcatg 9901 tgtaaagtaa ttcttttttg ccgtcgtcac acaatccaca tcaatgataa aatatgttta 9961 aggatcaatc acacggagtc ggtcataagg caaccgcaaa cgcaatgcaa actagcaagc 10021 aagcaggacc acaacaacaa ccattaccat cacaggcgac agcagcagca gctgcagcag 10081 cagcaaggca agcaaaggcc atctgcgcgt ggacttttcc aggcaccggc tttaagcgta 10141 attttcaatg attgttgtcc gtgtatcctc tcaccctttc accgcttgcg cgcacgtgag 10201 cagcacaact ggtaatgccc gaggacaata gacgaggaaa aagaagaaga aaaatgaaga 10261 gggagtgatg aagaagaaga gaagaataaa agaagacaga aaagaagata aaaaagtcaa 10321 aagacgacaa agacggaaaa aaaagaaacg cgcaatttta acaccagcaa cgacgacgaa 10381 aagggcgtca gctttgc // LOCUS Exported 12209 bp ds-DNA circular SYN 31-AUG-2017 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS pNOC-ARS-CRISPR-v2.dna SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 12209) AUTHORS . TITLE Direct Submission JOURNAL Exported Aug 31, 2017 from SnapGene Viewer 4.0.0 http://www.snapgene.com FEATURES Location/Qualifiers source 1..12209) /organism="synthetic DNA construct" /mol_type="other DNA" misc_feature 17..520) /label=CEN/ARS /note="S. cerevisiae CEN6 centromere fused to an autonomously replicating sequence" promoter 557..661) /gene="bla" /label=AmpR promoter CDS 662..1522) /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" rep_origin 1693..2281) /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" terminator 2349..2579) /label=35S terminator CDS complement(2586..3611)) /codon_start=1 /gene="aph(4)-Ia" /product="aminoglycoside phosphotransferase from E. coli" /label=HygR /note="confers resistance to hygromycin" /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK E" promoter 3636..4397) /label=LDSP promoter terminator 4415..5081) /label=LDSP terminator CDS complement(5091..5111)) /codon_start=1 /product="nuclear localization signal of SV40 large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS complement(5127..5153)) /codon_start=1 /product="HA (human influenza hemagglutinin) epitope tag" /label=HA /translation="YPYDVPDYA" CDS complement(5154..5669)) /codon_start=1 /label=Nlux /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW RLCERILAG" CDS complement(5706..9806)) /codon_start=1 /product="Cas9 (Csn1) endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system" /label=Cas9 /note="generates RNA-guided double strand breaks in DNA" /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD ATLIHQSITGLYETRIDLSQLGGD" CDS complement(9807..9827)) /codon_start=1 /product="nuclear localization signal of SV40 large T antigen" /label=SV40 NLS /translation="PKKKRKV" promoter 9838..11021) /label=Ribi misc_RNA 11050..11125) /label=g RNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" misc_feature 11130..11202) /label=HDV terminator 11209..12209) /label=CS terminator ORIGIN 1 accggtttct tagacggatc gcttgcctgt aacttacacg cgcctcgtat cttttaatga 61 tggaataatt tgggaattta ctctgtgttt atttattttt atgttttgta tttggatttt 121 agaaagtaaa taaagaaggt agaagagtta cggaatgaag aaaaaaaaat aaacaaaggt 181 ttaaaaaatt tcaacaaaaa gcgtacttta catatatatt tattagacaa gaaaagcaga 241 ttaaatagat atacattcga ttaacgataa gtaaaatgta aaatcacagg attttcgtgt 301 gtggtcttct acacagacaa gatgaaacaa ttcggcatta atacctgaga gcaggaagta 361 caagataaaa ggtagtattt gttggcgatc cccctagagt cttttacatc ttcggaaaac 421 aaaaactatt ttttctttaa tttctttttt tactttctat ttttaattta tatatttata 481 ttaaaaaatt taaattataa ttatttttat agcacgtgat gaaaaggacc caggtggcac 541 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat 601 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag 661 tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc 721 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc 781 acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc 841 cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc 901 ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt 961 ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt 1021 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat 1081 cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct 1141 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat 1201 gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc 1261 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg 1321 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc 1381 tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta 1441 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc 1501 ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga 1561 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat 1621 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat 1681 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa 1741 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa 1801 ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt 1861 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt 1921 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata 1981 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt 2041 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac 2101 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga 2161 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg 2221 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa 2281 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat 2341 gtttaaacag tagatgccga ccggagtccg caaaaatcac cagtctctct ctacaaatct 2401 atctctctct atttttctcc agaataatgt gtgagtagtt cccagataag ggaattaggg 2461 ttcttatagg gtttcgctca tgtgttgagc atataagaaa cccttagtat gtatttgtat 2521 ttgtaaaata cttctatcaa taaaatttct aattcctaaa accaaaatcc agtgacctga 2581 ctagcctatt cctttgccct cggacgagtg ctggggcgtc ggtttccact atcggcgagt 2641 acttctacac agccatcggt ccagacggcc gcgcttctgc gggcgatttg tgtacgcccg 2701 acagtcccgg ctccggatcg gacgattgcg tcgcatcgac cctgcgccca agctgcatca 2761 tcgaaattgc cgtcaaccaa gctctgatag agttggtcaa gaccaatgcg gagcatatac 2821 gcccggagcc gcggcgatcc tgcaagctcc ggatgcctcc gctcgaagta gcgcgtctgc 2881 tgctccatac aagccaacca cggcctccag aagaagatgt tggcgacctc gtattgggaa 2941 tccccgaaca tcgcctcgct ccagtcaatg accgctgtta tgcggccatt gtccgtcagg 3001 acattgttgg agccgaaatc cgcgtgcacg aggtgccgga cttcggggca gtcctcggcc 3061 caaagcatca gctcatcgag agcctgcgcg acggacgcac tgacggtgtc gtccatcaca 3121 gtttgccagt gatacacatg gggatcagca atcgcgcata tgaaatcacg ccatgtagtg 3181 tattgaccga ttccttgcgg tccgaatggg ccgaacccgc tcgtctggct aagatcggcc 3241 gcagcgatcg catccatggc ctccgcgacc ggctgcagaa cagcgggcag ttcggtttca 3301 ggcaggtctt gcaacgtgac accctgtgca cggcgggaga tgcaataggt caggctctcg 3361 ctgaattccc caatgtcaag cacttccgga atcgggagcg cggccgatgc aaagtgccga 3421 taaacataac gatctttgta gaaaccatcg gcgcagctat ttacccgcag gacatatcca 3481 cgccctccta catcgaagct gaaagcacga gattcttcgc cctccgagag ctgcatcagg 3541 tcggagacgc tgtcgaactt ttcgatcaga aacttctcga cagacgtcgc ggtgagttca 3601 ggctttttca tatcttattg ccccccgggg ccctcctgtg ttgatgcggg ctgagattgg 3661 tggtggtcta tcacgaatat gtgtgagggg taagtgcggt gttttgcgtg agattttaga 3721 atattgcccc gccccggggc aggccggcgt ggcggaacaa ccaggcacac gagcgcgaat 3781 ggtgataccg acggagtcaa aactttgtga caagtagctg caccatgggc agtggtgagc 3841 tttcagacgt ggtatcactg tccactagtt cacacacaga atgcgtgtcc aaaaggtcta 3901 gagccgtctc gcttgcgtct ctccgtcgaa gaacagtgaa gaggctcgtc acgtcgacca 3961 gacgacggga ggctggtcac catcgcagat gtctcccaca aagcagcacg gcaactccta 4021 ctccttcaca caatggaaga aaaggtggtc tgatggttct cagtggaaaa gaacgatatc 4081 aggctgaaaa aaatgatctg caggctccag attcctgaat cacgtcgact gtgacgaagc 4141 aaaccgcgtc gaacaacatc ggtcatgcca acgggtctcg tctctcgagc ccttttggcg 4201 gcgactaaga agtatgaagc ttcaggccgc aacgcgcgac acagcgtttt gtgtggtggg 4261 cctcggcatt gctctttgca tggcccagcg tgattagtgc gtggatttta agcccgagac 4321 cgaaggattg cgacatgtgc ctggctgtat aactcacgct tgctgctacg ctcgcctcct 4381 cctccatcca ctccatcgcg gccgccatgg cctctagtgg atcagcttgc atgcctgcag 4441 gagacgatat cacctcttct gtttccacga taaaaataga ctgctcattt cttcgtcgtc 4501 ttcatcgtct gctttttctg cttcgcctct gtctggggtc tgaaaccact acacacacac 4561 acaacactcg tactcccact ttcacaaaag cgtaagctca ccggcttttc ttacacgtac 4621 attttagtgg atcccatcac gccactacca cgcccgcggg ggatggaacg gaggggagag 4681 agagaagggg gaagcatgga tgaatgagac attgagggaa aggaggggag ggagcagtcc 4741 atcagggcgc tacctctctt gtcccccaaa ccctgttgag ccgttcaaca tgtttcatgt 4801 ttcctccctc cccccttccc tccctggcct ttccgcggag ccattcaagt gacgtctgga 4861 ccgcaccgta acaaaatcgt ttctatgggg ggtttgtttg acaaccacgt cttcagcgtt 4921 tttaaaaaaa aaagcgggca agccctctca ccctcactca tgcccatcct cctcctctcc 4981 tgcggaacat tcttacaaaa ggcgtaactc gacgacaact caaagaacga caaacatcaa 5041 tcccaaaaaa aaaatctcta ctcgtctctc ttggatcttt ccaattgtca gaccttcctc 5101 ttctttttcg ggctagccac catggacgcg tagtcgggca cgtcgtaggg gtatccggcc 5161 aggatgcgct cgcacaggcg ccagccggtt accccgttga tggtcacgcg gaacaacagc 5221 gagccatccg ggttgatcag ccgctcgtca atgatcttat tgccgttcca cagggtgccg 5281 gtcaccgtga tcttcttgcc gtcgaacacg gcaatgccct cgtaaggacg gccgaagtag 5341 tcaatcatgt tcggcgtcac accgtcgatg accagcgtcc cgtagtgcag tatcaccttg 5401 aagtggtgat cgtccacggg gtacacaacc ttgaagatct tctcgatctg gcccatttgg 5461 tccccgctca ggccctcgta gggaatgatg acgtggatgt cgatcttcag gccgttctcg 5521 ccgctcagga cgatgcgctg gattggggtc acggagacgc ccaggttctg gaaaagcgag 5581 gacacaccgc cctgctccag cacctggtcg aggttatagc cagctgtctg ccgccagtcg 5641 cccacgaagt cctcgagagt aaacaccatg ttaacggacc cgctgccgga cccgtcagcc 5701 ctgctgtctc caccgagctg agagaggtcg attcttgttt catagagccc cgtaattgac 5761 tgatgaatca gtgtggcgtc caggacctcc tttgtagagg tgtaccgctt tctgtctatg 5821 gtggtgtcga agtacttgaa ggctgcaggc gcgcccaagt tggtcagagt aaacaagtgg 5881 ataatgtttt ctgcctgctc cctgatgggc ttatccctgt gcttattgta agcagaaagc 5941 accttatcga ggttagcgtc ggcgaggatc actcttttgg agaattcgct tatttgctcg 6001 atgatctcat caaggtagtg tttgtgttgt tccacgaaca gctgcttctg ctcattatct 6061 tcgggagacc ctttgagctt ttcatagtgg ctggccagat acaagaaatt aacgtattta 6121 gagggcagtg ccagctcgtt acctttctgc agctcgcccg cactagcgag cattcgtttc 6181 cggccgtttt caagctcaaa gagagagtac ttgggaagct taatgatgag gtcttttttg 6241 acctctttat atcctttcgc ctcgagaaag tcgatggggt ttttttcgaa gcttgatcgc 6301 tccatgattg tgatgcccag cagttccttg acgcttttga gttttttaga cttccctttc 6361 tccactttgg ccacaaccag tacactgtaa gcgactgtag gagaatcgaa tccgccgtat 6421 ttcttggggt cccaatcttt tttgcgtgcg atcagcttgt cgctgttcct tttcgggagg 6481 atactttcct tggagaagcc tccggtctgt acttcggtct ttttaacgat gttcacctgc 6541 ggcatggaca ggaccttccg gactgtcgcg aaatccctac ccttgtccca cacgatttct 6601 cctgtttctc cgtttgtttc gataagtggt cgcttccgaa tctctccatt ggccagtgta 6661 atctcggtct tgaaaaaatt cataatattg ctgtaaaaga agtacttagc ggtggccttg 6721 cctatttcct gctcagactt tgcgatcatt ttcctaacat cgtacacttt atagtctccg 6781 taaacaaatt cagattcaag cttgggatat tttttgataa gtgcagtgcc taccactgca 6841 ttcaggtagg catcatgcgc atggtggtaa ttgttgatct ctctcacctt ataaaactga 6901 aagtcctttc tgaaatctga gaccagctta gacttcagag taataacttt cacctctcga 6961 atcagtttgt cattttcatc gtacttggtg ttcatgcgtg aatcgagaat ttgggccacg 7021 tgcttggtga tctggcgtgt ctcaacaagc tgccttttga tgaagccggc tttatccaac 7081 tcagacaggc cacctcgttc agccttagtc agattatcga acttccgttg tgtgatcagt 7141 ttggcgttca gcagctgccg ccaataattt ttcattttct tgacaacttc ttctgagggg 7201 acgttatcac tcttccctct atttttatcg gatcttgtca acactttatt atcaatagaa 7261 tcatctttga gaaaagactg gggcacgata tgatccacgt cgtagtcgga gagccgattg 7321 atgtccagtt cctgatccac gtacatgtcc ctgccgttct gcaggtagta caggtagagc 7381 ttctcattct gaagctgggt gttttcaact gggtgttcct taaggatttg ggaccccagt 7441 tcttttatac cctcttcaat cctcttcatc ctttccctac tgttcttctg tcccttctgg 7501 gtagtttggt tctctcgggc catctcgata acgatattct cgggcttatg ccttcccatt 7561 actttgacga gttcatccac gaccttaacg gtctgcagta ttcccttttt gatagctggg 7621 ctacctgcaa gattagcgat gtgctcgtga agactgtccc cctggccaga aacttgtgct 7681 ttctggatgt cctccttaaa ggtgagagag tcatcatgga tcaactgcat gaagttccgg 7741 ttggcaaatc catcggactt aagaaaatcc aggattgtct ttccactctg cttgtctcgg 7801 atcccattga tcagttttct tgacagccgc ccccatcctg tatatcggcg cctcttgagc 7861 tgtttcatga ctttgtcgtc gaagagatga gcgtaagttt tcaagcgttc ttcaatcatc 7921 tccctatctt caaacaacgt aagggtgagg acaatgtcct caagaatgtc ctcgttctcc 7981 tcattgtcca ggaagtcctt gtctttaatg attttcagga gatcgtgata cgttcccagg 8041 gatgcgttga agcgatcctc cactccgctg atttcaacag agtcgaaaca ttcaatcttt 8101 ttgaaatagt cttctttgag ctgtttcacg gtaactttcc ggttcgtctt gaagaggagg 8161 tccacgatag ctttcttctg ctctccagac aggaatgctg gctttctcat cccttctgtg 8221 acgtatttga ccttggtgag ctcgttataa actgtgaagt actcgtacag cagagagtgt 8281 ttaggaagca ccttttcgtt aggcagattt ttatcaaagt tagtcatcct ttcgatgaag 8341 gactgggcag aggccccctt atccacgact tcctcgaagt tccagggagt gatggtctct 8401 tctgatttgc gagtcatcca cgcgaatctg gaatttcccc gggcgagggg gcctacatag 8461 tagggtatcc gaaatgtgag gattttctca atcttttccc tgttatcttt caaaaagggg 8521 tagaaatcct cttgccgcct gaggatagcg tgcagttcgc ccaggtgaat ctggtggggg 8581 atgcttccat tgtcgaaagt gcgctgtttg cgcaacagat cttctctgtt aagctttacc 8641 agcagctcct cggtgccgtc cattttttcc aagatgggct taataaattt gtaaaattcc 8701 tcctggcttg ctccgccgtc aatgtatccg gcgtagccat ttttagactg atcgaagaaa 8761 atttccttgt acttctcagg cagttgctgt ctgacaaggg ccttcagcaa agtcaagtct 8821 tggtggtgct catcatagcg cttgatcata ctagcgctca gcggagcttt ggtgatctcc 8881 gtgttcactc gcagaatatc actcagcaga atggcgtctg acaggttctt tgccgccaaa 8941 aaaaggtctg cgtactggtc gccgatctgg gccagcagat tgtcgagatc atcatcgtag 9001 gtgtctttgc tcagttgaag cttggcatct tcggccaggt cgaagttaga tttaaagttg 9061 ggggtcagcc cgagtgacag ggcgataaga ttaccaaaca ggccgttctt cttctcccca 9121 gggagctgtg cgatgaggtt ttcgagccgc cgggatttgg acagcctagc gctcaggatt 9181 gctttggcgt caactccgga tgcgttgatc gggttctctt cgaaaagctg attgtaagtc 9241 tgaaccagtt ggataaagag tttgtcgaca tcgctgttgt ctgggttcag gtccccctcg 9301 atgaggaagt gtccccgaaa tttgatcata tgcgccagcg cgagatagat caaccgcaag 9361 tcagccttat cagtactgtc tacaagcttc ttcctcagat gatatatggt tgggtacttt 9421 tcatggtacg ccacctcgtc cacgatattg ccaaagattg ggtggcgctc gtgcttttta 9481 tcctcctcca ccaaaaagga ctcctccagc ctatggaaga aagagtcatc caccttagcc 9541 atctcattac taaagatctc ctgcaggtag cagatccgat tctttctgcg ggtatatctg 9601 cgccgtgctg ttcttttgag ccgcgtggct tcggccgtct ccccggagtc gaacaggagg 9661 gcgccaatga ggttcttctt tatgctgtgg cgatcggtat tgcccagaac tttgaatttt 9721 ttgctcggca ccttgtactc gtccgtaatg acggcccagc cgacgctgtt tgtgccgata 9781 tcgagcccaa tggagtactt cttgtccacc ttccgctttt tcttgggcat gctcgagggt 9841 tgcgtgtgta tctgtgtgca gtggatattg ttaccgagtt tggtgagcgt gagtccgtta 9901 gtgccctggt ggtggtggat taggagagtg ggtgactcgg tgtccatggc tttcttcgct 9961 cattatagga ggggaaagga atgagggagg gtggggagac cgcgtctgtt gttgaccacc 10021 gatttacttc ttgcctccct tcccctccct cccctcaatc cgtacgacac aaatagtagc 10081 cgagtgtctg ctgcagagcg catgattagt gtggtagaca acgagggagg gaaggatgta 10141 cagggcatgg cacggagaag cgatggtggc caggaagagg agaggtcgcg agaacaggat 10201 gtgttgcgaa tggataaaaa cagaaagcga tggctctggg cttcgaaagc aggggacatt 10261 aggacgtgta gaccatctcg acggatccct ctgtatctct gttgtgcgtg aatgttttct 10321 gtgcacgtgt agtgtgtgag agtagaaccc gggaactcga acagagaaaa gcatgggtgg 10381 ctgtggtgtg gaggcttcgt tcccaccaca tgcccttctc cttcgcctcg cctctccctg 10441 ccttcttcca cgcacccttg cgcccctcgt tctcaatacc tggctcactt ccaccattca 10501 aacaaccatc acgatacagg catttatcta tcgttgaaga cttcttcctc cggtagatct 10561 tagccaaggt aagaagaggg gcatgcagca aggagaaaga aatgatgcat gatgaggaat 10621 agaaggggag gagggaggga tatgatggga agcgaaagcg catattctgg tggtctgctg 10681 cctgatgggg acgcgtctag ctgtgacact gaggacggtg gctgctggtg gctgcgggcg 10741 ctgccttggt gatcaatggg agtaaaggga gggaaggagg tagcgtgaac ggatgacgcg 10801 gagaagttta ggggtctctt tacgtatcgc ccctgccgcc cgcctctctg cgataaatgt 10861 gcctgttacc ctgcagcctc tattcttcac tgtgttcctg ttttccaaca gcctctattc 10921 ttccctgtct tttgttgcag tggcgtcatc ctctctttgc cccagtcgtc gttctctcga 10981 ctcactcact ccccccctcc ttccctccct ccatccacag aatcgatgaa gagcaggcct 11041 tggctcttcg ttttagagct agaaatagca agttaaaata aggctagtcc gttatcaact 11101 tgaaaaagtg gcaccgagtc ggtgcttttg gccggcatgg tcccagcctc ctcgctggcg 11161 ccggctgggc aacatgcttc ggcatggcga atgggactcc tgggtaccat gggaaagaaa 11221 ggatgagaaa ggagaaagga catctagata accggcatat cacggtggtg tattagtgta 11281 gaatagtgaa gagaagactt gggaaaatgt gtaggaaagg ttgtttctgt gtatgtgggt 11341 tgggatgggt ggctgtttga gaaggaacag cgggcagggc gatgtagtgc tgaacgggca 11401 cggaaccact gagactgaag gaagtaggga gagagagggg caggggacgt gcactttaat 11461 ctttgcctcg gtagagtata cccatgcaag agtatgtggc cacctgtggt ggcttttggc 11521 caggtctggt gcagtgccaa tcatctccca tcaataatac aacttcagaa caacggcgca 11581 ttgatgggga gagagaaagt aaatttaagt aaggggtacg tagtagagga ttcaactgaa 11641 atattttcga ggagcggttg ggaagttgac cgattgaaag gagaagggag gggagcaggt 11701 gtgatagtca tgtgtaaagt aattcttttt tgccgtcgtc acacaatcca catcaatgat 11761 aaaatatgtt taaggatcaa tcacacggag tcggtcataa ggcaaccgca aacgcaatgc 11821 aaactagcaa gcaagcagga ccacaacaac aaccattacc atcacaggcg acagcagcag 11881 cagctgcagc agcagcaagg caagcaaagg ccatctgcgc gtggactttt ccaggcaccg 11941 gctttaagcg taattttcaa tgattgttgt ccgtgtatcc tctcaccctt tcaccgcttg 12001 cgcgcacgtg agcagcacaa ctggtaatgc ccgaggacaa tagacgagga aaaagaagaa 12061 gaaaaatgaa gagggagtga tgaagaagaa gagaagaata aaagaagaca gaaaagaaga 12121 taaaaaagtc aaaagacgac aaagacggaa aaaaaagaaa cgcgcaattt taacaccagc 12181 aacgacgacg aaaagggcgt cagctttgc //