LOCUS HDR_plasmid_in_p 9128 bp DNA circular SYN 11-NOV-2020 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS . SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 9128) AUTHORS . TITLE Direct Submission JOURNAL Exported May 2, 2023 from SnapGene 6.2.0 https://www.snapgene.com FEATURES Location/Qualifiers source 1..9128 /dnas_title="pKHR8ruvbl1KI" /mol_type="other DNA" /organism="synthetic DNA construct" rep_origin complement(6..461) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" promoter 626..644 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" misc_feature 699..716 /label=I-SceI misc_recomb 750..2221 /label=Left homology arm 5'UTR 750..844 CDS join(845..985,1608..1694,1851..1983) /dnas_title="ruvbl1 CDS" /codon_start=1 /label=ruvbl1 CDS /translation="MKIEEVKSTTKTQRIASHSHVKGLGLDEAGNAKQSASGLVGQES AREACGIITELIRSKKMAGRAILLAGPPGTGKTALALAMAQELGNKVPFCPMVGSEVY SSEIKKTEVLMENFRRAI" protein_bind 1753..1786 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." protein_bind 2228..2261 /label=FRT (minimal) /bound_moiety="FLP recombinase from the Saccharomyces cerevisiae 2u plasmid" /note="supports FLP-mediated excision but not integration (Turan and Bode, 2011)" polyA_signal 2325..2459 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" polyA_signal 2496..2703 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" CDS complement(2727..3446) /codon_start=1 /product="Venus YFP with monomerizing A206K mutation (Nagai et al., 2002)" /label=mVenus /note="mammalian codon-optimized" /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLT LKLICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFK DDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKN GIKANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDH MVLLEFVTAAGITLGMDELYK" misc_feature 3447..4011 /label=alpha-crystallin promoter protein_bind 4012..4045 /label=FRT (minimal) /bound_moiety="FLP recombinase from the Saccharomyces cerevisiae 2u plasmid" /note="supports FLP-mediated excision but not integration (Turan and Bode, 2011)" protein_bind 4047..4080 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." misc_recomb 4087..5008 /label=right homology arm misc_feature 5039..5930 /label=cmlc2 promoter CDS 5931..6641 /codon_start=1 /product="monomeric derivative of DsRed fluorescent protein (Shaner et al., 2004)" /label=mCherry /note="mammalian codon-optimized" /translation="MVSKGEDDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYE GTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVM NFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDG ALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQ YERAEGRHSTGGMDELYK" promoter complement(6657..6674) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" polyA_signal 6679..6813 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" misc_feature 6885..6902 /label=I-SceI promoter complement(6943..6961) /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" protein_bind 7006..7022 /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(7030..7060) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 7075..7096 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." rep_origin complement(7384..7972) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" CDS complement(8143..9003) /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGY IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVE YSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRL DRWEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPL LRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIA EIGASLIKHW" promoter complement(9004..9108) /gene="bla" /label=AmpR promoter exon 777..985 /note="ruvbl1 exon1" exon 1608..1694 /note="ruvbl1 exon 2" exon 1851..1983 /note="ruvbl1 exon 3" ORIGIN 1 cacctaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag 61 ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac 121 cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga 181 ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc 241 accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg 301 gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa 361 gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac 421 caccacaccc gccgcgctta atgcgccgct acagggcgcg tcccattcgc cattcaggct 481 gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa 541 agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg 601 ttgtaaaacg acggccagtg aattgtaata cgactcacta tagggcgaat tggagctcca 661 ccgcggtggc ggccaggtcc tttagtcggg tattcgggta gggataacag ggtaatggcg 721 gccgctctag aactagtgga tcccccgggg cgggtctctt gtgtttgatt ttaacggcct 781 ctcgtgagac aggcgtgtgg ttgttttatt cacgcacagc gcgttcgatt aattattaag 841 aaacatgaag atcgaagaag taaaaagcac aaccaagacg cagcgcatcg cttctcatag 901 ccatgtgaag ggcctcggtc tggatgaggc tggtaatgct aaacagtccg cgtccgggct 961 agtgggacaa gaaagcgcca gagaggtaac actagctaaa gaccaatttc cgtgtttatt 1021 tcattaatta agaaaagcaa acattttaat ttgataacct cgaatgattt gtatgtagct 1081 ggctatatgt gaagaataaa tacaaaactt ctaattcatt tgcttgtgtc aagagctgga 1141 tacaattgat tgtaaatacg ataaattcat atactttcat aggctatata tgaagagttc 1201 agatgcaaaa acctctaagt accgtctgaa acttccatta aagctgagca tttttatctc 1261 agcattctat attgattatt gatgagttat ttctctttaa agatcattaa attaacatac 1321 tttttaatta ttctttaatt tgtaattact gaacctacta acaggagcct gataaaaatg 1381 tttattttag aagaacattt cagacggctt ttagaagttt ttgcatctga actcttcata 1441 tatatatata tatatatata tatatggaat attttaatca cactttttaa acaaaataat 1501 gttttcatgt atgtgacatt ttctggcctg tagtttagcc ctgggcttta aaaaaaaaaa 1561 ctgatcctgt tacataaaca tctagaaaat tcttgcatat ttgttaggct tgtggcatca 1621 tcactgagct gattcgttcc aaaaagatgg ctggcagagc cattttgcta gccggaccgc 1681 ctggaacagg caaggtaagg gtagactggt tttatttaca ctgcactcta tatttataat 1741 ggtcaatata atataacttc gtatagcata cattatacga agttatagtt tcagagctgt 1801 tgtttaagat agtatgtata gtctgcccat ttgatttcta tccaccacag actgctctag 1861 cactggccat ggcacaggag ttgggaaata aagtgccatt ctgtcctatg gtgggcagcg 1921 aagtttattc atcagaaatc aagaagaccg aagtgctgat ggagaacttc agaagagcca 1981 taggtgagct ctcacaccac cataacctgt caaaaaagga tagaggaaaa gtttttttat 2041 atattcgcac attcagactt tctgcttaat aatataattt cataaaagta ttctttgcaa 2101 attttaaata gtgaaatcat attagcagtc aatgactatc aaagtacagc gttgttcaat 2161 caattacata gtgctaatat tgttttgaag tcatccttgc atgctatttt agacgcaata 2221 tgaattcgaa gttcctattc tctagaaagt ataggaactt cttaaaaaac ctcccacacc 2281 tccccctgaa cctgaaacat aaaatgaatg caattgttgt tgttaacttg tttattgcag 2341 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt 2401 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctggatca 2461 tcatcgatgg tacccaccgc ggtggcggcg atctctcccc agcatgcctg ctattgtctt 2521 cccaatcctc ccccttgctg tcctgcccca ccccaccccc cagaatagaa tgacacctac 2581 tcagacaatg cgatgcaatt tcctcatttt attaggaaag gacagtggga gtggcacctt 2641 ccagggtcaa ggaaggcacg ggggaggggc aaacaacaga tggctggcaa ctagaaggca 2701 cagtcgaggc tgatcagcgg gccgctttac ttgtacagct cgtccatgcc gagagtgatc 2761 ccggcggcgg tcacgaactc cagcaggacc atgtgatcgc gcttctcgtt ggggtctttg 2821 ctcagggcgg actggtagct caggtagtgg ttgtcgggca gcagcacggg gccgtcgccg 2881 atgggggtgt tctgctggta gtggtcggcg agctgcacgc cgccgtcctc gatgttgtgg 2941 cggatcttga agttggcctt gatgccgttc ttctgcttgt cggcggtgat atagacgttg 3001 tggctgttgt agttgtactc cagcttgtgc cccaggatgt tgccgtcctc cttgaagtcg 3061 atgcccttca gctcgatgcg gttcaccagg gtgtcgccct cgaacttcac ctcggcgcgg 3121 gtcttgtagt tgccgtcgtc cttgaagaag atggtgcgct cctggacgta gccttcgggc 3181 atggcggact tgaagaagtc gtgctgcttc atgtggtcgg ggtagcgggc gaagcactgc 3241 aggccgtagc ccagggtggt cacgagggtg ggccagggca cgggcagctt gccggtggtg 3301 cagatcagct tcagggtcag cttgccgtag gtggcatcgc cctcgccctc gccggacacg 3361 ctgaacttgt ggccgtttac gtcgccgtcc agctcgacca ggatgggcac caccccggtg 3421 aacagctcct cgcccttgct caccatgttg tcagacctgg taactcctta ctgtaactta 3481 ttgcacctga tgaacaaaag atgtgaactg aacgcaagta ggaggctaat agaggctagt 3541 acagtacaaa gatccccaaa tgatgtgtgc cggccttagt ggaggcagac ccctggttat 3601 atacctgtgc tgcagaaaga ggggcttaca aatggatctc tctggaatgg cattatctca 3661 tggtgggaac agtgttgtca gcagaatcag cggggtggac tctgacctga gaatgacagt 3721 ctagcactga atggctcaga caatactctt gtggccaact atcaccggaa aataagtgcc 3781 tgccaaaaca aaccagacca gatgaagcta catggctgta gaacgtcagt atttaatgtg 3841 ttgtggccac ttggttcacc agatttaaga gctaattttt ttaaatatca cagataatca 3901 atcgcttttt ttttttcttg actgaaaagc ttagactgga aaattctttg aaaaagtttg 3961 cactacagaa aataaataca taacatcctg cactgaatgc acactattaa tgaagttcct 4021 attctctaga aagtatagga acttcaataa cttcgtatag catacattat acgaagttat 4081 gatatctcaa cttgccgtgg taaacaaata tgggccatta atgaaacaaa tgtgcaaatc 4141 tcaaacatac tttatctaaa tcaaaaaagg ctgttggccg ttatcttgcg taatgagtga 4201 ttcctaccat cattaaatca attcaatcaa aactgctgat tcaattaaaa atgaagcaag 4261 tgactgtctt taatataaaa tcgagtcttt ggctcattcg atttgtttaa aaatagatta 4321 gttctggaac aatgcatacc atcgttgctc tgaaatgtgt aaaatgtttt ggtatgaccg 4381 ttcagaactg attatgttga caattgaaaa taacacagta tttttgtttt gaaatgtaaa 4441 cttactgttt tttgagctgt tgatattttt gcttttttac ttgaagtaaa gggtttatag 4501 tcctaggctt aaccgcagtg tttagactga atatgtattt gtttttataa tgagcaacat 4561 gcattaatgt cagctagcgt gtcattaaac agcaggacta agtgaatatc attttatttt 4621 agatgatata acacacccct acaataaaga agccatgtaa aatctgtatt ttcctttatg 4681 tattcctata gatttgagca tgaccttgta ctgttcttct agttggttca caaccctcaa 4741 ctctgaaacc ctcaagtatg actctgaaac aatgtagact atgtgatcaa atgagatatg 4801 catgaatgca cacattatta ctacgctgac agagatatat ggcatgaacc attgaaaata 4861 cacttctggc tttttattac gtaatgaaat atgtggtttt tgtaaaagtg tattggccag 4921 taaaaccgaa aaactattaa aagtgtttat atcaatctag tatctaaagt tctgttttgt 4981 tttttgatcg aacaggtctg cgtatcaaat caagcttatc gataccgtcg acctcgagaa 5041 atcagttgtg ttaaataaga gacattcaaa ataaatgtaa atgagctctc caaatcagca 5101 gacttaacat tctttaaaat gattgattca atagtgataa aaatcaggca tagccagttg 5161 taactttaga taaattacag aaaatgtcaa atacagagaa ccgattcttt tttatgatac 5221 atccaagcac acatttaaca caatccaggc aaaccccgaa tttcacagtc acaagcactg 5281 tttgtacaag agctttgcct aaggacacac agtctctata agtccaggtc gttggtttca 5341 ctcttatttt aaacatgtga catttttcct gccatcctgt cttaggctgc tgtttgcttc 5401 attccatgtc acattaaatt cctcagtagc accttttaca cacacagcca atcttttcca 5461 gaaaattcaa ttgctttgaa gagataatgt gtgaacaaat ccatttagaa aaggaaaatt 5521 aagaatttgt aaaatcatct gtaaattgtt ggcattcttc tgtatatgaa catcacatca 5581 tttacaggta aaggtctggt cattaattat atgacaattt actggtatta ttttgtgaaa 5641 ggggctattt tcaatgcgtt catccatcct tttcatccct caaatctctc attcacgtcc 5701 ccctccccat ctgcacactt tatctcattt tccaccctgc tggaatctga gcacttgtgc 5761 agttatcagg gctcctgtat ttaggaggct ctgggtgtcc atgtagggga cgaacagaaa 5821 cactgcagac ctttatagaa gaacaattga taagagtcct catacataaa gactccatta 5881 gtaagccagt gacccaggag cccagaccaa cagcaaagca gacagtgacc atggtgagca 5941 agggcgagga cgacaacatg gccatcatca aggagttcat gcgcttcaag gtgcacatgg 6001 agggctccgt gaacggccac gagttcgaga tcgagggcga gggcgagggc cgcccctacg 6061 agggcaccca gaccgccaag ctgaaggtga ccaagggcgg ccccctgccc ttcgcctggg 6121 acatcctgtc ccctcagttc atgtacggct ccaaggccta cgtgaagcac cccgccgaca 6181 tccccgacta cttgaagctg tccttccccg agggcttcaa gtgggagcgc gtgatgaact 6241 tcgaggacgg cggcgtggtg accgtgaccc aggactcctc cctgcaggac ggcgagttca 6301 tctacaaggt gaagctgcgc ggcaccaact tcccctccga cggccccgta atgcagaaga 6361 agaccatggg ctgggaggcc tcctccgagc ggatgtaccc cgaggacggc gccctgaagg 6421 gcgagatcaa gcagaggctg aagctgaagg acggcggcca ctacgacgcc gaggtcaaga 6481 ccacctacaa ggccaagaag cccgtgcagc tgcccggcgc ctacaacgtc aacatcaagc 6541 tggacatcac ctcccacaac gaggactaca ccatcgtgga acagtacgag cgcgccgagg 6601 gccgccactc caccggcggc atggacgagc tgtacaagta aggcgcgcct ctagaactat 6661 agtgagtcgt attacgtaga tccagacatg ataagataca ttgatgagtt tggacaaacc 6721 acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta 6781 tttgtaacca ttataagctg caataaacaa gttaacaaca acaattgcat tcattttatg 6841 tttcaggttc agggggaggt gtgggaggtt tttttcgagc cgccattacc ctgttatccc 6901 tacccgaata cccgactaaa ggaccggtac cagcttttgt tccctttagt gagggttaat 6961 ttcgagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac 7021 aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt 7081 gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc 7141 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg 7201 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt 7261 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa 7321 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc 7381 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag 7441 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt 7501 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg 7561 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg 7621 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg 7681 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac 7741 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg 7801 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt 7861 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg 7921 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc 7981 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt 8041 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt 8101 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag 8161 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt 8221 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc 8281 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc 8341 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg 8401 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac 8461 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg 8521 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc 8581 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact 8641 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc 8701 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat 8761 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc 8821 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac 8881 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa 8941 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact 9001 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg 9061 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg 9121 aaaagtgc //