LOCUS supplementary_fi 5618 bp DNA circular SYN 09-AUG-2023 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS . SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5618) AUTHORS Gal TITLE Direct Submission JOURNAL Exported Aug 9, 2023 from SnapGene 6.2.2 https://www.snapgene.com FEATURES Location/Qualifiers source 1..5618 /mol_type="other DNA" /organism="synthetic DNA construct" CDS complement(105..1187) /codon_start=1 /gene="lacI" /product="lac repressor" /label=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)." /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR ALADSLMQLARQVSRLESGQ" promoter complement(1188..1265) /gene="lacI" /label=lacI promoter promoter 1311..1329 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" protein_bind 1330..1354 /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)." misc_feature 1385..1414 /label=Homology to Phspnk misc_signal 1390..1400 /label=3xSTOP misc_signal 1403..1408 /label=RBS CDS 1418..1507 /codon_start=1 /product="two Strep-Tag(R)II moieties connected by a linker for enhanced binding to Strep-Tactin(R), an engineered form of streptavidin (Schmidt et al., 2013)" /label=Twin-Strep-tag /translation="SAWSHPQFEKGGGSGGGSGGSAWSHPQFEK" CDS 1538..2017 /codon_start=1 /label=dSARM1-TIR /translation="MAKTLDVFVSYRRSNGSQLASLLKVHLQLRGFSVFIDVERLEAGK FDNGLLNSIRQAKNFVLVLTPDALHRCINDEDCKDWVHRAIVAALNSNCNIIPIIDQQF DWPEVERLPEDMRSVAHFNGVNWIHDYQDACIDKLERFLRGEKNIDRIAAMVPGT" CDS 1547..1963 /codon_start=1 /label=TIR domain /translation="TLDVFVSYRRSNGSQLASLLKVHLQLRGFSVFIDVERLEAGKFDN GLLNSIRQAKNFVLVLTPDALHRCINDEDCKDWVHRAIVAALNSNCNIIPIIDQQFDWP EVERLPEDMRSVAHFNGVNWIHDYQDACIDKLERF" misc_feature 1790..1792 /label=catalytic E misc_feature 2018..2044 CDS 2043..2044 /gene="sfGFP(Sp)" /label=sfGFP(Sp) misc_feature 2045..2057 /label=MCS-2 /note="multiple cloning site 2" terminator 2081..2128 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" misc_feature 2310 terminator 2356..2383 /label=rrnB T2 terminator /note="transcription terminator T2 from the E. coli rrnB gene" terminator complement(2402..2488) /gene="Escherichia coli rrnB" /label=rrnB T1 terminator /note="transcription terminator T1 from the E. coli rrnB gene" terminator 2416..2485 /label=T1T2 terminator (from b.sub rrnB gene) /note="T1T2 terminator (from b.sub rrnB gene)" CDS complement(2526..3185) /codon_start=1 /gene="cat" /product="chloramphenicol acetyltransferase" /label=CmR /note="confers resistance to chloramphenicol" /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA" promoter complement(3186..3288) /label=cat promoter /note="promoter of the E. coli cat gene" promoter 3498..3526 /label=tet promoter /note="promoter for tetracycline efflux protein gene" promoter complement(3975..4008) /label=T7 Phi-3.8 promoter /note="weak promoter for bacteriophage T7 RNA polymerase" CDS complement(4006..4461) /codon_start=1 /product="lysozyme from bacteriophage T7" /label=T7 lysozyme /note="inhibitor of T7 RNA polymerase" /translation="MARVQFKQRESTDAIFVHCSATKPSQNVGVREIRQWHKEQGWLDV GYHFIIKRDGTVEAGRDEMAVGSHAKGYNHNSIGVCLVGGIDDKGKFDANFTPAQMQSL RSLLVTLLAKYEGAGLRAHHEVAPKACPSFDLKRWWEKNELVTSDRG" rep_origin complement(4687..5591) /direction=LEFT /label=p15a replicon /note="p15a replicon according to Sathiamoorthy & Shin 2012 Plos ONEhttp://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjo urnal.pone.0047259 " rep_origin complement(4954..5498) /direction=LEFT /label=p15A ori /note="Plasmids containing the medium-copy-number p15A origin of replication can be propagated in E. coli cells that contain a second plasmid with the ColE1 origin. " ORIGIN 1 cccgcgccca ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc 61 ccggtgccta atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc 121 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg 181 gtttgcgtat tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga 241 ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc 301 agcaggcgaa aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg 361 gtatcgtcgt atcccactac cgagatgtcc gcaccaacgc gcagcccgga ctcggtaatg 421 gcgcgcattg cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg 481 ccctcattca gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc 541 cgttccgcta tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc 601 agacgcgccg agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat 661 gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg 721 atgggtgtct ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc 781 acagcaatgg catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc 841 gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac 901 accaccacgc tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac 961 ggcgcgtgca gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc 1021 agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt 1081 tcccgcgttt tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa 1141 gagacaccgg catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg 1201 aattgactct cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg 1261 gtgtccggga tctcgacgct ctcccttatg cgactcctgc attaggaaat taatacgact 1321 cactataggg gaattgtgag cggataacaa ttcccctgta gaaataattt tgtttaactt 1381 taatctagct gattaactaa taaggaggac aaacatgtct gcatggtcac atcctcaatt 1441 tgaaaagggc ggtggatctg gtggggggag tggaggttca gcctggtccc acccacaatt 1501 tgagaagggc ggcggaagtt caggaggagg cgctagtatg gccaagactt tagacgtttt 1561 cgtatcatat cgcagatcaa acggatcaca attagcgtct ctccttaaag tgcaccttca 1621 attaagaggt ttcagcgtgt ttatcgatgt tgaacggctc gaagcgggta aattcgacaa 1681 tggcttgctt aattccatta gacaggcgaa aaatttcgtg ttggtcctca cccctgacgc 1741 tctccataga tgtataaacg acgaagattg taaggactgg gtacatcggg cgatcgtagc 1801 ggcgttgaac tcaaattgta atattatacc tataatagac caacaattcg actggccgga 1861 agtcgagaga ttacctgaag atatgcgctc tgttgcacac ttcaatggcg tcaactggat 1921 tcatgactac caggacgcct gtatagacaa acttgaacgc ttcctccgtg gtgaaaagaa 1981 tattgaccgt attgcggcta tggtcccagg aacctaataa tgagcactag tcaaggtcgg 2041 caattaatta acctaggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc 2101 ctctaaacgg gtcttgaggg gttttttgct gaaacctcag gcatttgaga agcacacggt 2161 cacactgctt ccggtagtca ataaaccggt aaaccagcaa tagacataag cggctattta 2221 acgaccctgc cctgaaccga cgaccgggtc gaatttgctt tcgaatttct gccattcatc 2281 cgcttattat cacttattca ggcgtagcaa ccaggcgttt aagggcacca ataaccaaga 2341 gtttgtagaa acgcaaaaag gccatccgtc aggatggcct tctgcttagc tagagcggcg 2401 gatttgtcct actcaggaga gcgttcaccg acaaacaaca gataaaacga aaggcccagt 2461 ctttcgactg agcctttcgt tttatttgat gcctcaagct agagagtcga attcccttaa 2521 aaaaattacg ccccgccctg ccactcatcg cagtactgtt gtaattcatt aagcattctg 2581 ccgacatgga agccatcaca gacggcatga tgaacctgaa tcgccagcgg catcagcacc 2641 ttgtcgcctt gcgtataata tttgcccata gtgaaaacgg gggcgaagaa gttgtccata 2701 ttggccacgt ttaaatcaaa actggtgaaa ctcacccagg gattggctga gacgaaaaac 2761 atattctcaa taaacccttt agggaaatag gccaggtttt caccgtaaca cgccacatct 2821 tgcgaatata tgtgtagaaa ctgccggaaa tcgtcgtggt attcactcca gagcgatgaa 2881 aacgtttcag tttgctcatg gaaaacggtg taacaagggt gaacactatc ccatatcacc 2941 agctcaccgt ctttcattgc catacggaac tccggatgag cattcatcag gcgggcaaga 3001 atgtgaataa aggccggata aaacttgtgc ttatttttct ttacggtctt taaaaaggcc 3061 gtaatatcca gctgaacggt ctggttatag gtacattgag caactgactg aaatgcctca 3121 aaatgttctt tacgatgcca ttgggatata tcaacggtgg tatatccagt gatttttttc 3181 tccattttag cttccttagc tcctgaaaat ctcgataact caaaaaatac gcccggtagt 3241 gatcttattt cattatggtg aaagttggaa cctcttacgt gccgatcaac gtctcatttt 3301 cgccaaaagt tggcccaggg cttcccggta tcaacaggga caccaggatt tatttattct 3361 gcgaagtgat cttccgtcac aggtatttat tcggcgcaaa gtgcgtcggg tgatgctgcc 3421 aacttactga tttagtgtat gatgcaaatg tagcacctga agtcagcccc atacgatata 3481 agttgtaatt ctcatgtttg acagcttatc atcgataagc tttaatgcgg tagtttatca 3541 cagttaaatt gctaacgcag tcaggcaccg tgtatgaaat ctaacaatgc gctcatcgtc 3601 atcctcggca ccgtcaccct ggatgctgta ggcataggct tggttatgcc ggtactgccg 3661 ggcctcttgc gggatatcgt ccattccgac agcatcgcca gtcactatgg cgtgctgcta 3721 gcgctatatg cgttgatgca atttctatgc gcacccgttc tcggagcact gtccgaccgc 3781 tttggccgcc gcccagtcct gctcgcttcg ctacttggag ccactatcga ctacgcgatc 3841 atggcgacca cacccgtcct gtggatccgg cccattggct gcctcccaca cttggatatg 3901 cctcctcgga gccttataga attgtttata agacttgcgc attatttgac ctccaatgcg 3961 aacaaaggga aaccgctgtg gtctcccttt agtgagttca attaattatc cacggtcaga 4021 agtgaccagt tcgttcttct cccaccaacg cttaaggtcg aacgaagggc aagccttcgg 4081 cgccacctca tgatgggcgc gaagaccagc gccttcgtac ttagccagca gtgtgacaag 4141 cagtgagcga agggattgca tttgggctgg cgtaaagtta gcgtcgaact tacctttatc 4201 gtcgatacca ccaacaaggc agacgccgat agagttgtgg ttgtaaccct tagcgtgaga 4261 gcctacagcc atctcatctc gtcctgcctc cacagtaccg tctcgcttga tgataaagtg 4321 gtatcccaca tcgagccaac cctgctcttt gtgccactgg cgaatctcac ggacaccaac 4381 attctgactt ggcttggtag ccgagcagtg aacaaagatt gcgtcagtag attcacgttg 4441 tttaaactgt acacgagcca ttatttcttt cctcctttcc tttttaatct atcaaagggg 4501 acccggatcc tctacgccgg acgcatcgtg gccggcatca ccggcgccac aggtgcggtt 4561 gctggcgcct atatcgccga catcaccgat ggggaagatc gggctggctt ctgtttctat 4621 cagctgtccc tcctgttcag ctactgacgg ggtggtgcgt aacggcaaaa gcaccgccgg 4681 acatcagcgc tagcggagtg tatactggct tactatgttg gcactgatga gggtgtcagt 4741 gaagtgcttc atgtggcagg agaaaaaagg ctgcaccggt gcgtcagcag aatatgtgat 4801 acaggatata ttccgcttcc tcgctcactg actcgctacg ctcggtcgtt cgactgcggc 4861 gagcggaaat ggcttacgaa cggggcggag atttcctgga agatgccagg aagatactta 4921 acagggaagt gagagggccg cggcaaagcc gtttttccat aggctccgcc cccctgacaa 4981 gcatcacgaa atctgacgct caaatcagtg gtggcgaaac ccgacaggac tataaagata 5041 ccaggcgttt cccctggcgg ctccctcgtg cgctctcctg ttcctgcctt tcggtttacc 5101 ggtgtcattc cgctgttatg gccgcgtttg tctcattcca cgcctgacac tcagttccgg 5161 gtaggcagtt cgctccaagc tggactgtat gcacgaaccc cccgttcagt ccgaccgctg 5221 cgccttatcc ggtaactatc gtcttgagtc caacccggaa agacatgcaa aagcaccact 5281 ggcagcagcc actggtaatt gatttagagg agttagtctt gaagtcatgc gccggttaag 5341 gctaaactga aaggacaagt tttggtgact gcgctcctcc aagccagtta cctcggttca 5401 aagagttggt agctcagaga accttcgaaa aaccgccctg caaggcggtt ttttcgtttt 5461 cagagcaaga gattacgcgc agaccaaaac gatctcaaga agatcatctt attaatcaga 5521 taaaatattt ctagatttca gtgcaattta tctcttcaaa tgtagcacct gaagtcagcc 5581 ccatacgata taagttgtaa ttctcatgtt agtcatgc //