LOCUS Exported 6725 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS pVRa42_4454 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6725) AUTHORS Rhodius VA, Segall-Shapiro TH, Sharon BD, Ghodasara A, Orlova E, Tabakh H, Burkhardt DH, Clancy K, Peterson TC, Gross CA, Voigt CA TITLE Design of orthogonal genetic switches based on a crosstalk map of sigmas, anti-sigmas, and promoters. JOURNAL Mol Syst Biol. 2013 Oct 29;9:702. doi: 10.1038/msb.2013.58. PUBMED 24169405 REFERENCE 2 (bases 1 to 6725) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6725 /organism="synthetic DNA construct" /mol_type="other DNA" CDS 1300..1317 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" primer_bind complement(1370..1388) /label=T7 Term /note="T7 terminator, reverse primer" terminator 1384..1431 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" rep_origin 1468..1923 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(1555..1574) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 1765..1786 /label=F1ori-F /note="F1 origin, forward primer" promoter 1950..2054 /gene="bla" /label=AmpR promoter CDS 2055..2915 /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind complement(2273..2292) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 3086..3674 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 3575..3594 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 3828..3845 /label=L4440 /note="L4440 vector, forward primer" misc_feature 3860..4002 /label=bom /note="basis of mobility region from pBR322" primer_bind complement(3928..3947) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 4088..4110 /label=pGEX 3' /note="pGEX vectors, reverse primer" CDS complement(4104..4295) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" CDS complement(5104..6186) /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" primer_bind 6148..6167 /label=LacI-R /note="LacI, reverse primer" promoter complement(6187..6264) /gene="lacI" /label=lacI promoter primer_bind 6470..6489 /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" primer_bind 6573..6592 /label=T7 /note="T7 promoter, forward primer" promoter 6573..6591 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" protein_bind 6592..6616 /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)." RBS 6631..6653 /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" CDS 6672..6689 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" CDS 6699..6722 /codon_start=1 /product="recognition and cleavage site for human rhinovirus 3C and PreScission proteases" /label=HRV 3C site /translation="LEVLFQGP" ORIGIN 1 atggcagcaa ccgatattag cgcactgctg gataccctgt ggcgtcgtga agcaagccgt 61 attattggtg cactggcacg tcagctgcgt gatgttggtc tggccgaaga actggcacag 121 gatgcactgg ttgcagcact ggaacattgg cctcgtaatg gtattccgga taatccggca 181 gcatggctga tgaccaccgc aaaacatcgt gcaattgatc gtctgcgtca ttatcagctg 241 cagcgtcgta aacaggatgc cctgagcttt gaaattgaac gtgaacagca ggcagcaagc 301 gcagcacagc tggttgatcc ggatgatgat ctgggtgatg atcagctgcg tctgatgttt 361 gttgcatgtc atccggcact gggcaccgat gcacgtgttg cactgaccct gcgtctgctg 421 ggtggtctga gtaccgatgc aattgcccgt gcatttctgg ttccggaagc aaccattgca 481 cagcgtattg ttcgtgcaaa acgtaccctg agcgataaac aggttccgtt tgaagttccg 541 cgtggtagcg aacgtcatgc acgtctggca agcgttctgg aagttctgta tctgattttt 601 aacgaaggct atgcagccag tgatggtgat gatgcccagc gtccggctct gtgtcatgaa 661 gcactgagcc tgattggtgt tctggcagag cagatgccga ccgcagccga agttcatgca 721 ctgcgtgcac tgatggcact gcaggccagc cgtagcgcag cccgtagtga tgcaagtggt 781 gcaccggttc tgctgctgga acaggatcgt agccgttggc atcagccgct gattcagctg 841 ggtctggcag ccctggcacg tgcacagcag ctgggtggcg gtgaaagcag ctatgccctg 901 caggcagcca ttgcagcatg tcatgttacc gcagcccagg cccaggatac cgattggcca 961 cgtattgcag ccctgtatac ccgtctggcc cagcgtgttc cgagtccggt tattgcactg 1021 aatcgtgcag ttgcgattgc aatggcagaa ggtccggcag ccggtctggt tctggttgaa 1081 gccctgcagc aggatagtgc actgcgccac tatcatctgc tgccgagcgt tcgtggtgat 1141 ctgctgtata aactgggtcg ttttgatgaa gcacgtgcag attttctgca tgccgcaacc 1201 ctggcaggta atgcacgtga taaaaccttt ctgctgaccc gtgcagatgc atgtagcggt 1261 ctgcctgcat aaggatccaa gcttgcggcc gcactcgagc accaccacca ccaccactga 1321 gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac cgctgagcaa 1381 taactagcat aaccccttgg ggcctctaaa cgggtcttga ggggtttttt gctgaaagga 1441 ggaactatat ccggattggc gaatgggacg cgccctgtag cggcgcatta agcgcggcgg 1501 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt 1561 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc 1621 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg 1681 attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga 1741 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc 1801 ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa 1861 aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgtttacaa 1921 tttcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa 1981 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt 2041 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg 2101 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag 2161 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg 2221 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg 2281 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt 2341 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga 2401 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac 2461 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc 2521 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc 2581 gtgacaccac gatgcctgca gcaatggcaa caacgttgcg caaactatta actggcgaac 2641 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag 2701 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg 2761 gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta 2821 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg 2881 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata 2941 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt 3001 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc 3061 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct 3121 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa 3181 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag 3241 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc 3301 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg 3361 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca 3421 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat 3481 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg 3541 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc 3601 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc 3661 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc 3721 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg 3781 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga 3841 gcgaggaagc ggaagagcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt 3901 cacaccgcat atatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca 3961 gtatacactc cgctatcgct acgtgactgg gtcatggctg cgccccgaca cccgccaaca 4021 cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg 4081 accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg 4141 cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca 4201 tccgcgtcca gctcgttgag tttctccaga agcgttaatg tctggcttct gataaagcgg 4261 gccatgttaa gggcggtttt ttcctgtttg gtcactgatg cctccgtgta agggggattt 4321 ctgttcatgg gggtaatgat accgatgaaa cgagagagga tgctcacgat acgggttact 4381 gatgatgaac atgcccggtt actggaacgt tgtgagggta aacaactggc ggtatggatg 4441 cggcgggacc agagaaaaat cactcagggt caatgccagc gcttcgttaa tacagatgta 4501 ggtgttccac agggtagcca gcagcatcct gcgatgcaga tccggaacat aatggtgcag 4561 ggcgctgact tccgcgtttc cagactttac gaaacacgga aaccgaagac cattcatgtt 4621 gttgctcagg tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt 4681 gattcattct gctaaccagt aaggcaaccc cgccagccta gccgggtcct caacgacagg 4741 agcacgatca tgcgcacccg tggggccgcc atgccggcga taatggcctg cttctcgccg 4801 aaacgtttgg tggcgggacc agtgacgaag gcttgagcga gggcgtgcaa gattccgaat 4861 accgcaagcg acaggccgat catcgtcgcg ctccagcgaa agcggtcctc gccgaaaatg 4921 acccagagcg ctgccggcac ctgtcctacg agttgcatga taaagaagac agtcataagt 4981 gcggcgacga tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt gaaggctctc 5041 aagggcatcg gtcgagatcc cggtgcctaa tgagtgagct aacttacatt aattgcgttg 5101 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc 5161 caacgcgcgg ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc ttttcaccag 5221 tgagacgggc aacagctgat tgcccttcac cgcctggccc tgagagagtt gcagcaagcg 5281 gtccacgctg gtttgcccca gcaggcgaaa atcctgtttg atggtggtta acggcgggat 5341 ataacatgag ctgtcttcgg tatcgtcgta tcccactacc gagatatccg caccaacgcg 5401 cagcccggac tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag 5461 catcgcagtg ggaacgatgc cctcattcag catttgcatg gtttgttgaa aaccggacat 5521 ggcactccag tcgccttccc gttccgctat cggctgaatt tgattgcgag tgagatattt 5581 atgccagcca gccagacgca gacgcgccga gacagaactt aatgggcccg ctaacagcgc 5641 gatttgctgg tgacccaatg cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg 5701 ggagaaaata atactgttga tgggtgtctg gtcagagaca tcaagaaata acgccggaac 5761 attagtgcag gcagcttcca cagcaatggc atcctggtca tccagcggat agttaatgat 5821 cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc 5881 gcttcgttct accatcgaca ccaccacgct ggcacccagt tgatcggcgc gagatttaat 5941 cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag 6001 caacgactgt ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc 6061 catcgccgct tccacttttt cccgcgtttt cgcagaaacg tggctggcct ggttcaccac 6121 gcgggaaacg gtctgataag agacaccggc atactctgcg acatcgtata acgttactgg 6181 tttcacattc accaccctga attgactctc ttccgggcgc tatcatgcca taccgcgaaa 6241 ggttttgcgc cattcgatgg tgtccgggat ctcgacgctc tcccttatgc gactcctgca 6301 ttaggaagca gcccagtagt aggttgaggc cgttgagcac cgccgccgca aggaatggtg 6361 catgcaagga gatggcgccc aacagtcccc cggccacggg gcctgccacc atacccacgc 6421 cgaaacaagc gctcatgagc ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg 6481 cgatataggc gccagcaacc gcacctgtgg cgccggtgat gccggccacg atgcgtccgg 6541 cgtagaggat cgagatctcg atcccgcgaa attaatacga ctcactatag gggaattgtg 6601 agcggataac aattcccctc tagaaataat tttgtttaac tttaagaagg agatatacca 6661 tgggcagcag ccatcatcat catcatcaca gcagcggcct ggaagttctg ttccaggggc 6721 cccat //