LOCUS Exported 6002 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS pVRa20_2913 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6002) 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 6002) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6002 /organism="synthetic DNA construct" /mol_type="other DNA" CDS 577..594 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" primer_bind complement(647..665) /label=T7 Term /note="T7 terminator, reverse primer" terminator 661..708 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" rep_origin 745..1200 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(832..851) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 1042..1063 /label=F1ori-F /note="F1 origin, forward primer" promoter 1227..1331 /gene="bla" /label=AmpR promoter CDS 1332..2192 /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(1550..1569) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 2363..2951 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 2852..2871 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 3105..3122 /label=L4440 /note="L4440 vector, forward primer" misc_feature 3137..3279 /label=bom /note="basis of mobility region from pBR322" primer_bind complement(3205..3224) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 3365..3387 /label=pGEX 3' /note="pGEX vectors, reverse primer" CDS complement(3381..3572) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" CDS complement(4381..5463) /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 5425..5444 /label=LacI-R /note="LacI, reverse primer" promoter complement(5464..5541) /gene="lacI" /label=lacI promoter primer_bind 5747..5766 /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" primer_bind 5850..5869 /label=T7 /note="T7 promoter, forward primer" promoter 5850..5868 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" protein_bind 5869..5893 /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 5908..5930 /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" CDS 5949..5966 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" CDS 5976..5999 /codon_start=1 /product="recognition and cleavage site for human rhinovirus 3C and PreScission proteases" /label=HRV 3C site /translation="LEVLFQGP" ORIGIN 1 atggaacgtg atcatgaagc actgctggtt gcacgtgttg cagatggtga tcgtgcagca 61 tttggtgaac tgctgaaaac ccatcagcag ccgctggttc gttatgcacg tcgtatgctg 121 agcgatccgg gtgcagcaga tgatgttgtt caagaaacct ttctgcgtct gtggacccgt 181 gcaggtagct ataaaagcag cagcgcacgt ctgaccacct ggctgcataa tattgcacat 241 aatctgtgcg tggatagctt tcgtcgtaat gcccgtctga gctttaccga tgatgaaagc 301 cagctggaag ttaccaccgc aggtccggaa ggtgatgcag aaagcaccga tagcgcacat 361 cgtgttcgta ccgcactgga aggtctgccg gaacgtcagc gtagcgcact gctgatgtgt 421 cattatcagg gtctgagcaa tcgtgaagca gcaaccgttc tggatgttag cgttgatgca 481 ctggaaagcc tgctggcacg tgcgcgtcgt cgtctgcgtg aagaactgct gtcacatgtt 541 gaaagctaag gatccaagct tgcggccgca ctcgagcacc accaccacca ccactgagat 601 ccggctgcta acaaagcccg aaaggaagct gagttggctg ctgccaccgc tgagcaataa 661 ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct gaaaggagga 721 actatatccg gattggcgaa tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg 781 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg 841 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg 901 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt 961 agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt 1021 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta 1081 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa 1141 atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg tttacaattt 1201 caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac 1261 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa 1321 aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat 1381 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc 1441 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga 1501 gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg 1561 cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc 1621 agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag 1681 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc 1741 tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg 1801 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg 1861 acaccacgat gcctgcagca atggcaacaa cgttgcgcaa actattaact ggcgaactac 1921 ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac 1981 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg 2041 agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg 2101 tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg 2161 agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac 2221 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg 2281 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg 2341 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc 2401 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc 2461 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt 2521 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc 2581 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact 2641 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac 2701 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag 2761 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg 2821 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg 2881 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga 2941 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt 3001 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct 3061 ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg 3121 aggaagcgga agagcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac 3181 accgcatata tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta 3241 tacactccgc tatcgctacg tgactgggtc atggctgcgc cccgacaccc gccaacaccc 3301 gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc 3361 gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag 3421 ctgcggtaaa gctcatcagc gtggtcgtga agcgattcac agatgtctgc ctgttcatcc 3481 gcgtccagct cgttgagttt ctccagaagc gttaatgtct ggcttctgat aaagcgggcc 3541 atgttaaggg cggttttttc ctgtttggtc actgatgcct ccgtgtaagg gggatttctg 3601 ttcatggggg taatgatacc gatgaaacga gagaggatgc tcacgatacg ggttactgat 3661 gatgaacatg cccggttact ggaacgttgt gagggtaaac aactggcggt atggatgcgg 3721 cgggaccaga gaaaaatcac tcagggtcaa tgccagcgct tcgttaatac agatgtaggt 3781 gttccacagg gtagccagca gcatcctgcg atgcagatcc ggaacataat ggtgcagggc 3841 gctgacttcc gcgtttccag actttacgaa acacggaaac cgaagaccat tcatgttgtt 3901 gctcaggtcg cagacgtttt gcagcagcag tcgcttcacg ttcgctcgcg tatcggtgat 3961 tcattctgct aaccagtaag gcaaccccgc cagcctagcc gggtcctcaa cgacaggagc 4021 acgatcatgc gcacccgtgg ggccgccatg ccggcgataa tggcctgctt ctcgccgaaa 4081 cgtttggtgg cgggaccagt gacgaaggct tgagcgaggg cgtgcaagat tccgaatacc 4141 gcaagcgaca ggccgatcat cgtcgcgctc cagcgaaagc ggtcctcgcc gaaaatgacc 4201 cagagcgctg ccggcacctg tcctacgagt tgcatgataa agaagacagt cataagtgcg 4261 gcgacgatag tcatgccccg cgcccaccgg aaggagctga ctgggttgaa ggctctcaag 4321 ggcatcggtc gagatcccgg tgcctaatga gtgagctaac ttacattaat tgcgttgcgc 4381 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa 4441 cgcgcgggga gaggcggttt gcgtattggg cgccagggtg gtttttcttt tcaccagtga 4501 gacgggcaac agctgattgc ccttcaccgc ctggccctga gagagttgca gcaagcggtc 4561 cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata 4621 acatgagctg tcttcggtat cgtcgtatcc cactaccgag atatccgcac caacgcgcag 4681 cccggactcg gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg caaccagcat 4741 cgcagtggga acgatgccct cattcagcat ttgcatggtt tgttgaaaac cggacatggc 4801 actccagtcg ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga gatatttatg 4861 ccagccagcc agacgcagac gcgccgagac agaacttaat gggcccgcta acagcgcgat 4921 ttgctggtga cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt cttcatggga 4981 gaaaataata ctgttgatgg gtgtctggtc agagacatca agaaataacg ccggaacatt 5041 agtgcaggca gcttccacag caatggcatc ctggtcatcc agcggatagt taatgatcag 5101 cccactgacg cgttgcgcga gaagattgtg caccgccgct ttacaggctt cgacgccgct 5161 tcgttctacc atcgacacca ccacgctggc acccagttga tcggcgcgag atttaatcgc 5221 cgcgacaatt tgcgacggcg cgtgcagggc cagactggag gtggcaacgc caatcagcaa 5281 cgactgtttg cccgccagtt gttgtgccac gcggttggga atgtaattca gctccgccat 5341 cgccgcttcc actttttccc gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg 5401 ggaaacggtc tgataagaga caccggcata ctctgcgaca tcgtataacg ttactggttt 5461 cacattcacc accctgaatt gactctcttc cgggcgctat catgccatac cgcgaaaggt 5521 tttgcgccat tcgatggtgt ccgggatctc gacgctctcc cttatgcgac tcctgcatta 5581 ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat 5641 gcaaggagat ggcgcccaac agtcccccgg ccacggggcc tgccaccata cccacgccga 5701 aacaagcgct catgagcccg aagtggcgag cccgatcttc cccatcggtg atgtcggcga 5761 tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt 5821 agaggatcga gatctcgatc ccgcgaaatt aatacgactc actatagggg aattgtgagc 5881 ggataacaat tcccctctag aaataatttt gtttaacttt aagaaggaga tataccatgg 5941 gcagcagcca tcatcatcat catcacagca gcggcctgga agttctgttc caggggcccc 6001 at //