LOCUS Exported 6038 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS pVRa15_436 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6038) 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 6038) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6038 /organism="synthetic DNA construct" /mol_type="other DNA" CDS 613..630 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" primer_bind complement(683..701) /label=T7 Term /note="T7 terminator, reverse primer" terminator 697..744 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" rep_origin 781..1236 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(868..887) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 1078..1099 /label=F1ori-F /note="F1 origin, forward primer" promoter 1263..1367 /gene="bla" /label=AmpR promoter CDS 1368..2228 /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(1586..1605) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 2399..2987 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 2888..2907 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 3141..3158 /label=L4440 /note="L4440 vector, forward primer" misc_feature 3173..3315 /label=bom /note="basis of mobility region from pBR322" primer_bind complement(3241..3260) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 3401..3423 /label=pGEX 3' /note="pGEX vectors, reverse primer" CDS complement(3417..3608) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" CDS complement(4417..5499) /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 5461..5480 /label=LacI-R /note="LacI, reverse primer" promoter complement(5500..5577) /gene="lacI" /label=lacI promoter primer_bind 5783..5802 /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" primer_bind 5886..5905 /label=T7 /note="T7 promoter, forward primer" promoter 5886..5904 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" protein_bind 5905..5929 /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 5944..5966 /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" CDS 5985..6002 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" CDS 6012..6035 /codon_start=1 /product="recognition and cleavage site for human rhinovirus 3C and PreScission proteases" /label=HRV 3C site /translation="LEVLFQGP" ORIGIN 1 atgacccaga ccccgaaagc accggcaaaa catcgtgatc cgcgtgatga actgccggaa 61 catctgcctg cactgcgtgc atttgcaatt agcctgaccc gtaatgttgc agttgcagat 121 gatctggttc aggataccat tgttaaagcc tggaccaact tcgataaatt tacggaaggc 181 accaatctgc gtgcctggct gtttaccatt ctgcgtaata ccttctatag cgataaacgt 241 aaacatcgtc gtgaagttcc agatccggaa ggtgttcatg cagcaagcct gtttgttaaa 301 ccggcacatg atggtcatct ggcatttagc gattttagcg cagcatttga tcagctgagc 361 cctgaacacc gtgaagtgct gattctggtt ggtgcaagcg gttttgccta tgaagatgca 421 gcacagatga tgggtgttgc cgttggcacc gttaaaagcc gtgcaaatcg tgcacgtgcc 481 cgtctggcag aactgctggg tctggaaaaa ggtgaagaaa tttttagcgg tgttgatggt 541 cagaccctgg cagttatgag ccgtagcggt atgaccgcag cataaggatc caagcttgcg 601 gccgcactcg agcaccacca ccaccaccac tgagatccgg ctgctaacaa agcccgaaag 661 gaagctgagt tggctgctgc caccgctgag caataactag cataacccct tggggcctct 721 aaacgggtct tgaggggttt tttgctgaaa ggaggaacta tatccggatt ggcgaatggg 781 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg 841 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca 901 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta 961 gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc 1021 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg 1081 gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat 1141 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta 1201 acgcgaattt taacaaaata ttaacgttta caatttcagg tggcactttt cggggaaatg 1261 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga 1321 gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac 1381 atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc 1441 cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca 1501 tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc 1561 caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg 1621 ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac 1681 cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca 1741 taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg 1801 agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac 1861 cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gcagcaatgg 1921 caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat 1981 taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg 2041 ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg 2101 cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc 2161 aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc 2221 attggtaact gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt 2281 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt 2341 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt 2401 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag 2461 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca 2521 gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca 2581 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg 2641 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg 2701 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct 2761 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga 2821 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc 2881 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg 2941 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg 3001 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt 3061 tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc 3121 gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcctgatgc 3181 ggtattttct ccttacgcat ctgtgcggta tttcacaccg catatatggt gcactctcag 3241 tacaatctgc tctgatgccg catagttaag ccagtataca ctccgctatc gctacgtgac 3301 tgggtcatgg ctgcgccccg acacccgcca acacccgctg acgcgccctg acgggcttgt 3361 ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag 3421 aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc atcagcgtgg 3481 tcgtgaagcg attcacagat gtctgcctgt tcatccgcgt ccagctcgtt gagtttctcc 3541 agaagcgtta atgtctggct tctgataaag cgggccatgt taagggcggt tttttcctgt 3601 ttggtcactg atgcctccgt gtaaggggga tttctgttca tgggggtaat gataccgatg 3661 aaacgagaga ggatgctcac gatacgggtt actgatgatg aacatgcccg gttactggaa 3721 cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg accagagaaa aatcactcag 3781 ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc cacagggtag ccagcagcat 3841 cctgcgatgc agatccggaa cataatggtg cagggcgctg acttccgcgt ttccagactt 3901 tacgaaacac ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga cgttttgcag 3961 cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat tctgctaacc agtaaggcaa 4021 ccccgccagc ctagccgggt cctcaacgac aggagcacga tcatgcgcac ccgtggggcc 4081 gccatgccgg cgataatggc ctgcttctcg ccgaaacgtt tggtggcggg accagtgacg 4141 aaggcttgag cgagggcgtg caagattccg aataccgcaa gcgacaggcc gatcatcgtc 4201 gcgctccagc gaaagcggtc ctcgccgaaa atgacccaga gcgctgccgg cacctgtcct 4261 acgagttgca tgataaagaa gacagtcata agtgcggcga cgatagtcat gccccgcgcc 4321 caccggaagg agctgactgg gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc 4381 taatgagtga gctaacttac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga 4441 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt 4501 attgggcgcc agggtggttt ttcttttcac cagtgagacg ggcaacagct gattgccctt 4561 caccgcctgg ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg 4621 aaaatcctgt ttgatggtgg ttaacggcgg gatataacat gagctgtctt cggtatcgtc 4681 gtatcccact accgagatat ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat 4741 tgcgcccagc gccatctgat cgttggcaac cagcatcgca gtgggaacga tgccctcatt 4801 cagcatttgc atggtttgtt gaaaaccgga catggcactc cagtcgcctt cccgttccgc 4861 tatcggctga atttgattgc gagtgagata tttatgccag ccagccagac gcagacgcgc 4921 cgagacagaa cttaatgggc ccgctaacag cgcgatttgc tggtgaccca atgcgaccag 4981 atgctccacg cccagtcgcg taccgtcttc atgggagaaa ataatactgt tgatgggtgt 5041 ctggtcagag acatcaagaa ataacgccgg aacattagtg caggcagctt ccacagcaat 5101 ggcatcctgg tcatccagcg gatagttaat gatcagccca ctgacgcgtt gcgcgagaag 5161 attgtgcacc gccgctttac aggcttcgac gccgcttcgt tctaccatcg acaccaccac 5221 gctggcaccc agttgatcgg cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg 5281 cagggccaga ctggaggtgg caacgccaat cagcaacgac tgtttgcccg ccagttgttg 5341 tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc gcttccactt tttcccgcgt 5401 tttcgcagaa acgtggctgg cctggttcac cacgcgggaa acggtctgat aagagacacc 5461 ggcatactct gcgacatcgt ataacgttac tggtttcaca ttcaccaccc tgaattgact 5521 ctcttccggg cgctatcatg ccataccgcg aaaggttttg cgccattcga tggtgtccgg 5581 gatctcgacg ctctccctta tgcgactcct gcattaggaa gcagcccagt agtaggttga 5641 ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc 5701 ccccggccac ggggcctgcc accataccca cgccgaaaca agcgctcatg agcccgaagt 5761 ggcgagcccg atcttcccca tcggtgatgt cggcgatata ggcgccagca accgcacctg 5821 tggcgccggt gatgccggcc acgatgcgtc cggcgtagag gatcgagatc tcgatcccgc 5881 gaaattaata cgactcacta taggggaatt gtgagcggat aacaattccc ctctagaaat 5941 aattttgttt aactttaaga aggagatata ccatgggcag cagccatcat catcatcatc 6001 acagcagcgg cctggaagtt ctgttccagg ggccccat //