LOCUS       sequence-74451-e        6035 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6035)
  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 6035)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Mol Syst 
            Biol."; date: "2013-10-29"; volume: "9:702. doi"; pages: " 
            10.1038/msb.2013.58"
COMMENT     Sequence Label: pVRa20_992
FEATURES             Location/Qualifiers
     source          1..6035
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             610..627
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(680..698)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      694..741
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      778..1233
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(865..884)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1075..1096
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1260..1364
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1365..1433,1434..2225)
                     /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(1583..1602)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2396..2984
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2885..2904
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3138..3155
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3170..3312
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3238..3257)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3398..3420
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3414..3605)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4414..5496)
                     /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     5458..5477
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5497..5574)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5780..5799
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5883..5902
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5883..5901
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5902..5926
                     /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             5941..5963
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5982..5999
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             6009..6032
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
ORIGIN
        1 atgaatgaaa ccgatcctga tctggaactg ctgaaacgta ttggtaataa tgatgcacag
       61 gccgttaaag aaatggttac ccgtaaactg cctcgtctgc tggcactggc aagtcgcctg
      121 ctgggtgatg cagatgaagc acgtgatatt gcacaagaaa gttttctgcg catttggaaa
      181 caggcagcaa gctggcgtag cgaacaggca cgttttgata cctggctgca tcgtgttgca
      241 ctgaatctgt gttatgatcg tctgcgtcgt cgtaaagaac atgtgccggt tgatagcgaa
      301 catgcctgtg aagcactgga tacccgtccg gcaccggatg aacagctgga agcaagcgca
      361 cagagccgtc gtatggcaca ggcactggat cagctgccgg atcgtcagcg tgaagcaatt
      421 gttctgcagt attatcaaga actgagcaat accgaagcag cagcactgat gcaaattagc
      481 gttgaagccc tggaaagcct gctgagccgt gcacgtcgta atctgcgtag ccatctggcc
      541 gaagcaccgg gtgcagatct gagcggtcgt cgcaaaccgt aaggatccaa gcttgcggcc
      601 gcactcgagc accaccacca ccaccactga gatccggctg ctaacaaagc ccgaaaggaa
      661 gctgagttgg ctgctgccac cgctgagcaa taactagcat aaccccttgg ggcctctaaa
      721 cgggtcttga ggggtttttt gctgaaagga ggaactatat ccggattggc gaatgggacg
      781 cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta
      841 cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt
      901 tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg
      961 ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat
     1021 cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac
     1081 tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag
     1141 ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg
     1201 cgaattttaa caaaatatta acgtttacaa tttcaggtgg cacttttcgg ggaaatgtgc
     1261 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
     1321 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt
     1381 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag
     1441 aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg
     1501 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa
     1561 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc
     1621 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag
     1681 tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa
     1741 ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc
     1801 taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg
     1861 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgca gcaatggcaa
     1921 caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa
     1981 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg
     2041 gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag
     2101 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg
     2161 caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt
     2221 ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt
     2281 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
     2341 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     2401 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     2461 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     2521 gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga
     2581 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     2641 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     2701 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     2761 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     2821 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     2881 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     2941 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     3001 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
     3061 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca
     3121 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt
     3181 attttctcct tacgcatctg tgcggtattt cacaccgcat atatggtgca ctctcagtac
     3241 aatctgctct gatgccgcat agttaagcca gtatacactc cgctatcgct acgtgactgg
     3301 gtcatggctg cgccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg
     3361 ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg
     3421 ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg
     3481 tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca gctcgttgag tttctccaga
     3541 agcgttaatg tctggcttct gataaagcgg gccatgttaa gggcggtttt ttcctgtttg
     3601 gtcactgatg cctccgtgta agggggattt ctgttcatgg gggtaatgat accgatgaaa
     3661 cgagagagga tgctcacgat acgggttact gatgatgaac atgcccggtt actggaacgt
     3721 tgtgagggta aacaactggc ggtatggatg cggcgggacc agagaaaaat cactcagggt
     3781 caatgccagc gcttcgttaa tacagatgta ggtgttccac agggtagcca gcagcatcct
     3841 gcgatgcaga tccggaacat aatggtgcag ggcgctgact tccgcgtttc cagactttac
     3901 gaaacacgga aaccgaagac cattcatgtt gttgctcagg tcgcagacgt tttgcagcag
     3961 cagtcgcttc acgttcgctc gcgtatcggt gattcattct gctaaccagt aaggcaaccc
     4021 cgccagccta gccgggtcct caacgacagg agcacgatca tgcgcacccg tggggccgcc
     4081 atgccggcga taatggcctg cttctcgccg aaacgtttgg tggcgggacc agtgacgaag
     4141 gcttgagcga gggcgtgcaa gattccgaat accgcaagcg acaggccgat catcgtcgcg
     4201 ctccagcgaa agcggtcctc gccgaaaatg acccagagcg ctgccggcac ctgtcctacg
     4261 agttgcatga taaagaagac agtcataagt gcggcgacga tagtcatgcc ccgcgcccac
     4321 cggaaggagc tgactgggtt gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa
     4381 tgagtgagct aacttacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     4441 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     4501 gggcgccagg gtggtttttc ttttcaccag tgagacgggc aacagctgat tgcccttcac
     4561 cgcctggccc tgagagagtt gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa
     4621 atcctgtttg atggtggtta acggcgggat ataacatgag ctgtcttcgg tatcgtcgta
     4681 tcccactacc gagatatccg caccaacgcg cagcccggac tcggtaatgg cgcgcattgc
     4741 gcccagcgcc atctgatcgt tggcaaccag catcgcagtg ggaacgatgc cctcattcag
     4801 catttgcatg gtttgttgaa aaccggacat ggcactccag tcgccttccc gttccgctat
     4861 cggctgaatt tgattgcgag tgagatattt atgccagcca gccagacgca gacgcgccga
     4921 gacagaactt aatgggcccg ctaacagcgc gatttgctgg tgacccaatg cgaccagatg
     4981 ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata atactgttga tgggtgtctg
     5041 gtcagagaca tcaagaaata acgccggaac attagtgcag gcagcttcca cagcaatggc
     5101 atcctggtca tccagcggat agttaatgat cagcccactg acgcgttgcg cgagaagatt
     5161 gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct accatcgaca ccaccacgct
     5221 ggcacccagt tgatcggcgc gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag
     5281 ggccagactg gaggtggcaa cgccaatcag caacgactgt ttgcccgcca gttgttgtgc
     5341 cacgcggttg ggaatgtaat tcagctccgc catcgccgct tccacttttt cccgcgtttt
     5401 cgcagaaacg tggctggcct ggttcaccac gcgggaaacg gtctgataag agacaccggc
     5461 atactctgcg acatcgtata acgttactgg tttcacattc accaccctga attgactctc
     5521 ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg tgtccgggat
     5581 ctcgacgctc tcccttatgc gactcctgca ttaggaagca gcccagtagt aggttgaggc
     5641 cgttgagcac cgccgccgca aggaatggtg catgcaagga gatggcgccc aacagtcccc
     5701 cggccacggg gcctgccacc atacccacgc cgaaacaagc gctcatgagc ccgaagtggc
     5761 gagcccgatc ttccccatcg gtgatgtcgg cgatataggc gccagcaacc gcacctgtgg
     5821 cgccggtgat gccggccacg atgcgtccgg cgtagaggat cgagatctcg atcccgcgaa
     5881 attaatacga ctcactatag gggaattgtg agcggataac aattcccctc tagaaataat
     5941 tttgtttaac tttaagaagg agatatacca tgggcagcag ccatcatcat catcatcaca
     6001 gcagcggcct ggaagttctg ttccaggggc cccat
//
