LOCUS       sequence-74438-e        6125 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 6125)
  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 6125)
  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: pVRa14_1324
FEATURES             Location/Qualifiers
     source          1..6125
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             700..717
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(770..788)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      784..831
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      868..1323
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(955..974)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1165..1186
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1350..1454
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1455..1523,1524..2315)
                     /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(1673..1692)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2486..3074
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2975..2994
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3228..3245
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3260..3402
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3328..3347)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3488..3510
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3504..3695)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4504..5586)
                     /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     5548..5567
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5587..5664)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5870..5889
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5973..5992
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5973..5991
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5992..6016
                     /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             6031..6053
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             6072..6089
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             6099..6122
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
ORIGIN
        1 atgaatgata ccgcagcaga tcatagccat gcagatggtc cgaccaccgg tcatgcacat
       61 accgcaacct ttagcagtga tgccgatggt caggcatgga cccctccgac ctgggaagaa
      121 attgttagca cccatagcgg tcgtgtttat cgtctggcat atcgtctgac cggtaatcag
      181 catgatgcag aagatctgac ccaagaagtt tttgttcgcg tttttcgtag cctgagcacc
      241 tatacaccgg gtacatttga aggttggctg catcgtatta ccaccaacct gtttctggat
      301 atggttcgtc gtaaacagcg tattcgtttt gatgcactgg gtgatgatgc agcagaacgt
      361 ctgccgagcc gtgaaccgac tccgcagcag ctgtttcatg atgcacattt tgatgcggat
      421 gttcagcagg cactggatac cctggcaccg gaatttcgtg cagcagttgt tctgtgtgat
      481 attgaaggtc tgtcctatga agaaatcgca gcaaccctgg gtgttaaact gggcaccgtt
      541 cgtagccgta ttcatcgtgg tcgtagccag ctgcgtaaag cactggcaca tcgtagcccg
      601 aaagcacgtg cagatcgtcg tagtctggca gccggtgttc cggctctggg tggtggtggt
      661 acaagcgcat aaggatccaa gcttgcggcc gcactcgagc accaccacca ccaccactga
      721 gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac cgctgagcaa
      781 taactagcat aaccccttgg ggcctctaaa cgggtcttga ggggtttttt gctgaaagga
      841 ggaactatat ccggattggc gaatgggacg cgccctgtag cggcgcatta agcgcggcgg
      901 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt
      961 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
     1021 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
     1081 attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
     1141 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc
     1201 ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa
     1261 aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgtttacaa
     1321 tttcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     1381 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     1441 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     1501 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     1561 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     1621 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
     1681 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt
     1741 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
     1801 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
     1861 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
     1921 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     1981 gtgacaccac gatgcctgca gcaatggcaa caacgttgcg caaactatta actggcgaac
     2041 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
     2101 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
     2161 gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
     2221 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     2281 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata
     2341 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
     2401 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     2461 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     2521 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     2581 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag
     2641 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     2701 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     2761 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     2821 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     2881 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     2941 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     3001 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     3061 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     3121 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
     3181 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga
     3241 gcgaggaagc ggaagagcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt
     3301 cacaccgcat atatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca
     3361 gtatacactc cgctatcgct acgtgactgg gtcatggctg cgccccgaca cccgccaaca
     3421 cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg
     3481 accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg
     3541 cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca
     3601 tccgcgtcca gctcgttgag tttctccaga agcgttaatg tctggcttct gataaagcgg
     3661 gccatgttaa gggcggtttt ttcctgtttg gtcactgatg cctccgtgta agggggattt
     3721 ctgttcatgg gggtaatgat accgatgaaa cgagagagga tgctcacgat acgggttact
     3781 gatgatgaac atgcccggtt actggaacgt tgtgagggta aacaactggc ggtatggatg
     3841 cggcgggacc agagaaaaat cactcagggt caatgccagc gcttcgttaa tacagatgta
     3901 ggtgttccac agggtagcca gcagcatcct gcgatgcaga tccggaacat aatggtgcag
     3961 ggcgctgact tccgcgtttc cagactttac gaaacacgga aaccgaagac cattcatgtt
     4021 gttgctcagg tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt
     4081 gattcattct gctaaccagt aaggcaaccc cgccagccta gccgggtcct caacgacagg
     4141 agcacgatca tgcgcacccg tggggccgcc atgccggcga taatggcctg cttctcgccg
     4201 aaacgtttgg tggcgggacc agtgacgaag gcttgagcga gggcgtgcaa gattccgaat
     4261 accgcaagcg acaggccgat catcgtcgcg ctccagcgaa agcggtcctc gccgaaaatg
     4321 acccagagcg ctgccggcac ctgtcctacg agttgcatga taaagaagac agtcataagt
     4381 gcggcgacga tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt gaaggctctc
     4441 aagggcatcg gtcgagatcc cggtgcctaa tgagtgagct aacttacatt aattgcgttg
     4501 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     4561 caacgcgcgg ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc ttttcaccag
     4621 tgagacgggc aacagctgat tgcccttcac cgcctggccc tgagagagtt gcagcaagcg
     4681 gtccacgctg gtttgcccca gcaggcgaaa atcctgtttg atggtggtta acggcgggat
     4741 ataacatgag ctgtcttcgg tatcgtcgta tcccactacc gagatatccg caccaacgcg
     4801 cagcccggac tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag
     4861 catcgcagtg ggaacgatgc cctcattcag catttgcatg gtttgttgaa aaccggacat
     4921 ggcactccag tcgccttccc gttccgctat cggctgaatt tgattgcgag tgagatattt
     4981 atgccagcca gccagacgca gacgcgccga gacagaactt aatgggcccg ctaacagcgc
     5041 gatttgctgg tgacccaatg cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg
     5101 ggagaaaata atactgttga tgggtgtctg gtcagagaca tcaagaaata acgccggaac
     5161 attagtgcag gcagcttcca cagcaatggc atcctggtca tccagcggat agttaatgat
     5221 cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc
     5281 gcttcgttct accatcgaca ccaccacgct ggcacccagt tgatcggcgc gagatttaat
     5341 cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag
     5401 caacgactgt ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc
     5461 catcgccgct tccacttttt cccgcgtttt cgcagaaacg tggctggcct ggttcaccac
     5521 gcgggaaacg gtctgataag agacaccggc atactctgcg acatcgtata acgttactgg
     5581 tttcacattc accaccctga attgactctc ttccgggcgc tatcatgcca taccgcgaaa
     5641 ggttttgcgc cattcgatgg tgtccgggat ctcgacgctc tcccttatgc gactcctgca
     5701 ttaggaagca gcccagtagt aggttgaggc cgttgagcac cgccgccgca aggaatggtg
     5761 catgcaagga gatggcgccc aacagtcccc cggccacggg gcctgccacc atacccacgc
     5821 cgaaacaagc gctcatgagc ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg
     5881 cgatataggc gccagcaacc gcacctgtgg cgccggtgat gccggccacg atgcgtccgg
     5941 cgtagaggat cgagatctcg atcccgcgaa attaatacga ctcactatag gggaattgtg
     6001 agcggataac aattcccctc tagaaataat tttgtttaac tttaagaagg agatatacca
     6061 tgggcagcag ccatcatcat catcatcaca gcagcggcct ggaagttctg ttccaggggc
     6121 cccat
//
