LOCUS       sequence-74495-e        6725 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 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 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: pVRa42_4454
FEATURES             Location/Qualifiers
     source          1..6725
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     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             join(2055..2123,2124..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
//
