LOCUS       sequence-74437-e        6095 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 6095)
  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 6095)
  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: pVRa12_808
FEATURES             Location/Qualifiers
     source          1..6095
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             670..687
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(740..758)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      754..801
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      838..1293
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(925..944)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1135..1156
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1320..1424
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1425..1493,1494..2285)
                     /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(1643..1662)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2456..3044
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2945..2964
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3198..3215
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3230..3372
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3298..3317)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3458..3480
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3474..3665)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4474..5556)
                     /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     5518..5537
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5557..5634)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5840..5859
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5943..5962
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5943..5961
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5962..5986
                     /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             6001..6023
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             6042..6059
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             6069..6092
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
ORIGIN
        1 atgctggatt ttcgtcagcc gaatcgtacc aaacaagaat ttgaagaact ggcactggca
       61 catctggacc cgctgtatag cgcagcactg cgtctgacca aaaatgaacg tgatgcagaa
      121 gatctggttc aggatacctg tatgcgtgca tatcgctttt tcgataaatt tgaacgtggc
      181 accaacatta aagcctggct gtttaaaatt ctgaccaaca ccttcattaa ccgctatcgt
      241 cgtaaagtta aagaacgtac cgttgttgaa ggtgtggaac gtgaagcagt gcatgaacgt
      301 tttgttagcc gtgatgcaac cgattttgca gcaaatccgg aacagtattt ttttgatcgt
      361 ctgctgagtg atgatgttct gcgtgcaatt gatgcactgc cgattgattt tcgcctggtt
      421 gttattctgg ccgatctgca agagtttagc tataaagaaa tcgccgagat tctggaatgt
      481 ccggttggca ccgttatgag ccgtctgttt cgtggtcgta aactgctgca gaaaaatctg
      541 cgtgaatatg ccgaaggtca gggtgttttt cgtcatgatg gtgaaccggt taatgcaccg
      601 gcagatctgg aagaatatcg tcatcgtaaa aaaaccggct aaggatccaa gcttgcggcc
      661 gcactcgagc accaccacca ccaccactga gatccggctg ctaacaaagc ccgaaaggaa
      721 gctgagttgg ctgctgccac cgctgagcaa taactagcat aaccccttgg ggcctctaaa
      781 cgggtcttga ggggtttttt gctgaaagga ggaactatat ccggattggc gaatgggacg
      841 cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta
      901 cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt
      961 tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg
     1021 ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat
     1081 cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac
     1141 tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag
     1201 ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg
     1261 cgaattttaa caaaatatta acgtttacaa tttcaggtgg cacttttcgg ggaaatgtgc
     1321 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
     1381 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt
     1441 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag
     1501 aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg
     1561 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa
     1621 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc
     1681 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag
     1741 tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa
     1801 ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc
     1861 taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg
     1921 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgca gcaatggcaa
     1981 caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa
     2041 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg
     2101 gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag
     2161 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg
     2221 caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt
     2281 ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt
     2341 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
     2401 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     2461 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     2521 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     2581 gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga
     2641 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     2701 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     2761 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     2821 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     2881 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     2941 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     3001 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     3061 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
     3121 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca
     3181 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt
     3241 attttctcct tacgcatctg tgcggtattt cacaccgcat atatggtgca ctctcagtac
     3301 aatctgctct gatgccgcat agttaagcca gtatacactc cgctatcgct acgtgactgg
     3361 gtcatggctg cgccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg
     3421 ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg
     3481 ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg
     3541 tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca gctcgttgag tttctccaga
     3601 agcgttaatg tctggcttct gataaagcgg gccatgttaa gggcggtttt ttcctgtttg
     3661 gtcactgatg cctccgtgta agggggattt ctgttcatgg gggtaatgat accgatgaaa
     3721 cgagagagga tgctcacgat acgggttact gatgatgaac atgcccggtt actggaacgt
     3781 tgtgagggta aacaactggc ggtatggatg cggcgggacc agagaaaaat cactcagggt
     3841 caatgccagc gcttcgttaa tacagatgta ggtgttccac agggtagcca gcagcatcct
     3901 gcgatgcaga tccggaacat aatggtgcag ggcgctgact tccgcgtttc cagactttac
     3961 gaaacacgga aaccgaagac cattcatgtt gttgctcagg tcgcagacgt tttgcagcag
     4021 cagtcgcttc acgttcgctc gcgtatcggt gattcattct gctaaccagt aaggcaaccc
     4081 cgccagccta gccgggtcct caacgacagg agcacgatca tgcgcacccg tggggccgcc
     4141 atgccggcga taatggcctg cttctcgccg aaacgtttgg tggcgggacc agtgacgaag
     4201 gcttgagcga gggcgtgcaa gattccgaat accgcaagcg acaggccgat catcgtcgcg
     4261 ctccagcgaa agcggtcctc gccgaaaatg acccagagcg ctgccggcac ctgtcctacg
     4321 agttgcatga taaagaagac agtcataagt gcggcgacga tagtcatgcc ccgcgcccac
     4381 cggaaggagc tgactgggtt gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa
     4441 tgagtgagct aacttacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     4501 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     4561 gggcgccagg gtggtttttc ttttcaccag tgagacgggc aacagctgat tgcccttcac
     4621 cgcctggccc tgagagagtt gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa
     4681 atcctgtttg atggtggtta acggcgggat ataacatgag ctgtcttcgg tatcgtcgta
     4741 tcccactacc gagatatccg caccaacgcg cagcccggac tcggtaatgg cgcgcattgc
     4801 gcccagcgcc atctgatcgt tggcaaccag catcgcagtg ggaacgatgc cctcattcag
     4861 catttgcatg gtttgttgaa aaccggacat ggcactccag tcgccttccc gttccgctat
     4921 cggctgaatt tgattgcgag tgagatattt atgccagcca gccagacgca gacgcgccga
     4981 gacagaactt aatgggcccg ctaacagcgc gatttgctgg tgacccaatg cgaccagatg
     5041 ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata atactgttga tgggtgtctg
     5101 gtcagagaca tcaagaaata acgccggaac attagtgcag gcagcttcca cagcaatggc
     5161 atcctggtca tccagcggat agttaatgat cagcccactg acgcgttgcg cgagaagatt
     5221 gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct accatcgaca ccaccacgct
     5281 ggcacccagt tgatcggcgc gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag
     5341 ggccagactg gaggtggcaa cgccaatcag caacgactgt ttgcccgcca gttgttgtgc
     5401 cacgcggttg ggaatgtaat tcagctccgc catcgccgct tccacttttt cccgcgtttt
     5461 cgcagaaacg tggctggcct ggttcaccac gcgggaaacg gtctgataag agacaccggc
     5521 atactctgcg acatcgtata acgttactgg tttcacattc accaccctga attgactctc
     5581 ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg tgtccgggat
     5641 ctcgacgctc tcccttatgc gactcctgca ttaggaagca gcccagtagt aggttgaggc
     5701 cgttgagcac cgccgccgca aggaatggtg catgcaagga gatggcgccc aacagtcccc
     5761 cggccacggg gcctgccacc atacccacgc cgaaacaagc gctcatgagc ccgaagtggc
     5821 gagcccgatc ttccccatcg gtgatgtcgg cgatataggc gccagcaacc gcacctgtgg
     5881 cgccggtgat gccggccacg atgcgtccgg cgtagaggat cgagatctcg atcccgcgaa
     5941 attaatacga ctcactatag gggaattgtg agcggataac aattcccctc tagaaataat
     6001 tttgtttaac tttaagaagg agatatacca tgggcagcag ccatcatcat catcatcaca
     6061 gcagcggcct ggaagttctg ttccaggggc cccat
//
