LOCUS       sequence-74491-e        5984 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 5984)
  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 5984)
  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: pVRa38_1442
FEATURES             Location/Qualifiers
     source          1..5984
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             559..576
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(629..647)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      643..690
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      727..1182
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(814..833)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1024..1045
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1209..1313
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1314..1382,1383..2174)
                     /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(1532..1551)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2345..2933
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2834..2853
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3087..3104
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3119..3261
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3187..3206)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3347..3369
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3363..3554)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4363..5445)
                     /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     5407..5426
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5446..5523)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5729..5748
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5832..5851
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5832..5850
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5851..5875
                     /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             5890..5912
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5931..5948
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             5958..5981
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
ORIGIN
        1 atgcctgcag aaccggcaga tgatccgcgt agctggtcag cagatgaagc agttagcgca
       61 ctgtatgcag cacattggcg tcgttttgtt gcactggcaa gcagcctgat gggtgaaagc
      121 gcaagcgctg aagaggttgt tgcagatgca tttgttaccc tgcatggtcg ttggaatcgt
      181 ctgagcgata aaggtagcgc agttgcatat ctgcgtacca gcgttgttaa tggtgcacgt
      241 gatgttctgc gtcatcgtgt tgttaccgat cgtcgtcgtc cgctgcctga tccggcaccg
      301 gatggtccgg aagaacatgc agttcgtgca gccgaacatc gtatggttct gggtgcactg
      361 gatgcactgc cgacccgtca gcgtgaagtt ctggttctgc gttatcaggg tgaaatgagc
      421 gaagaagata ttgcagcagc actgggtatt agccgtggtg cagttaaaac ccatgcacat
      481 cgtggtctga gcgcactgcg taccgcactg agcctggaag aaaccccgta aggatccaag
      541 cttgcggccg cactcgagca ccaccaccac caccactgag atccggctgc taacaaagcc
      601 cgaaaggaag ctgagttggc tgctgccacc gctgagcaat aactagcata accccttggg
      661 gcctctaaac gggtcttgag gggttttttg ctgaaaggag gaactatatc cggattggcg
      721 aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
      781 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc
      841 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
      901 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta
      961 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
     1021 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg
     1081 atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa
     1141 aatttaacgc gaattttaac aaaatattaa cgtttacaat ttcaggtggc acttttcggg
     1201 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
     1261 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
     1321 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
     1381 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
     1441 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
     1501 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg
     1561 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
     1621 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
     1681 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
     1741 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
     1801 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgcag
     1861 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
     1921 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
     1981 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta
     2041 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
     2101 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
     2161 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
     2221 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
     2281 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     2341 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     2401 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     2461 gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc
     2521 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     2581 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     2641 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     2701 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     2761 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     2821 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     2881 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     2941 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc
     3001 ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg
     3061 ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc
     3121 tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata tatggtgcac
     3181 tctcagtaca atctgctctg atgccgcata gttaagccag tatacactcc gctatcgcta
     3241 cgtgactggg tcatggctgc gccccgacac ccgccaacac ccgctgacgc gccctgacgg
     3301 gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg
     3361 tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc agctgcggta aagctcatca
     3421 gcgtggtcgt gaagcgattc acagatgtct gcctgttcat ccgcgtccag ctcgttgagt
     3481 ttctccagaa gcgttaatgt ctggcttctg ataaagcggg ccatgttaag ggcggttttt
     3541 tcctgtttgg tcactgatgc ctccgtgtaa gggggatttc tgttcatggg ggtaatgata
     3601 ccgatgaaac gagagaggat gctcacgata cgggttactg atgatgaaca tgcccggtta
     3661 ctggaacgtt gtgagggtaa acaactggcg gtatggatgc ggcgggacca gagaaaaatc
     3721 actcagggtc aatgccagcg cttcgttaat acagatgtag gtgttccaca gggtagccag
     3781 cagcatcctg cgatgcagat ccggaacata atggtgcagg gcgctgactt ccgcgtttcc
     3841 agactttacg aaacacggaa accgaagacc attcatgttg ttgctcaggt cgcagacgtt
     3901 ttgcagcagc agtcgcttca cgttcgctcg cgtatcggtg attcattctg ctaaccagta
     3961 aggcaacccc gccagcctag ccgggtcctc aacgacagga gcacgatcat gcgcacccgt
     4021 ggggccgcca tgccggcgat aatggcctgc ttctcgccga aacgtttggt ggcgggacca
     4081 gtgacgaagg cttgagcgag ggcgtgcaag attccgaata ccgcaagcga caggccgatc
     4141 atcgtcgcgc tccagcgaaa gcggtcctcg ccgaaaatga cccagagcgc tgccggcacc
     4201 tgtcctacga gttgcatgat aaagaagaca gtcataagtg cggcgacgat agtcatgccc
     4261 cgcgcccacc ggaaggagct gactgggttg aaggctctca agggcatcgg tcgagatccc
     4321 ggtgcctaat gagtgagcta acttacatta attgcgttgc gctcactgcc cgctttccag
     4381 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     4441 ttgcgtattg ggcgccaggg tggtttttct tttcaccagt gagacgggca acagctgatt
     4501 gcccttcacc gcctggccct gagagagttg cagcaagcgg tccacgctgg tttgccccag
     4561 caggcgaaaa tcctgtttga tggtggttaa cggcgggata taacatgagc tgtcttcggt
     4621 atcgtcgtat cccactaccg agatatccgc accaacgcgc agcccggact cggtaatggc
     4681 gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc atcgcagtgg gaacgatgcc
     4741 ctcattcagc atttgcatgg tttgttgaaa accggacatg gcactccagt cgccttcccg
     4801 ttccgctatc ggctgaattt gattgcgagt gagatattta tgccagccag ccagacgcag
     4861 acgcgccgag acagaactta atgggcccgc taacagcgcg atttgctggt gacccaatgc
     4921 gaccagatgc tccacgccca gtcgcgtacc gtcttcatgg gagaaaataa tactgttgat
     4981 gggtgtctgg tcagagacat caagaaataa cgccggaaca ttagtgcagg cagcttccac
     5041 agcaatggca tcctggtcat ccagcggata gttaatgatc agcccactga cgcgttgcgc
     5101 gagaagattg tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta ccatcgacac
     5161 caccacgctg gcacccagtt gatcggcgcg agatttaatc gccgcgacaa tttgcgacgg
     5221 cgcgtgcagg gccagactgg aggtggcaac gccaatcagc aacgactgtt tgcccgccag
     5281 ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc atcgccgctt ccactttttc
     5341 ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg cgggaaacgg tctgataaga
     5401 gacaccggca tactctgcga catcgtataa cgttactggt ttcacattca ccaccctgaa
     5461 ttgactctct tccgggcgct atcatgccat accgcgaaag gttttgcgcc attcgatggt
     5521 gtccgggatc tcgacgctct cccttatgcg actcctgcat taggaagcag cccagtagta
     5581 ggttgaggcc gttgagcacc gccgccgcaa ggaatggtgc atgcaaggag atggcgccca
     5641 acagtccccc ggccacgggg cctgccacca tacccacgcc gaaacaagcg ctcatgagcc
     5701 cgaagtggcg agcccgatct tccccatcgg tgatgtcggc gatataggcg ccagcaaccg
     5761 cacctgtggc gccggtgatg ccggccacga tgcgtccggc gtagaggatc gagatctcga
     5821 tcccgcgaaa ttaatacgac tcactatagg ggaattgtga gcggataaca attcccctct
     5881 agaaataatt ttgtttaact ttaagaagga gatataccat gggcagcagc catcatcatc
     5941 atcatcacag cagcggcctg gaagttctgt tccaggggcc ccat
//
