LOCUS       sequence-74472-e        5963 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 5963)
  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 5963)
  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: pVRa31_2963
FEATURES             Location/Qualifiers
     source          1..5963
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             538..555
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(608..626)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      622..669
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      706..1161
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(793..812)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1003..1024
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1188..1292
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1293..1361,1362..2153)
                     /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(1511..1530)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2324..2912
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2813..2832
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3066..3083
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3098..3240
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3166..3185)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3326..3348
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3342..3533)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4342..5424)
                     /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     5386..5405
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5425..5502)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5708..5727
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5811..5830
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5811..5829
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5830..5854
                     /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             5869..5891
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5910..5927
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             5937..5960
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
ORIGIN
        1 atgagcgata gcaatgatat tgaactggca gccaatggtg ataaacatgc atttcgtcgt
       61 ctgtatgaac gttatgttcg tggtgtttat ggtatggttt ttcgtctgac cgcagatcgt
      121 agcctggcag atgatctggt tcaagaggtt tttgttcagg catggcgtag cctgccgaaa
      181 tttgatggtc gtgcaaaatt tagcacctgg ctgtttagcc tggccaataa tgttggtatt
      241 accgaaatgc gtaaacagag ccgttggttt aaacgtaatg aacgtgccat tgatcacaat
      301 acccaggatg aaaccgatag ctcagatatt gatctgggtg aactggataa actgattatt
      361 cgtctgccgg aacagacccg tcaggtgttt gttctgcatg cactgcagga ttatagccat
      421 cagcagattg ccgatctgct gcatattgca gaaggcacca gcaaaagcca gttttttcgt
      481 gctaaaaaac tgatgcaaga atggctgtaa ggatccaagc ttgcggccgc actcgagcac
      541 caccaccacc accactgaga tccggctgct aacaaagccc gaaaggaagc tgagttggct
      601 gctgccaccg ctgagcaata actagcataa ccccttgggg cctctaaacg ggtcttgagg
      661 ggttttttgc tgaaaggagg aactatatcc ggattggcga atgggacgcg ccctgtagcg
      721 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg
      781 ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc
      841 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc
      901 tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga
      961 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa
     1021 ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg attttgccga
     1081 tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca
     1141 aaatattaac gtttacaatt tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta
     1201 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
     1261 aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc
     1321 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga
     1381 aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca
     1441 acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt
     1501 ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg
     1561 gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc
     1621 atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata
     1681 acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt
     1741 tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag
     1801 ccataccaaa cgacgagcgt gacaccacga tgcctgcagc aatggcaaca acgttgcgca
     1861 aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg
     1921 aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg
     1981 ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag
     2041 atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg
     2101 aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag
     2161 accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga
     2221 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
     2281 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     2341 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     2401 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     2461 caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     2521 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     2581 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     2641 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     2701 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     2761 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     2821 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     2881 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     2941 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg
     3001 tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg
     3061 agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat tttctcctta
     3121 cgcatctgtg cggtatttca caccgcatat atggtgcact ctcagtacaa tctgctctga
     3181 tgccgcatag ttaagccagt atacactccg ctatcgctac gtgactgggt catggctgcg
     3241 ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc
     3301 gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca
     3361 tcaccgaaac gcgcgaggca gctgcggtaa agctcatcag cgtggtcgtg aagcgattca
     3421 cagatgtctg cctgttcatc cgcgtccagc tcgttgagtt tctccagaag cgttaatgtc
     3481 tggcttctga taaagcgggc catgttaagg gcggtttttt cctgtttggt cactgatgcc
     3541 tccgtgtaag ggggatttct gttcatgggg gtaatgatac cgatgaaacg agagaggatg
     3601 ctcacgatac gggttactga tgatgaacat gcccggttac tggaacgttg tgagggtaaa
     3661 caactggcgg tatggatgcg gcgggaccag agaaaaatca ctcagggtca atgccagcgc
     3721 ttcgttaata cagatgtagg tgttccacag ggtagccagc agcatcctgc gatgcagatc
     3781 cggaacataa tggtgcaggg cgctgacttc cgcgtttcca gactttacga aacacggaaa
     3841 ccgaagacca ttcatgttgt tgctcaggtc gcagacgttt tgcagcagca gtcgcttcac
     3901 gttcgctcgc gtatcggtga ttcattctgc taaccagtaa ggcaaccccg ccagcctagc
     3961 cgggtcctca acgacaggag cacgatcatg cgcacccgtg gggccgccat gccggcgata
     4021 atggcctgct tctcgccgaa acgtttggtg gcgggaccag tgacgaaggc ttgagcgagg
     4081 gcgtgcaaga ttccgaatac cgcaagcgac aggccgatca tcgtcgcgct ccagcgaaag
     4141 cggtcctcgc cgaaaatgac ccagagcgct gccggcacct gtcctacgag ttgcatgata
     4201 aagaagacag tcataagtgc ggcgacgata gtcatgcccc gcgcccaccg gaaggagctg
     4261 actgggttga aggctctcaa gggcatcggt cgagatcccg gtgcctaatg agtgagctaa
     4321 cttacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     4381 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt
     4441 ggtttttctt ttcaccagtg agacgggcaa cagctgattg cccttcaccg cctggccctg
     4501 agagagttgc agcaagcggt ccacgctggt ttgccccagc aggcgaaaat cctgtttgat
     4561 ggtggttaac ggcgggatat aacatgagct gtcttcggta tcgtcgtatc ccactaccga
     4621 gatatccgca ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc ccagcgccat
     4681 ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc tcattcagca tttgcatggt
     4741 ttgttgaaaa ccggacatgg cactccagtc gccttcccgt tccgctatcg gctgaatttg
     4801 attgcgagtg agatatttat gccagccagc cagacgcaga cgcgccgaga cagaacttaa
     4861 tgggcccgct aacagcgcga tttgctggtg acccaatgcg accagatgct ccacgcccag
     4921 tcgcgtaccg tcttcatggg agaaaataat actgttgatg ggtgtctggt cagagacatc
     4981 aagaaataac gccggaacat tagtgcaggc agcttccaca gcaatggcat cctggtcatc
     5041 cagcggatag ttaatgatca gcccactgac gcgttgcgcg agaagattgt gcaccgccgc
     5101 tttacaggct tcgacgccgc ttcgttctac catcgacacc accacgctgg cacccagttg
     5161 atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga
     5221 ggtggcaacg ccaatcagca acgactgttt gcccgccagt tgttgtgcca cgcggttggg
     5281 aatgtaattc agctccgcca tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg
     5341 gctggcctgg ttcaccacgc gggaaacggt ctgataagag acaccggcat actctgcgac
     5401 atcgtataac gttactggtt tcacattcac caccctgaat tgactctctt ccgggcgcta
     5461 tcatgccata ccgcgaaagg ttttgcgcca ttcgatggtg tccgggatct cgacgctctc
     5521 ccttatgcga ctcctgcatt aggaagcagc ccagtagtag gttgaggccg ttgagcaccg
     5581 ccgccgcaag gaatggtgca tgcaaggaga tggcgcccaa cagtcccccg gccacggggc
     5641 ctgccaccat acccacgccg aaacaagcgc tcatgagccc gaagtggcga gcccgatctt
     5701 ccccatcggt gatgtcggcg atataggcgc cagcaaccgc acctgtggcg ccggtgatgc
     5761 cggccacgat gcgtccggcg tagaggatcg agatctcgat cccgcgaaat taatacgact
     5821 cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt tgtttaactt
     5881 taagaaggag atataccatg ggcagcagcc atcatcatca tcatcacagc agcggcctgg
     5941 aagttctgtt ccaggggccc cat
//
