LOCUS       sequence-74421-e        6002 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 6002)
  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 6002)
  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: pVRa01_3473
FEATURES             Location/Qualifiers
     source          1..6002
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             577..594
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(647..665)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      661..708
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      745..1200
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(832..851)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1042..1063
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1227..1331
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1332..1400,1401..2192)
                     /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(1550..1569)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2363..2951
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2852..2871
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3105..3122
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3137..3279
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3205..3224)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3365..3387
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3381..3572)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4381..5463)
                     /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     5425..5444
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5464..5541)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5747..5766
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5850..5869
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5850..5868
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5869..5893
                     /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             5908..5930
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5949..5966
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             5976..5999
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
ORIGIN
        1 atgtttgaaa aaagtgatga acagctgatc gaaaaagccc tgaaaggtca taaaaaaagc
       61 tggtttagcc tgctgaaacg ctatgaaaaa ccggtgtatc attatggtct gcgtatgacc
      121 ggtaataatg cagatgcact ggatctgctg caggatgttt ttattagcgt gtttcgtaat
      181 ctggcaacct atcgtggtga tggtagcttt aaaggttggc tgtttcgtat tgcccattat
      241 cgttgcatcg aattttatcg tcgtaaacgt ccgatgcagg gttttgatga tgcaccggat
      301 attccgtgtg ataccgcacc ggttgatcag tatattgaaa acacccagcg taatgcacag
      361 ctggttagcg ccatgcaaca actgccgctg gcacagaaaa gcgttattga actgaaattt
      421 ttcggccact tcacctttga agaaattgca gaacagaccg gtatgagcgt taataccgca
      481 aaaagccgtc tgtatagcgc actgagcaaa atgaaaaccc tgatggaagt ggattacgtt
      541 gaactgtaag gatccaagct tgcggccgca ctcgagcacc accaccacca ccactgagat
      601 ccggctgcta acaaagcccg aaaggaagct gagttggctg ctgccaccgc tgagcaataa
      661 ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct gaaaggagga
      721 actatatccg gattggcgaa tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg
      781 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg
      841 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
      901 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
      961 agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
     1021 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta
     1081 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa
     1141 atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg tttacaattt
     1201 caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
     1261 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
     1321 aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat
     1381 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc
     1441 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga
     1501 gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg
     1561 cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc
     1621 agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag
     1681 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc
     1741 tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg
     1801 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg
     1861 acaccacgat gcctgcagca atggcaacaa cgttgcgcaa actattaact ggcgaactac
     1921 ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac
     1981 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg
     2041 agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg
     2101 tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg
     2161 agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac
     2221 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg
     2281 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     2341 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     2401 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     2461 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt
     2521 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     2581 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     2641 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     2701 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     2761 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     2821 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     2881 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     2941 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     3001 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct
     3061 ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg
     3121 aggaagcgga agagcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac
     3181 accgcatata tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta
     3241 tacactccgc tatcgctacg tgactgggtc atggctgcgc cccgacaccc gccaacaccc
     3301 gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc
     3361 gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag
     3421 ctgcggtaaa gctcatcagc gtggtcgtga agcgattcac agatgtctgc ctgttcatcc
     3481 gcgtccagct cgttgagttt ctccagaagc gttaatgtct ggcttctgat aaagcgggcc
     3541 atgttaaggg cggttttttc ctgtttggtc actgatgcct ccgtgtaagg gggatttctg
     3601 ttcatggggg taatgatacc gatgaaacga gagaggatgc tcacgatacg ggttactgat
     3661 gatgaacatg cccggttact ggaacgttgt gagggtaaac aactggcggt atggatgcgg
     3721 cgggaccaga gaaaaatcac tcagggtcaa tgccagcgct tcgttaatac agatgtaggt
     3781 gttccacagg gtagccagca gcatcctgcg atgcagatcc ggaacataat ggtgcagggc
     3841 gctgacttcc gcgtttccag actttacgaa acacggaaac cgaagaccat tcatgttgtt
     3901 gctcaggtcg cagacgtttt gcagcagcag tcgcttcacg ttcgctcgcg tatcggtgat
     3961 tcattctgct aaccagtaag gcaaccccgc cagcctagcc gggtcctcaa cgacaggagc
     4021 acgatcatgc gcacccgtgg ggccgccatg ccggcgataa tggcctgctt ctcgccgaaa
     4081 cgtttggtgg cgggaccagt gacgaaggct tgagcgaggg cgtgcaagat tccgaatacc
     4141 gcaagcgaca ggccgatcat cgtcgcgctc cagcgaaagc ggtcctcgcc gaaaatgacc
     4201 cagagcgctg ccggcacctg tcctacgagt tgcatgataa agaagacagt cataagtgcg
     4261 gcgacgatag tcatgccccg cgcccaccgg aaggagctga ctgggttgaa ggctctcaag
     4321 ggcatcggtc gagatcccgg tgcctaatga gtgagctaac ttacattaat tgcgttgcgc
     4381 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa
     4441 cgcgcgggga gaggcggttt gcgtattggg cgccagggtg gtttttcttt tcaccagtga
     4501 gacgggcaac agctgattgc ccttcaccgc ctggccctga gagagttgca gcaagcggtc
     4561 cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata
     4621 acatgagctg tcttcggtat cgtcgtatcc cactaccgag atatccgcac caacgcgcag
     4681 cccggactcg gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg caaccagcat
     4741 cgcagtggga acgatgccct cattcagcat ttgcatggtt tgttgaaaac cggacatggc
     4801 actccagtcg ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga gatatttatg
     4861 ccagccagcc agacgcagac gcgccgagac agaacttaat gggcccgcta acagcgcgat
     4921 ttgctggtga cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt cttcatggga
     4981 gaaaataata ctgttgatgg gtgtctggtc agagacatca agaaataacg ccggaacatt
     5041 agtgcaggca gcttccacag caatggcatc ctggtcatcc agcggatagt taatgatcag
     5101 cccactgacg cgttgcgcga gaagattgtg caccgccgct ttacaggctt cgacgccgct
     5161 tcgttctacc atcgacacca ccacgctggc acccagttga tcggcgcgag atttaatcgc
     5221 cgcgacaatt tgcgacggcg cgtgcagggc cagactggag gtggcaacgc caatcagcaa
     5281 cgactgtttg cccgccagtt gttgtgccac gcggttggga atgtaattca gctccgccat
     5341 cgccgcttcc actttttccc gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg
     5401 ggaaacggtc tgataagaga caccggcata ctctgcgaca tcgtataacg ttactggttt
     5461 cacattcacc accctgaatt gactctcttc cgggcgctat catgccatac cgcgaaaggt
     5521 tttgcgccat tcgatggtgt ccgggatctc gacgctctcc cttatgcgac tcctgcatta
     5581 ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat
     5641 gcaaggagat ggcgcccaac agtcccccgg ccacggggcc tgccaccata cccacgccga
     5701 aacaagcgct catgagcccg aagtggcgag cccgatcttc cccatcggtg atgtcggcga
     5761 tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt
     5821 agaggatcga gatctcgatc ccgcgaaatt aatacgactc actatagggg aattgtgagc
     5881 ggataacaat tcccctctag aaataatttt gtttaacttt aagaaggaga tataccatgg
     5941 gcagcagcca tcatcatcat catcacagca gcggcctgga agttctgttc caggggcccc
     6001 at
//
