LOCUS       sequence-74483-e        6062 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 6062)
  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 6062)
  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: pVRa34_1384
FEATURES             Location/Qualifiers
     source          1..6062
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             637..654
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(707..725)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      721..768
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      805..1260
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(892..911)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1102..1123
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1287..1391
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1392..1460,1461..2252)
                     /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(1610..1629)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2423..3011
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2912..2931
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3165..3182
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3197..3339
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3265..3284)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3425..3447
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3441..3632)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4441..5523)
                     /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     5485..5504
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5524..5601)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5807..5826
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5910..5929
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5910..5928
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5929..5953
                     /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             5968..5990
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             6009..6026
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             6036..6059
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
ORIGIN
        1 atgaataccc gtacccgtcc gaccaccaca ccggttccgc ctgttgaaga aacccgttat
       61 gaagaagaac tggcacacgg tctggttaaa gcagatgaag atgcatttgc agccatttat
      121 cgtcgttggg gtagcctggt tcataccctg gcaacccgta gcctgggtga tgcacatgaa
      181 gcagaagatg ttacccagca ggtttttgtt ggtgcatggc gtggtcgtca tggttttcgt
      241 ccggaacgtg gcaccctggg agcgtggctg gttggtatta cccgtcgtaa agttgttgat
      301 gcactggcag cacgcacccg tcgtctgagc ctggttgaaa gcgcaggtca ggatattaca
      361 ccggcacgtc tggttcagcc tgcactggat gaagttctgg atcgtgttct gctggttgaa
      421 gcactgagcc gtctgccgca ggcacagcgt gatgttctgt gtatggcatt ttatgaagat
      481 ctgacccagg cacagattgc agaacgtacc ggtgttccgc tgggcaccgt taaaagccat
      541 gcacgtcgtg gtctgcatcg tctgcgtacc gcagttggtc cgggtgcagc acatgatacc
      601 tgtgtttaag gatccaagct tgcggccgca ctcgagcacc accaccacca ccactgagat
      661 ccggctgcta acaaagcccg aaaggaagct gagttggctg ctgccaccgc tgagcaataa
      721 ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct gaaaggagga
      781 actatatccg gattggcgaa tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg
      841 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg
      901 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
      961 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
     1021 agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
     1081 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta
     1141 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa
     1201 atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg tttacaattt
     1261 caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
     1321 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
     1381 aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat
     1441 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc
     1501 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga
     1561 gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg
     1621 cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc
     1681 agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag
     1741 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc
     1801 tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg
     1861 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg
     1921 acaccacgat gcctgcagca atggcaacaa cgttgcgcaa actattaact ggcgaactac
     1981 ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac
     2041 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg
     2101 agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg
     2161 tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg
     2221 agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac
     2281 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg
     2341 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     2401 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     2461 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     2521 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt
     2581 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     2641 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     2701 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     2761 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     2821 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     2881 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     2941 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     3001 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     3061 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct
     3121 ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg
     3181 aggaagcgga agagcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac
     3241 accgcatata tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta
     3301 tacactccgc tatcgctacg tgactgggtc atggctgcgc cccgacaccc gccaacaccc
     3361 gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc
     3421 gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag
     3481 ctgcggtaaa gctcatcagc gtggtcgtga agcgattcac agatgtctgc ctgttcatcc
     3541 gcgtccagct cgttgagttt ctccagaagc gttaatgtct ggcttctgat aaagcgggcc
     3601 atgttaaggg cggttttttc ctgtttggtc actgatgcct ccgtgtaagg gggatttctg
     3661 ttcatggggg taatgatacc gatgaaacga gagaggatgc tcacgatacg ggttactgat
     3721 gatgaacatg cccggttact ggaacgttgt gagggtaaac aactggcggt atggatgcgg
     3781 cgggaccaga gaaaaatcac tcagggtcaa tgccagcgct tcgttaatac agatgtaggt
     3841 gttccacagg gtagccagca gcatcctgcg atgcagatcc ggaacataat ggtgcagggc
     3901 gctgacttcc gcgtttccag actttacgaa acacggaaac cgaagaccat tcatgttgtt
     3961 gctcaggtcg cagacgtttt gcagcagcag tcgcttcacg ttcgctcgcg tatcggtgat
     4021 tcattctgct aaccagtaag gcaaccccgc cagcctagcc gggtcctcaa cgacaggagc
     4081 acgatcatgc gcacccgtgg ggccgccatg ccggcgataa tggcctgctt ctcgccgaaa
     4141 cgtttggtgg cgggaccagt gacgaaggct tgagcgaggg cgtgcaagat tccgaatacc
     4201 gcaagcgaca ggccgatcat cgtcgcgctc cagcgaaagc ggtcctcgcc gaaaatgacc
     4261 cagagcgctg ccggcacctg tcctacgagt tgcatgataa agaagacagt cataagtgcg
     4321 gcgacgatag tcatgccccg cgcccaccgg aaggagctga ctgggttgaa ggctctcaag
     4381 ggcatcggtc gagatcccgg tgcctaatga gtgagctaac ttacattaat tgcgttgcgc
     4441 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa
     4501 cgcgcgggga gaggcggttt gcgtattggg cgccagggtg gtttttcttt tcaccagtga
     4561 gacgggcaac agctgattgc ccttcaccgc ctggccctga gagagttgca gcaagcggtc
     4621 cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata
     4681 acatgagctg tcttcggtat cgtcgtatcc cactaccgag atatccgcac caacgcgcag
     4741 cccggactcg gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg caaccagcat
     4801 cgcagtggga acgatgccct cattcagcat ttgcatggtt tgttgaaaac cggacatggc
     4861 actccagtcg ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga gatatttatg
     4921 ccagccagcc agacgcagac gcgccgagac agaacttaat gggcccgcta acagcgcgat
     4981 ttgctggtga cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt cttcatggga
     5041 gaaaataata ctgttgatgg gtgtctggtc agagacatca agaaataacg ccggaacatt
     5101 agtgcaggca gcttccacag caatggcatc ctggtcatcc agcggatagt taatgatcag
     5161 cccactgacg cgttgcgcga gaagattgtg caccgccgct ttacaggctt cgacgccgct
     5221 tcgttctacc atcgacacca ccacgctggc acccagttga tcggcgcgag atttaatcgc
     5281 cgcgacaatt tgcgacggcg cgtgcagggc cagactggag gtggcaacgc caatcagcaa
     5341 cgactgtttg cccgccagtt gttgtgccac gcggttggga atgtaattca gctccgccat
     5401 cgccgcttcc actttttccc gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg
     5461 ggaaacggtc tgataagaga caccggcata ctctgcgaca tcgtataacg ttactggttt
     5521 cacattcacc accctgaatt gactctcttc cgggcgctat catgccatac cgcgaaaggt
     5581 tttgcgccat tcgatggtgt ccgggatctc gacgctctcc cttatgcgac tcctgcatta
     5641 ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat
     5701 gcaaggagat ggcgcccaac agtcccccgg ccacggggcc tgccaccata cccacgccga
     5761 aacaagcgct catgagcccg aagtggcgag cccgatcttc cccatcggtg atgtcggcga
     5821 tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt
     5881 agaggatcga gatctcgatc ccgcgaaatt aatacgactc actatagggg aattgtgagc
     5941 ggataacaat tcccctctag aaataatttt gtttaacttt aagaaggaga tataccatgg
     6001 gcagcagcca tcatcatcat catcacagca gcggcctgga agttctgttc caggggcccc
     6061 at
//
