LOCUS       sequence-74475-e        6008 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 6008)
  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 6008)
  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: pVRa32_3724
FEATURES             Location/Qualifiers
     source          1..6008
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             583..600
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(653..671)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      667..714
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      751..1206
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(838..857)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1048..1069
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1233..1337
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1338..1406,1407..2198)
                     /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(1556..1575)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2369..2957
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2858..2877
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3111..3128
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3143..3285
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3211..3230)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3371..3393
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3387..3578)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4387..5469)
                     /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     5431..5450
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5470..5547)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5753..5772
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5856..5875
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5856..5874
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5875..5899
                     /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             5914..5936
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5955..5972
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             5982..6005
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
ORIGIN
        1 atgtttcaga aaattgtgat cctggatagc acccagcctc gtcagccgag cagcagcgca
       61 ggtattcgtc agatgaccgc agatcagatt cagatgctgc gtgcatttat tcagaaacgt
      121 gttatgaatc cggatgatgt ggatgatatt ctgcagtgtg tttttctgga agcactgcgt
      181 aacgaacata aatttcagca tgcaagcaaa ccgcagacct ggctgtgtgg tattgcactg
      241 aacctgattc gtaaccattt ccgtaaaatg tatcgccagc cgtatcaaga aagctgggaa
      301 gatgaagttc atagcgaact ggaaggtcat ggtgatgtta gccatcaggt tgaaggtcat
      361 cgtcagctgg cacgtgttat tcaggcaatt gattgtctgc cgagcaatat gcagaaagtt
      421 ctggaagtta gcctggaaat ggatggcaat taccaagaaa ccgcaaatag cctgggtgtt
      481 ccgattggca ccgttcgtag ccgtctgagc cgtgcacgtg tgcagctgaa acagcagatt
      541 gatccgtttg cataaggatc caagcttgcg gccgcactcg agcaccacca ccaccaccac
      601 tgagatccgg ctgctaacaa agcccgaaag gaagctgagt tggctgctgc caccgctgag
      661 caataactag cataacccct tggggcctct aaacgggtct tgaggggttt tttgctgaaa
      721 ggaggaacta tatccggatt ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg
      781 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc
      841 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa
      901 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac
      961 ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt
     1021 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca
     1081 accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt
     1141 taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgttta
     1201 caatttcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
     1261 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
     1321 attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg
     1381 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg
     1441 aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc
     1501 ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat
     1561 gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact
     1621 attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca
     1681 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact
     1741 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg
     1801 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg
     1861 agcgtgacac cacgatgcct gcagcaatgg caacaacgtt gcgcaaacta ttaactggcg
     1921 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg
     1981 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag
     2041 ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc
     2101 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga
     2161 tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat
     2221 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc
     2281 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     2341 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     2401 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     2461 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc
     2521 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
     2581 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     2641 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     2701 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     2761 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     2821 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     2881 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     2941 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     3001 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta
     3061 ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag
     3121 tgagcgagga agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta
     3181 tttcacaccg catatatggt gcactctcag tacaatctgc tctgatgccg catagttaag
     3241 ccagtataca ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg acacccgcca
     3301 acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct
     3361 gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg
     3421 aggcagctgc ggtaaagctc atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt
     3481 tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta atgtctggct tctgataaag
     3541 cgggccatgt taagggcggt tttttcctgt ttggtcactg atgcctccgt gtaaggggga
     3601 tttctgttca tgggggtaat gataccgatg aaacgagaga ggatgctcac gatacgggtt
     3661 actgatgatg aacatgcccg gttactggaa cgttgtgagg gtaaacaact ggcggtatgg
     3721 atgcggcggg accagagaaa aatcactcag ggtcaatgcc agcgcttcgt taatacagat
     3781 gtaggtgttc cacagggtag ccagcagcat cctgcgatgc agatccggaa cataatggtg
     3841 cagggcgctg acttccgcgt ttccagactt tacgaaacac ggaaaccgaa gaccattcat
     3901 gttgttgctc aggtcgcaga cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc
     3961 ggtgattcat tctgctaacc agtaaggcaa ccccgccagc ctagccgggt cctcaacgac
     4021 aggagcacga tcatgcgcac ccgtggggcc gccatgccgg cgataatggc ctgcttctcg
     4081 ccgaaacgtt tggtggcggg accagtgacg aaggcttgag cgagggcgtg caagattccg
     4141 aataccgcaa gcgacaggcc gatcatcgtc gcgctccagc gaaagcggtc ctcgccgaaa
     4201 atgacccaga gcgctgccgg cacctgtcct acgagttgca tgataaagaa gacagtcata
     4261 agtgcggcga cgatagtcat gccccgcgcc caccggaagg agctgactgg gttgaaggct
     4321 ctcaagggca tcggtcgaga tcccggtgcc taatgagtga gctaacttac attaattgcg
     4381 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     4441 ggccaacgcg cggggagagg cggtttgcgt attgggcgcc agggtggttt ttcttttcac
     4501 cagtgagacg ggcaacagct gattgccctt caccgcctgg ccctgagaga gttgcagcaa
     4561 gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg
     4621 gatataacat gagctgtctt cggtatcgtc gtatcccact accgagatat ccgcaccaac
     4681 gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc gccatctgat cgttggcaac
     4741 cagcatcgca gtgggaacga tgccctcatt cagcatttgc atggtttgtt gaaaaccgga
     4801 catggcactc cagtcgcctt cccgttccgc tatcggctga atttgattgc gagtgagata
     4861 tttatgccag ccagccagac gcagacgcgc cgagacagaa cttaatgggc ccgctaacag
     4921 cgcgatttgc tggtgaccca atgcgaccag atgctccacg cccagtcgcg taccgtcttc
     4981 atgggagaaa ataatactgt tgatgggtgt ctggtcagag acatcaagaa ataacgccgg
     5041 aacattagtg caggcagctt ccacagcaat ggcatcctgg tcatccagcg gatagttaat
     5101 gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac aggcttcgac
     5161 gccgcttcgt tctaccatcg acaccaccac gctggcaccc agttgatcgg cgcgagattt
     5221 aatcgccgcg acaatttgcg acggcgcgtg cagggccaga ctggaggtgg caacgccaat
     5281 cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt aattcagctc
     5341 cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa acgtggctgg cctggttcac
     5401 cacgcgggaa acggtctgat aagagacacc ggcatactct gcgacatcgt ataacgttac
     5461 tggtttcaca ttcaccaccc tgaattgact ctcttccggg cgctatcatg ccataccgcg
     5521 aaaggttttg cgccattcga tggtgtccgg gatctcgacg ctctccctta tgcgactcct
     5581 gcattaggaa gcagcccagt agtaggttga ggccgttgag caccgccgcc gcaaggaatg
     5641 gtgcatgcaa ggagatggcg cccaacagtc ccccggccac ggggcctgcc accataccca
     5701 cgccgaaaca agcgctcatg agcccgaagt ggcgagcccg atcttcccca tcggtgatgt
     5761 cggcgatata ggcgccagca accgcacctg tggcgccggt gatgccggcc acgatgcgtc
     5821 cggcgtagag gatcgagatc tcgatcccgc gaaattaata cgactcacta taggggaatt
     5881 gtgagcggat aacaattccc ctctagaaat aattttgttt aactttaaga aggagatata
     5941 ccatgggcag cagccatcat catcatcatc acagcagcgg cctggaagtt ctgttccagg
     6001 ggccccat
//
