LOCUS       sequence-74493-e        6092 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 6092)
  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 6092)
  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: pVRa40_3198
FEATURES             Location/Qualifiers
     source          1..6092
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             667..684
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(737..755)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      751..798
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      835..1290
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(922..941)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1132..1153
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1317..1421
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1422..1490,1491..2282)
                     /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(1640..1659)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2453..3041
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2942..2961
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3195..3212
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3227..3369
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3295..3314)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3455..3477
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3471..3662)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4471..5553)
                     /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     5515..5534
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5554..5631)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5837..5856
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5940..5959
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5940..5958
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5959..5983
                     /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             5998..6020
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             6039..6056
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             6066..6089
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
ORIGIN
        1 atggttgatc cgggtgttag tccgggttgt gttcgttttg ttaccctgga aattagcccg
       61 agcatgacca tgcagggtga acgtctggat gcagttgttg ccgaagcagt tgccggtgat
      121 cgtaatgcac tgcgtgaagt tctggaaacc attcgtccga ttgttgttcg ttattgtcgt
      181 gcacgtgttg gcaccgttga acgtagcggt ctgagcgcag atgatgttgc acaagaagtt
      241 tgtctggcaa ccattaccgc actgcctcgt tatcgtgatc gtggtcgtcc gtttctggca
      301 tttctgtatg gtattgcagc acataaagtt gcagatgcac atcgtgcagc aggtcgtgat
      361 cgcgcatatc cggcagaaac cctgccggaa cgttggtcag cagatgcagg tccggaacaa
      421 atggcaattg aagcagatag cgttacccgt atgaatgaac tgctggaaat tctgcctgca
      481 aaacagcgtg aaattctgat tctgcgtgtt gttgttggtc tgtcagcaga agaaaccgca
      541 gcagcagttg gtagcaccac cggtgcagtt cgtgttgcac agcatcgtgc actgcagcgt
      601 ctgaaagatg aaattgttgc agccggtgat tatgcataag gatccaagct tgcggccgca
      661 ctcgagcacc accaccacca ccactgagat ccggctgcta acaaagcccg aaaggaagct
      721 gagttggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg
      781 gtcttgaggg gttttttgct gaaaggagga actatatccg gattggcgaa tgggacgcgc
      841 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
      901 ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
      961 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
     1021 tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc
     1081 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct
     1141 tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga
     1201 ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
     1261 attttaacaa aatattaacg tttacaattt caggtggcac ttttcgggga aatgtgcgcg
     1321 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat
     1381 aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc
     1441 gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa
     1501 cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac
     1561 tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga
     1621 tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag
     1681 agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca
     1741 cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca
     1801 tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa
     1861 ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc
     1921 tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgcagca atggcaacaa
     1981 cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag
     2041 actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct
     2101 ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac
     2161 tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa
     2221 ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt
     2281 aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat
     2341 ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg
     2401 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     2461 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     2521 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     2581 cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact
     2641 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     2701 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     2761 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     2821 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
     2881 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     2941 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
     3001 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
     3061 ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc
     3121 ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc
     3181 gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg atgcggtatt
     3241 ttctccttac gcatctgtgc ggtatttcac accgcatata tggtgcactc tcagtacaat
     3301 ctgctctgat gccgcatagt taagccagta tacactccgc tatcgctacg tgactgggtc
     3361 atggctgcgc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc
     3421 ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt
     3481 tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtga
     3541 agcgattcac agatgtctgc ctgttcatcc gcgtccagct cgttgagttt ctccagaagc
     3601 gttaatgtct ggcttctgat aaagcgggcc atgttaaggg cggttttttc ctgtttggtc
     3661 actgatgcct ccgtgtaagg gggatttctg ttcatggggg taatgatacc gatgaaacga
     3721 gagaggatgc tcacgatacg ggttactgat gatgaacatg cccggttact ggaacgttgt
     3781 gagggtaaac aactggcggt atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa
     3841 tgccagcgct tcgttaatac agatgtaggt gttccacagg gtagccagca gcatcctgcg
     3901 atgcagatcc ggaacataat ggtgcagggc gctgacttcc gcgtttccag actttacgaa
     3961 acacggaaac cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag
     4021 tcgcttcacg ttcgctcgcg tatcggtgat tcattctgct aaccagtaag gcaaccccgc
     4081 cagcctagcc gggtcctcaa cgacaggagc acgatcatgc gcacccgtgg ggccgccatg
     4141 ccggcgataa tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt gacgaaggct
     4201 tgagcgaggg cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat cgtcgcgctc
     4261 cagcgaaagc ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg tcctacgagt
     4321 tgcatgataa agaagacagt cataagtgcg gcgacgatag tcatgccccg cgcccaccgg
     4381 aaggagctga ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg tgcctaatga
     4441 gtgagctaac ttacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     4501 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     4561 cgccagggtg gtttttcttt tcaccagtga gacgggcaac agctgattgc ccttcaccgc
     4621 ctggccctga gagagttgca gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc
     4681 ctgtttgatg gtggttaacg gcgggatata acatgagctg tcttcggtat cgtcgtatcc
     4741 cactaccgag atatccgcac caacgcgcag cccggactcg gtaatggcgc gcattgcgcc
     4801 cagcgccatc tgatcgttgg caaccagcat cgcagtggga acgatgccct cattcagcat
     4861 ttgcatggtt tgttgaaaac cggacatggc actccagtcg ccttcccgtt ccgctatcgg
     4921 ctgaatttga ttgcgagtga gatatttatg ccagccagcc agacgcagac gcgccgagac
     4981 agaacttaat gggcccgcta acagcgcgat ttgctggtga cccaatgcga ccagatgctc
     5041 cacgcccagt cgcgtaccgt cttcatggga gaaaataata ctgttgatgg gtgtctggtc
     5101 agagacatca agaaataacg ccggaacatt agtgcaggca gcttccacag caatggcatc
     5161 ctggtcatcc agcggatagt taatgatcag cccactgacg cgttgcgcga gaagattgtg
     5221 caccgccgct ttacaggctt cgacgccgct tcgttctacc atcgacacca ccacgctggc
     5281 acccagttga tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc
     5341 cagactggag gtggcaacgc caatcagcaa cgactgtttg cccgccagtt gttgtgccac
     5401 gcggttggga atgtaattca gctccgccat cgccgcttcc actttttccc gcgttttcgc
     5461 agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc tgataagaga caccggcata
     5521 ctctgcgaca tcgtataacg ttactggttt cacattcacc accctgaatt gactctcttc
     5581 cgggcgctat catgccatac cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc
     5641 gacgctctcc cttatgcgac tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt
     5701 tgagcaccgc cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac agtcccccgg
     5761 ccacggggcc tgccaccata cccacgccga aacaagcgct catgagcccg aagtggcgag
     5821 cccgatcttc cccatcggtg atgtcggcga tataggcgcc agcaaccgca cctgtggcgc
     5881 cggtgatgcc ggccacgatg cgtccggcgt agaggatcga gatctcgatc ccgcgaaatt
     5941 aatacgactc actatagggg aattgtgagc ggataacaat tcccctctag aaataatttt
     6001 gtttaacttt aagaaggaga tataccatgg gcagcagcca tcatcatcat catcacagca
     6061 gcggcctgga agttctgttc caggggcccc at
//
