LOCUS       sequence-464071-        6576 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cumate and vanillate inducible toggle switch reported by mKate 
            and/or sfGFP fluorescence signals; has RBS3 at R1 site and RBS2 at 
            R2 site..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6576)
  AUTHORS   Chan DTC, Winter L, Bjerg J, Krsmanovic S, Baldwin GS, Bernstein HC
  TITLE     Fine Tuning Genetic Circuits via Host Context and RBS Modulation
  JOURNAL   bioRxiv 2024.07.20.604438
REFERENCE   2  (bases 1 to 6576)
  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: "bioRxiv 
            2024.07.20.604438"
COMMENT     Sequence Label: pVCS8
FEATURES             Location/Qualifiers
     source          1..6576
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    133..160
                     /label=CuO
                     /bound_moiety="CymR"
                     /note="CymR-binding P2 operator sequence from the p-cmt 
                     operon of Pseudomonas putida (Mullick et al., 2006)"
     protein_bind    191..218
                     /label=CuO
                     /bound_moiety="CymR"
                     /note="CymR-binding P2 operator sequence from the p-cmt 
                     operon of Pseudomonas putida (Mullick et al., 2006)"
     RBS             337..348
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     RBS             1129..1140
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     RBS             2802..2813
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             2820..3530
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /translation="MRKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGTKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELY"
     terminator      3598..3692
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     complement(3813..3832)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             3824..4639
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     oriT            4806..4914
                     /note="incP origin of transfer"
     CDS             complement(4925..5587)
                     /codon_start=1
                     /product="replication protein for the broad-host-range 
                     plasmid pBBR1 from Bordetella bronchiseptica"
                     /label=pBBR1 Rep
                     /translation="MATQSREIGIQAKNKPGHWVQTERKAHEAWAGLIARKPTAAMLLH
                     HLVAQMGHQNAVVVSQKTLSKLIGRSLRTVQYAVKDLVAERWISVVKLNGPGTVSAYVV
                     NDRVAWGQPRDQLRLSVFSAAVVVDHDDQDESLLGHGDLRRIPTLYPGEQQLPTGPGEE
                     PPSQPGIPGMEPDLPALTETEEWERRGQQRLPMPDEPCFLDDGEPLEPPTRVTLPRR"
     rep_origin      5588..6359
                     /label=pBBR1 oriV
                     /note="replication origin of the broad-host-range plasmid 
                     pBBR1 from Bordetella bronchiseptica; requires the pBBR1 
                     Rep protein for replication"
     terminator      complement(6474..6560)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
ORIGIN
        1 ctcgggtaag aactcgcact tcgtggaaac actattatct ggtgggtctc tgtccctcgg
       61 taccaaattc cagaaaagag acgctttcga gcgtcttttt tcgttttggt ccgtgcctac
      121 tctggaaaat ctaacaaaca gacaatctgg tctgtttgta ttatggaaaa tttttctgta
      181 taatagattc aacaaacaga caatctggtc tgtttgtatt atagcgctca acgggtgtgc
      241 ttcccgttct gatgagtccg tgaggacgaa agcgcctcta caaataattt tgtttaaggc
      301 tcgtgttact attggctgag ataagggtag cagaaaaaag aggagaaata gtccatggac
      361 atgcctcgta ttaaaccggg tcagcgtgtt atgatggcac tgcgtaaaat gattgcaagc
      421 ggtgaaatca aaagtggtga acgtattgca gaaattccga ccgcagcagc actgggtgtt
      481 agccgtatgc cggttcgtat cgcactgcgt tcactggaac aagaaggtct ggttgttcgt
      541 ctgggtgcac gtggttatgc agcccgtggt gttagcagcg atcagattcg tgatgcaatt
      601 gaagttcgtg gtgttctgga aggttttgca gcacgtcgtc tggcagaacg tggtatgacc
      661 gcagaaaccc atgcacgttt tgttgtactg attgcagaag gtgaagcact gtttgcagcc
      721 ggtcgcctga atggtgaaga tctggatcgt tatgccgcat ataatcaggc atttcatgat
      781 accctggtta gcgcagcagg taatggtgca gttgaaagcg cactggcacg taatggtttt
      841 gaaccgtttg cagcagccgg tgcactggcc ctggatctga tggacctgtc tgccgaatat
      901 gaacatctgc tggcagcaca tcgtcagcat caggcagttc tggatgcagt tagctgtggt
      961 gatgccgaag gtgcagaacg tattatgcgt gatcatgcac tggcagcaat tcgtaatgca
     1021 aaagtttttg aagcagcagc aagcgcaggc gcaccgctgg gtgcagcatg gtcaattcgt
     1081 gcagattgag gctcggtatc tcgtggtctg acggtaaaat ctattgtaaa agaggagaaa
     1141 tagtccatgt cagaattaat taaagaaaat atgcacatga aattatatat ggaaggtact
     1201 gtcaacaatc atcatttcaa atgcacatcc gaaggtgaag gtaaaccata tgaaggcaca
     1261 caaacaatgc gcatcaaagc agttgaaggt ggacccctgc cctttgcgtt tgacattctc
     1321 gcaacgagct ttatgtacgg gtctaaaact tttatcaatc acacccaagg cattcctgac
     1381 ttttttaaac agtcctttcc tgaaggcttt acctgggaac gtgtaacaac ttatgaagat
     1441 ggcggtgtac ttacagcaac tcaagatacg agtttacaag atggctgtct gatttacaat
     1501 gttaaaatcc gtggcgtaaa tttcccgagt aacggacccg taatgcaaaa aaaaactctt
     1561 ggttgggaag catcaacaga aaccttatat cctgcggacg gtggcttaga aggacgcgca
     1621 gacatggcac tgaaattagt tggaggcggt catttaatct gcaacctgaa aacaacctat
     1681 cgttccaaaa aacccgctaa aaaccttaaa atgcctggag tatactatgt tgatcgtcgc
     1741 ttagaacgta ttaaagaagc tgataaagaa acctacgttg aacaacatga agtagccgta
     1801 gcccgttatt gtgaccttcc gtcgaaatta ggacatcgtt gaggctcgtt acttacgaca
     1861 ctccgagaca gtcagagggt atttattgaa ctagtccgac gaacaataag gcctccctaa
     1921 cggggggcct tttttattga taacaaaagt gcctactctg gaaaatctat tggatccaat
     1981 tgacagctag ctcagtccta ggtaccattg gatccaatag tagtcaccgg ctgtgcttgc
     2041 cggtctgatg agcctgtgaa ggcgaaacta cctctacaaa taattttgtt taaggctcgt
     2101 gttactattg gctgagataa gggtagcaga aaaaagaggg gaaatagtcc atgagcccga
     2161 aacgtcgtac ccaggcagaa cgtgcaatgg aaacccaggg taaactgatt gcagcagcac
     2221 tgggtgttct gcgtgaaaaa ggttatgcag gttttcgtat tgcagatgtt ccgggtgcag
     2281 ccggtgttag ccgtggtgca cagagccatc attttccgac caaactggaa ctgctgctgg
     2341 caacctttga atggctgtat gagcagatta ccgaacgtag ccgtgcacgt ctggcaaaac
     2401 tgaaaccgga agatgatgtt attcagcaga tgctggatga tgcagcagaa ttttttctgg
     2461 atgatgattt tagcatcggc ctggatctga ttgttgcagc agatcgtgat ccggcactgc
     2521 gtgaaggtat tcagcgtacc gttgaacgta atcgttttgt tgttgaagat atgtggctgg
     2581 gtgtgctggt gagccgtggt ctgagccgtg atgatgccga agatattctg tggctgattt
     2641 ttaacagcgt tcgtggtctg gtagttcgta gcctgtggca gaaagataaa gaacgttttg
     2701 aacgtgtgcg taatagcacc ctggaaattg cacgtgaacg ttatgcaaaa ttcaaacgtt
     2761 gaggctcggt atctcgtggt ctgacggtaa aatctattgt aaaagaggag aaatagtcca
     2821 tgcgtaaagg cgaagagctg ttcactggtg tcgtccctat tctggtggaa ctggatggtg
     2881 atgtcaacgg tcataagttt tccgtgcgtg gcgagggtga aggtgacgca actaatggta
     2941 aactgacgct gaagttcatc tgtactactg gtaaactgcc ggtaccttgg ccgactctgg
     3001 taacgacgct gacttatggt gttcagtgct ttgctcgtta tccggaccat atgaagcagc
     3061 atgacttctt caagtccgcc atgccggaag gctatgtgca ggaacgcacg atttccttta
     3121 aggatgacgg cacgtacaaa acgcgtgcgg aagtgaaatt tgaaggcgat actctggtaa
     3181 accgcattga gctgaaaggc attgacttta aagaagacgg caatatcctg ggccataagc
     3241 tggaatacaa ttttaacagc cacaatgttt acatcaccgc cgataaacaa aaaaatggca
     3301 ctaaagcgaa ttttaaaatt cgccacaacg tggaggatgg cagcgtgcag ctggctgatc
     3361 actaccagca aaacactcca atcggtgatg gtcctgttct gctgccagac aatcactatc
     3421 tgagcacgca aagcgttctg tctaaagatc cgaacgagaa acgcgatcat atggttctgc
     3481 tggagttcgt aaccgcagcg ggcatcacgc atggtatgga tgaactgtac ggctcgggag
     3541 acctatcggt aataacagtc caatctggtg taacttcgga atcgtccact agtcttggac
     3601 tcctgttgat agatccagta atgacctcag aactccatct ggatttgttc agaacgctcg
     3661 gttgccgccg ggcgtttttt attggtgaga atccaggggt ccccaataat tacgatttaa
     3721 atttgtgtct caaaatctct gatgttacat tgcacaagat aaaaatatat catcatgaac
     3781 aataaaactg tctgcttaca taaacagtaa tacaaggggt gttatgagcc atattcagcg
     3841 tgaaacgagc tgtagccgtc cgcgtctgaa cagcaacatg gatgcggatc tgtatggcta
     3901 taaatgggcg cgtgataacg tgggtcagag cggcgcgacc atttatcgtc tgtatggcaa
     3961 accggatgcg ccggaactgt ttctgaaaca tggcaaaggc agcgtggcga acgatgtgac
     4021 cgatgaaatg gtgcgtctga actggctgac cgaatttatg ccgctgccga ccattaaaca
     4081 ttttattcgc accccggatg atgcgtggct gctgaccacc gcgattccgg gcaaaaccgc
     4141 gtttcaggtg ctggaagaat atccggatag cggcgaaaac attgtggatg cgctggccgt
     4201 gtttctgcgt cgtctgcata gcattccggt gtgcaactgc ccgtttaaca gcgatcgtgt
     4261 gtttcgtctg gcccaggcgc agagccgtat gaacaacggc ctggtggatg cgagcgattt
     4321 tgatgatgaa cgtaacggct ggccggtgga acaggtgtgg aaagaaatgc ataaactgct
     4381 gccgtttagc ccggatagcg tggtgaccca cggcgatttt agcctggata acctgatttt
     4441 cgatgaaggc aaactgattg gctgcattga tgtgggccgt gtgggcattg cggatcgtta
     4501 tcaggatctg gccattctgt ggaactgcct gggcgaattt agcccgagcc tgcaaaaacg
     4561 tctgtttcag aaatatggca ttgataatcc ggatatgaac aaactgcaat ttcatctgat
     4621 gctggatgaa tttttctaat aattaattgg accgcggtcc gcgcgttgtc cttttccgct
     4681 gcataaccct gcttcggggt cattatagcg attttttcgg tatatccatc ctttttcgca
     4741 cgatatacag gattttgcca aagggttcgt gtagactttc cttggtgtat ccaacggcgt
     4801 cagccgggca ggataggtga agtaggccca cccgcgagcg ggtgttcctt cttcactgtc
     4861 ccttattcgc acctggcggt gctcaacggg aatcctgctc tgcgaggctg gccgtaggcc
     4921 ggccctaccg gcgcggcagc gttacccgtg tcggcggctc caacggctcg ccatcgtcca
     4981 gaaaacacgg ctcatcgggc atcggcaggc gctgctgccc gcgccgttcc cattcctccg
     5041 tttcggtcaa ggctggcagg tctggttcca tgcccggaat gccgggctgg ctgggcggct
     5101 cctcgccggg gccggtcggt agttgctgct cgcccggata cagggtcggg atgcggcgca
     5161 ggtcgccatg ccccaacagc gattcgtcct ggtcgtcgtg atcaaccacc acggcggcac
     5221 tgaacaccga caggcgcaac tggtcgcggg gctggcccca cgccacgcgg tcattgacca
     5281 cgtaggccga cacggtgccg gggccgttga gcttcacgac ggagatccag cgctcggcca
     5341 ccaagtcctt gactgcgtat tggaccgtcc gcaaagaacg tccgatgagc ttggaaagtg
     5401 tcttctggct gaccaccacg gcgttctggt ggcccatctg cgccacgagg tgatgcagca
     5461 gcattgccgc cgtgggtttc ctcgcaataa gcccggccca cgcctcatgc gctttgcgtt
     5521 ccgtttgcac ccagtgaccg ggcttgttct tggcttgaat gccgatttct ctggactgcg
     5581 tggccatgct tatctccatg cggtaggggt gccgcacggt tgcggcacca tgcgcaatca
     5641 gctgcaactt ttcggcagcg cgacaacaat tatgcgttgc gtaaaagtgg cagtcaatta
     5701 cagattttct ttaacctacg caatgagcta ttgcgggggg tgccgcaatg agctgttgcg
     5761 tacccccctt ttttaagttg ttgattttta agtctttcgc atttcgccct atatctagtt
     5821 ctttggtgcc caaagaaggg cacccctgcg gggttccccc acgccttcgg cgcggctccc
     5881 cctccggcaa aaagtggccc ctccggggct tgttgatcga ctgcgcggcc ttcggccttg
     5941 cccaaggtgg cgctgccccc ttggaacccc cgcactcgcc gccgtgaggc tcggggggca
     6001 ggcgggcggg cttcgccctt cgactgcccc cactcgcata ggcttgggtc gttccaggcg
     6061 cgtcaaggcc aagccgctgc gcggtcgctg cgcgagcctt gacccgcctt ccacttggtg
     6121 tccaaccggc aagcgaagcg cgcaggccgc aggccggagg cttttcccca gagaaaatta
     6181 aaaaaattga tggggcaagg ccgcaggccg cgcagttgga gccggtgggt atgtggtcga
     6241 aggctgggta gccggtgggc aatccctgtg gtcaagctcg tgggcaggcg cagcctgtcc
     6301 atcagcttgt ccagcagggt tgtccacggg ccgagcgaag cgagccagcc ggtggccgct
     6361 cgcggccatc gtccacatat ccacgggctg gcaagggagc gcagcgaccg cgcagggcga
     6421 agcccggaga gcaagcccgt agggggggcg cgcccagctg tctagggcgg cggatttgtc
     6481 ctactcagga gagcgttcac cgacaaacaa cagataaaac gaaaggccca gtctttcgac
     6541 tgagcctttc gttttatttg atgcctttaa ttaagg
//
