LOCUS       sequence-464053-        6582 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cumate and vanillate inducible toggle switch reported by mKate 
            and/or sfGFP fluorescence signals; has RBS2 at R1 site and RBS1 at 
            R2 site..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6582)
  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 6582)
  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: pVCS4
FEATURES             Location/Qualifiers
     source          1..6582
                     /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             1129..1140
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     RBS             2808..2819
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             2826..3536
                     /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      3604..3698
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     complement(3819..3838)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             3830..4645
                     /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            4812..4920
                     /note="incP origin of transfer"
     CDS             complement(4931..5593)
                     /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      5594..6365
                     /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(6480..6566)
                     /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 aggggaaata 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 ctagtccctc ggtaccaaat tccagaaaag
     1921 agacgctttc gagcgtcttt tttcgttttg gtccgtgcct actctggaaa atctattgga
     1981 tccaattgac agctagctca gtcctaggta ccattggatc caatagcgct caacgggtgt
     2041 gcttcccgtt ctgatgagtc cgtgaggacg aaagcgcctc tacaaataat tttgtttaag
     2101 gctcgtgtta ctattggctg agataagggt agcagaaaat cacacaggac tagtccatga
     2161 gcccgaaacg tcgtacccag gcagaacgtg caatggaaac ccagggtaaa ctgattgcag
     2221 cagcactggg tgttctgcgt gaaaaaggtt atgcaggttt tcgtattgca gatgttccgg
     2281 gtgcagccgg tgttagccgt ggtgcacaga gccatcattt tccgaccaaa ctggaactgc
     2341 tgctggcaac ctttgaatgg ctgtatgagc agattaccga acgtagccgt gcacgtctgg
     2401 caaaactgaa accggaagat gatgttattc agcagatgct ggatgatgca gcagaatttt
     2461 ttctggatga tgattttagc atcggcctgg atctgattgt tgcagcagat cgtgatccgg
     2521 cactgcgtga aggtattcag cgtaccgttg aacgtaatcg ttttgttgtt gaagatatgt
     2581 ggctgggtgt gctggtgagc cgtggtctga gccgtgatga tgccgaagat attctgtggc
     2641 tgatttttaa cagcgttcgt ggtctggtag ttcgtagcct gtggcagaaa gataaagaac
     2701 gttttgaacg tgtgcgtaat agcaccctgg aaattgcacg tgaacgttat gcaaaattca
     2761 aacgttgagg ctcggtatct cgtggtctga cggtaaaatc tattgtaaaa gaggagaaat
     2821 agtccatgcg taaaggcgaa gagctgttca ctggtgtcgt ccctattctg gtggaactgg
     2881 atggtgatgt caacggtcat aagttttccg tgcgtggcga gggtgaaggt gacgcaacta
     2941 atggtaaact gacgctgaag ttcatctgta ctactggtaa actgccggta ccttggccga
     3001 ctctggtaac gacgctgact tatggtgttc agtgctttgc tcgttatccg gaccatatga
     3061 agcagcatga cttcttcaag tccgccatgc cggaaggcta tgtgcaggaa cgcacgattt
     3121 cctttaagga tgacggcacg tacaaaacgc gtgcggaagt gaaatttgaa ggcgatactc
     3181 tggtaaaccg cattgagctg aaaggcattg actttaaaga agacggcaat atcctgggcc
     3241 ataagctgga atacaatttt aacagccaca atgtttacat caccgccgat aaacaaaaaa
     3301 atggcactaa agcgaatttt aaaattcgcc acaacgtgga ggatggcagc gtgcagctgg
     3361 ctgatcacta ccagcaaaac actccaatcg gtgatggtcc tgttctgctg ccagacaatc
     3421 actatctgag cacgcaaagc gttctgtcta aagatccgaa cgagaaacgc gatcatatgg
     3481 ttctgctgga gttcgtaacc gcagcgggca tcacgcatgg tatggatgaa ctgtacggct
     3541 cgggagacct atcggtaata acagtccaat ctggtgtaac ttcggaatcg tccactagtc
     3601 ttggactcct gttgatagat ccagtaatga cctcagaact ccatctggat ttgttcagaa
     3661 cgctcggttg ccgccgggcg ttttttattg gtgagaatcc aggggtcccc aataattacg
     3721 atttaaattt gtgtctcaaa atctctgatg ttacattgca caagataaaa atatatcatc
     3781 atgaacaata aaactgtctg cttacataaa cagtaataca aggggtgtta tgagccatat
     3841 tcagcgtgaa acgagctgta gccgtccgcg tctgaacagc aacatggatg cggatctgta
     3901 tggctataaa tgggcgcgtg ataacgtggg tcagagcggc gcgaccattt atcgtctgta
     3961 tggcaaaccg gatgcgccgg aactgtttct gaaacatggc aaaggcagcg tggcgaacga
     4021 tgtgaccgat gaaatggtgc gtctgaactg gctgaccgaa tttatgccgc tgccgaccat
     4081 taaacatttt attcgcaccc cggatgatgc gtggctgctg accaccgcga ttccgggcaa
     4141 aaccgcgttt caggtgctgg aagaatatcc ggatagcggc gaaaacattg tggatgcgct
     4201 ggccgtgttt ctgcgtcgtc tgcatagcat tccggtgtgc aactgcccgt ttaacagcga
     4261 tcgtgtgttt cgtctggccc aggcgcagag ccgtatgaac aacggcctgg tggatgcgag
     4321 cgattttgat gatgaacgta acggctggcc ggtggaacag gtgtggaaag aaatgcataa
     4381 actgctgccg tttagcccgg atagcgtggt gacccacggc gattttagcc tggataacct
     4441 gattttcgat gaaggcaaac tgattggctg cattgatgtg ggccgtgtgg gcattgcgga
     4501 tcgttatcag gatctggcca ttctgtggaa ctgcctgggc gaatttagcc cgagcctgca
     4561 aaaacgtctg tttcagaaat atggcattga taatccggat atgaacaaac tgcaatttca
     4621 tctgatgctg gatgaatttt tctaataatt aattggaccg cggtccgcgc gttgtccttt
     4681 tccgctgcat aaccctgctt cggggtcatt atagcgattt tttcggtata tccatccttt
     4741 ttcgcacgat atacaggatt ttgccaaagg gttcgtgtag actttccttg gtgtatccaa
     4801 cggcgtcagc cgggcaggat aggtgaagta ggcccacccg cgagcgggtg ttccttcttc
     4861 actgtccctt attcgcacct ggcggtgctc aacgggaatc ctgctctgcg aggctggccg
     4921 taggccggcc ctaccggcgc ggcagcgtta cccgtgtcgg cggctccaac ggctcgccat
     4981 cgtccagaaa acacggctca tcgggcatcg gcaggcgctg ctgcccgcgc cgttcccatt
     5041 cctccgtttc ggtcaaggct ggcaggtctg gttccatgcc cggaatgccg ggctggctgg
     5101 gcggctcctc gccggggccg gtcggtagtt gctgctcgcc cggatacagg gtcgggatgc
     5161 ggcgcaggtc gccatgcccc aacagcgatt cgtcctggtc gtcgtgatca accaccacgg
     5221 cggcactgaa caccgacagg cgcaactggt cgcggggctg gccccacgcc acgcggtcat
     5281 tgaccacgta ggccgacacg gtgccggggc cgttgagctt cacgacggag atccagcgct
     5341 cggccaccaa gtccttgact gcgtattgga ccgtccgcaa agaacgtccg atgagcttgg
     5401 aaagtgtctt ctggctgacc accacggcgt tctggtggcc catctgcgcc acgaggtgat
     5461 gcagcagcat tgccgccgtg ggtttcctcg caataagccc ggcccacgcc tcatgcgctt
     5521 tgcgttccgt ttgcacccag tgaccgggct tgttcttggc ttgaatgccg atttctctgg
     5581 actgcgtggc catgcttatc tccatgcggt aggggtgccg cacggttgcg gcaccatgcg
     5641 caatcagctg caacttttcg gcagcgcgac aacaattatg cgttgcgtaa aagtggcagt
     5701 caattacaga ttttctttaa cctacgcaat gagctattgc ggggggtgcc gcaatgagct
     5761 gttgcgtacc cccctttttt aagttgttga tttttaagtc tttcgcattt cgccctatat
     5821 ctagttcttt ggtgcccaaa gaagggcacc cctgcggggt tcccccacgc cttcggcgcg
     5881 gctccccctc cggcaaaaag tggcccctcc ggggcttgtt gatcgactgc gcggccttcg
     5941 gccttgccca aggtggcgct gcccccttgg aacccccgca ctcgccgccg tgaggctcgg
     6001 ggggcaggcg ggcgggcttc gcccttcgac tgcccccact cgcataggct tgggtcgttc
     6061 caggcgcgtc aaggccaagc cgctgcgcgg tcgctgcgcg agccttgacc cgccttccac
     6121 ttggtgtcca accggcaagc gaagcgcgca ggccgcaggc cggaggcttt tccccagaga
     6181 aaattaaaaa aattgatggg gcaaggccgc aggccgcgca gttggagccg gtgggtatgt
     6241 ggtcgaaggc tgggtagccg gtgggcaatc cctgtggtca agctcgtggg caggcgcagc
     6301 ctgtccatca gcttgtccag cagggttgtc cacgggccga gcgaagcgag ccagccggtg
     6361 gccgctcgcg gccatcgtcc acatatccac gggctggcaa gggagcgcag cgaccgcgca
     6421 gggcgaagcc cggagagcaa gcccgtaggg ggggcgcgcc cagctgtcta gggcggcgga
     6481 tttgtcctac tcaggagagc gttcaccgac aaacaacaga taaaacgaaa ggcccagtct
     6541 ttcgactgag cctttcgttt tatttgatgc ctttaattaa gg
//
