LOCUS       sequence-464094-        6373 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cumate and vanillate inducible toggle switch reported by mKate 
            and/or sfGFP fluorescence signals; has RBS1 at R1 site and RBS1 at 
            R2 site..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6373)
  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 6373)
  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: pVCS1
FEATURES             Location/Qualifiers
     source          1..6373
                     /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             2801..2812
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             2819..3529
                     /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      3597..3691
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     complement(3812..3831)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             3823..4638
                     /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"
     CDS             complement(4722..5384)
                     /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      5385..6156
                     /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(6271..6357)
                     /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 cagaaaatca cacaggacta 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 ctccagacag tcagagggta tttattgaac tagtccgacg aacaataagg cctccctaac
     1921 ggggggcctt ttttattgat aacaaaagtg cctactctgg aaaatctatt ggatccaatt
     1981 gacagctagc tcagtcctag gtaccattgg atccaatagt agtcaccggc tgtgcttgcc
     2041 ggtctgatga gcctgtgaag gcgaaactac ctctacaaat aattttgttt aaggctcgtg
     2101 ttactattgg ctgagataag ggtagcagaa aatcacacag gactagtcca tgagcccgaa
     2161 acgtcgtacc caggcagaac gtgcaatgga aacccagggt aaactgattg cagcagcact
     2221 gggtgttctg cgtgaaaaag gttatgcagg ttttcgtatt gcagatgttc cgggtgcagc
     2281 cggtgttagc cgtggtgcac agagccatca ttttccgacc aaactggaac tgctgctggc
     2341 aacctttgaa tggctgtatg agcagattac cgaacgtagc cgtgcacgtc tggcaaaact
     2401 gaaaccggaa gatgatgtta ttcagcagat gctggatgat gcagcagaat tttttctgga
     2461 tgatgatttt agcatcggcc tggatctgat tgttgcagca gatcgtgatc cggcactgcg
     2521 tgaaggtatt cagcgtaccg ttgaacgtaa tcgttttgtt gttgaagata tgtggctggg
     2581 tgtgctggtg agccgtggtc tgagccgtga tgatgccgaa gatattctgt ggctgatttt
     2641 taacagcgtt cgtggtctgg tagttcgtag cctgtggcag aaagataaag aacgttttga
     2701 acgtgtgcgt aatagcaccc tggaaattgc acgtgaacgt tatgcaaaat tcaaacgttg
     2761 aggctcggta tctcgtggtc tgacggtaaa atctattgta aaagaggaga aatagtccat
     2821 gcgtaaaggc gaagagctgt tcactggtgt cgtccctatt ctggtggaac tggatggtga
     2881 tgtcaacggt cataagtttt ccgtgcgtgg cgagggtgaa ggtgacgcaa ctaatggtaa
     2941 actgacgctg aagttcatct gtactactgg taaactgccg gtaccttggc cgactctggt
     3001 aacgacgctg acttatggtg ttcagtgctt tgctcgttat ccggaccata tgaagcagca
     3061 tgacttcttc aagtccgcca tgccggaagg ctatgtgcag gaacgcacga tttcctttaa
     3121 ggatgacggc acgtacaaaa cgcgtgcgga agtgaaattt gaaggcgata ctctggtaaa
     3181 ccgcattgag ctgaaaggca ttgactttaa agaagacggc aatatcctgg gccataagct
     3241 ggaatacaat tttaacagcc acaatgttta catcaccgcc gataaacaaa aaaatggcac
     3301 taaagcgaat tttaaaattc gccacaacgt ggaggatggc agcgtgcagc tggctgatca
     3361 ctaccagcaa aacactccaa tcggtgatgg tcctgttctg ctgccagaca atcactatct
     3421 gagcacgcaa agcgttctgt ctaaagatcc gaacgagaaa cgcgatcata tggttctgct
     3481 ggagttcgta accgcagcgg gcatcacgca tggtatggat gaactgtacg gctcgggaga
     3541 cctatcggta ataacagtcc aatctggtgt aacttcggaa tcgtccacta gtcttggact
     3601 cctgttgata gatccagtaa tgacctcaga actccatctg gatttgttca gaacgctcgg
     3661 ttgccgccgg gcgtttttta ttggtgagaa tccaggggtc cccaataatt acgatttaaa
     3721 tttgtgtctc aaaatctctg atgttacatt gcacaagata aaaatatatc atcatgaaca
     3781 ataaaactgt ctgcttacat aaacagtaat acaaggggtg ttatgagcca tattcagcgt
     3841 gaaacgagct gtagccgtcc gcgtctgaac agcaacatgg atgcggatct gtatggctat
     3901 aaatgggcgc gtgataacgt gggtcagagc ggcgcgacca tttatcgtct gtatggcaaa
     3961 ccggatgcgc cggaactgtt tctgaaacat ggcaaaggca gcgtggcgaa cgatgtgacc
     4021 gatgaaatgg tgcgtctgaa ctggctgacc gaatttatgc cgctgccgac cattaaacat
     4081 tttattcgca ccccggatga tgcgtggctg ctgaccaccg cgattccggg caaaaccgcg
     4141 tttcaggtgc tggaagaata tccggatagc ggcgaaaaca ttgtggatgc gctggccgtg
     4201 tttctgcgtc gtctgcatag cattccggtg tgcaactgcc cgtttaacag cgatcgtgtg
     4261 tttcgtctgg cccaggcgca gagccgtatg aacaacggcc tggtggatgc gagcgatttt
     4321 gatgatgaac gtaacggctg gccggtggaa caggtgtgga aagaaatgca taaactgctg
     4381 ccgtttagcc cggatagcgt ggtgacccac ggcgatttta gcctggataa cctgattttc
     4441 gatgaaggca aactgattgg ctgcattgat gtgggccgtg tgggcattgc ggatcgttat
     4501 caggatctgg ccattctgtg gaactgcctg ggcgaattta gcccgagcct gcaaaaacgt
     4561 ctgtttcaga aatatggcat tgataatccg gatatgaaca aactgcaatt tcatctgatg
     4621 ctggatgaat ttttctaata attaattgga ccgcggtccg cgcgttgtcc ttttccgctg
     4681 cataaccctg cttcggggtc attataggcc gtaggccggc cctaccggcg cggcagcgtt
     4741 acccgtgtcg gcggctccaa cggctcgcca tcgtccagaa aacacggctc atcgggcatc
     4801 ggcaggcgct gctgcccgcg ccgttcccat tcctccgttt cggtcaaggc tggcaggtct
     4861 ggttccatgc ccggaatgcc gggctggctg ggcggctcct cgccggggcc ggtcggtagt
     4921 tgctgctcgc ccggatacag ggtcgggatg cggcgcaggt cgccatgccc caacagcgat
     4981 tcgtcctggt cgtcgtgatc aaccaccacg gcggcactga acaccgacag gcgcaactgg
     5041 tcgcggggct ggccccacgc cacgcggtca ttgaccacgt aggccgacac ggtgccgggg
     5101 ccgttgagct tcacgacgga gatccagcgc tcggccacca agtccttgac tgcgtattgg
     5161 accgtccgca aagaacgtcc gatgagcttg gaaagtgtct tctggctgac caccacggcg
     5221 ttctggtggc ccatctgcgc cacgaggtga tgcagcagca ttgccgccgt gggtttcctc
     5281 gcaataagcc cggcccacgc ctcatgcgct ttgcgttccg tttgcaccca gtgaccgggc
     5341 ttgttcttgg cttgaatgcc gatttctctg gactgcgtgg ccatgcttat ctccatgcgg
     5401 taggggtgcc gcacggttgc ggcaccatgc gcaatcagct gcaacttttc ggcagcgcga
     5461 caacaattat gcgttgcgta aaagtggcag tcaattacag attttcttta acctacgcaa
     5521 tgagctattg cggggggtgc cgcaatgagc tgttgcgtac cccccttttt taagttgttg
     5581 atttttaagt ctttcgcatt tcgccctata tctagttctt tggtgcccaa agaagggcac
     5641 ccctgcgggg ttcccccacg ccttcggcgc ggctccccct ccggcaaaaa gtggcccctc
     5701 cggggcttgt tgatcgactg cgcggccttc ggccttgccc aaggtggcgc tgcccccttg
     5761 gaacccccgc actcgccgcc gtgaggctcg gggggcaggc gggcgggctt cgcccttcga
     5821 ctgcccccac tcgcataggc ttgggtcgtt ccaggcgcgt caaggccaag ccgctgcgcg
     5881 gtcgctgcgc gagccttgac ccgccttcca cttggtgtcc aaccggcaag cgaagcgcgc
     5941 aggccgcagg ccggaggctt ttccccagag aaaattaaaa aaattgatgg ggcaaggccg
     6001 caggccgcgc agttggagcc ggtgggtatg tggtcgaagg ctgggtagcc ggtgggcaat
     6061 ccctgtggtc aagctcgtgg gcaggcgcag cctgtccatc agcttgtcca gcagggttgt
     6121 ccacgggccg agcgaagcga gccagccggt ggccgctcgc ggccatcgtc cacatatcca
     6181 cgggctggca agggagcgca gcgaccgcgc agggcgaagc ccggagagca agcccgtagg
     6241 gggggcgcgc ccagctgtct agggcggcgg atttgtccta ctcaggagag cgttcaccga
     6301 caaacaacag ataaaacgaa aggcccagtc tttcgactga gcctttcgtt ttatttgatg
     6361 cctttaatta agg
//
