LOCUS       sequence-315021-        7663 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Broad-Host range expression vector, RK2 and pUC origin, CymR(AM) 
            repressor, PCymRC promoter, mRFP, Hygromycin Resistance Marker. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7663)
  AUTHORS   Schuster LA, Reisch CR
  TITLE     A plasmid toolbox for controlled gene expression across the 
            Proteobacteria.
  JOURNAL   Nucleic Acids Res. 2021 Jun 14. pii: 6298615. doi: 
            10.1093/nar/gkab496.
  PUBMED    34125913
REFERENCE   2  (bases 1 to 7663)
  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: "Nucleic 
            Acids Res. 2021 Jun 14. pii: 6298615. doi: 10.1093/nar/gkab496."
COMMENT     Sequence Label: pKCyR8
FEATURES             Location/Qualifiers
     source          1..7663
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(249..1397)
                     /codon_start=1
                     /product="trans-acting replication protein that binds to 
                     and activates oriV"
                     /label=trfA
                     /translation="MNRTFDRKAYRQELIDAGFSAEDAETIASRTVMRAPRETFQSVGS
                     MVQQATAKIERDSVQLAPPALPAPSAAVERSRRLEQEAAGLAKSMTIDTRGTMTTKKRK
                     TAGEDLAKQVSEAKQAALLKHTKQQIKEMQLSLFDIAPWPDTMRAMPNDTARSALFTTR
                     NKKIPREALQNKVIFHVNKDVKITYTGVELRADDDELVWQQVLEYAKRTPIGEPITFTF
                     YELCQDLGWSINGRYYTKAEECLSRLQATAMGFTSDRVGHLESVSLLHRFRVLDRGKKT
                     SRCQVLIDEEIVVLFAGDHYTKFIWEKYRKLSPTARRMFDYFSSHREPYPLKLETFRLM
                     CGSDSTRVKKWREQVGEACEELRGSGLVEHAWVNDDLVHCKR"
     CDS             complement(1669..2040)
                     /codon_start=1
                     /gene="traJ"
                     /product="oriT-recognizing protein"
                     /label=traJ
                     /translation="MADETKPTRKGSPPIKVYCLPDERRAIEEKAAAAGMSL*AYLLAV
                     GQGYKITGVVDYEHVRELARINGDLGRLGGLLKLWLTDDPRTARFGDATILALLAKIEE
                     KQDELGKVMMGVVRPRAEP"
     oriT            2073..2182
                     /note="incP origin of transfer"
     rep_origin      2335..2966
                     /label=oriV
                     /note="incP origin of replication"
     rep_origin      3429..4017
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3918..3937
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4335..4357
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4349..4366
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4350..4366
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    5169..5196
                     /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    5227..5254
                     /label=CuO
                     /bound_moiety="CymR"
                     /note="CymR-binding P2 operator sequence from the p-cmt 
                     operon of Pseudomonas putida (Mullick et al., 2006)"
     misc_RNA        5258..5307
                     /label=sTRSV HHRz
                     /note="hammerhead ribozyme from the tobacco ringspot virus 
                     satellite RNA (Khvorova et al., 2003)"
     RBS             5352..5363
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             5370..6047
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     CDS             complement(6486..7511)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             complement(6486..7511)
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     promoter        complement(7512..7583)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ggtggcactt ttcggggatc gtggaaacga taggcctatg ccatgcgggt caaggcgact
       61 tccggcaagc tatacgcgcc ctagaattgt caattttaat cctctgttta tcggcagttc
      121 gtagagcgcg ccgtgcgtcc cgagcgatac tgagcgaagc aagtgcgtcg agcagtgccc
      181 gcttgttcct gaaatgccag taaagcgctg gctgctgaac ccccagccgg aactgacccc
      241 acaaggccct agcgtttgca atgcaccagg tcatcattga cccaggcgtg ttccaccagg
      301 ccgctgcctc gcaactcttc gcaggcttcg ccgacctgct cgcgccactt cttcacgcgg
      361 gtggaatccg atccgcacat gaggcggaag gtttccagct tgagcgggta cggctcccgg
      421 tgcgagctga aatagtcgaa catccgtcgg gccgtcggcg acagcttgcg gtacttctcc
      481 catatgaatt tcgtgtagtg gtcgccagca aacagcacga cgatttcctc gtcgatcagg
      541 acctggcaac gggacgtttt cttgccacgg tccaggacgc ggaagcggtg cagcagcgac
      601 accgattcca ggtgcccaac gcggtcggac gtgaagccca tcgccgtcgc ctgtaggcgc
      661 gacaggcatt cctcggcctt cgtgtaatac cggccattga tcgaccagcc caggtcctgg
      721 caaagctcgt agaacgtgaa ggtgatcggc tcgccgatag gggtgcgctt cgcgtactcc
      781 aacacctgct gccacaccag ttcgtcatcg tcggcccgca gctcgacgcc ggtgtaggtg
      841 atcttcacgt ccttgttgac gtggaaaatg accttgtttt gcagcgcctc gcgcgggatt
      901 ttcttgttgc gcgtggtgaa cagggcagag cgggccgtgt cgtttggcat cgctcgcatc
      961 gtgtccggcc acggcgcaat atcgaacaag gaaagctgca tttccttgat ctgctgcttc
     1021 gtgtgtttca gcaacgcggc ctgcttggcc tcgctgacct gttttgccag gtcctcgccg
     1081 gcggtttttc gcttcttggt cgtcatagtt cctcgcgtgt cgatggtcat cgacttcgcc
     1141 aaacctgccg cctcctgttc gagacgacgc gaacgctcca cggcggccga tggcgcgggc
     1201 agggcagggg gagccagttg cacgctgtcg cgctcgatct tggccgtagc ttgctggacc
     1261 atcgagccga cggactggaa ggtttcgcgg ggcgcacgca tgacggtgcg gcttgcgatg
     1321 gtttcggcat cctcggcgga aaaccccgcg tcgatcagtt cttgcctgta tgccttccgg
     1381 tcaaacgtcc gattcattca ccctccttgc gggattgccc cgactcacgc cggggcaatg
     1441 tgcccttatt cctgatttga cccgcctggt gccttggtgt ccagataatc caccttatcg
     1501 gcaatgaagt cggtcccgta gaccgtctgg ccgtccttct cgtacttggt attccgaatc
     1561 ttgccctgca cgaataccag ctccgcgaag tcgctcttct tgatggagcg catggggacg
     1621 tgcttggcaa tcacgcgcac cccccggccg ttttagcggc taaaaaagtc atggctctgc
     1681 cctcgggcgg accacgccca tcatgacctt gccaagctcg tcctgcttct cttcgatctt
     1741 cgccagcagg gcgaggatcg tggcatcacc gaaccgcgcc gtgcgcgggt cgtcggtgag
     1801 ccagagtttc agcaggccgc ccaggcggcc caggtcgcca ttgatgcggg ccagctcgcg
     1861 gacgtgctca tagtccacga cgcccgtgat tttgtagccc tggccgacgg ccagcaggta
     1921 ggcctacagg ctcatgccgg ccgccgccgc cttttcctca atcgctcttc gttcgtctgg
     1981 aaggcagtac accttgatag gtgggctgcc cttcctggtt ggcttggttt catcagccat
     2041 ccgcttgccc tcatctgtta cgccggcggt agccggccag cctcgcagag caggattccc
     2101 gttgagcacc gccaggtgcg aataagggac agtgaagaag gaacacccgc tcgcgggtgg
     2161 gcctacttca cctatcctgc ccggctgacg ccgttggata caccaaggaa agtctacacg
     2221 aaccctttgg caaaatcctg tatatcgtgc gaaaaaggat ggatataccg aaaaaatcgc
     2281 tataatgacc ccgaagcagg gttatgcagc ggaaaagatc cgtcgaccct ttccgacgct
     2341 caccgggctg gttgccctcg ccgctgggct ggcggccgtc tatggccctg caaacgcgcc
     2401 agaaacgccg tcgaagccgt gtgcgagaca ccgcggccgc cggcgttgtg gatacctcgc
     2461 ggaaaacttg gccctcactg acagatgagg ggcggacgtt gacacttgag gggccgactc
     2521 acccggcgcg gcgttgacag atgaggggca ggctcgattt cggccggcga cgtggagctg
     2581 gccagcctcg caaatcggcg aaaacgcctg attttacgcg agtttcccac agatgatgtg
     2641 gacaagcctg gggataagtg ccctgcggta ttgacacttg aggggcgcga ctactgacag
     2701 atgaggggcg cgatccttga cacttgaggg gcagagtgct gacagatgag gggcgcacct
     2761 attgacattt gaggggctgt ccacaggcag aaaatccagc atttgcaagg gtttccgccc
     2821 gtttttcggc caccgctaac ctgtctttta acctgctttt aaaccaatat ttataaacct
     2881 tgtttttaac cagggctgcg ccctgtgcgc gtgaccgcgc acgccgaagg ggggtgcccc
     2941 cccttctcga accctcccgg cccgctaacg cgggcctccc atccccccag gggctgcgcc
     3001 cctcggccgc gaacggcctc accccaaaaa tggcagccaa gctgaccact tctgcgctcg
     3061 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc
     3121 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg
     3181 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
     3241 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta
     3301 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
     3361 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     3421 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     3481 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     3541 actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc
     3601 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     3661 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     3721 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     3781 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     3841 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     3901 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     3961 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     4021 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttcg tttaagggca
     4081 ccaataactg ccttaaaaaa attacgcccc gccctgccac tcatcgcagt acggcctatt
     4141 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc
     4201 ttacaatttc cattcgccat tcaggctgcg caactgtttg gaagggcgat cggtgcgggc
     4261 ctcttcgcta ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat taagttgggt
     4321 aacgccaggg ttttcccagt cacgacgttg taaaacgacg gccagtgatc aacgtttgaa
     4381 ttttgcataa cgttcacgtg caatttccag ggtgctatta cgcacacgtt caaaacgttc
     4441 tttatctttc tgccacaggc tacgaactac cagaccacga acgctgttaa aaatcagcca
     4501 cagaatatct tcggcatcat cacggctcag accacggctc accagcacac ccagccacat
     4561 atcttcaaca acaaaacgat tacgttcaac ggtacgctga ataccttcac gcagtgccgg
     4621 atcacgatct gctgcaacaa tcagatccag gccgatgcta aaatcatcat ccagaaaaaa
     4681 ttctgctgca tcatccagca tctgctgaat aacatcatct tccggtttca gttttgccag
     4741 acgtgcacgg ctacgttcgg taatctgctc atacagccat tcaaaggttg ccagcagcag
     4801 ttccagtttg gtcggaaaat gatggctctg tgcaccacgg ctaacaccgg ctgcacccgg
     4861 aacatctgca atacgaaaac ctgcataacc tttttcacgc agaacaccca gtgctgctgc
     4921 aatcagttta ccctgggttt ccattgcacg ttctgcctgg gtacgacgtt tcgggctcat
     4981 aagaatctcc gtttggtatt tactatgaat attccattgg gcgctatcat gccataccgc
     5041 gaaaggtttt gcgccattcg atggcgcgcc gccccaatta ttgaaggcct ccctaacggg
     5101 gggccttttt ttgtttctgg tctcccgctt aacgatcgtt ggctgggata catatgtcaa
     5161 tgtaccggaa caaacagaca atctggtctg tttgtattat ggaaaatttt tctgtataat
     5221 agattcaaca aacagacaat ctggtctgtt tgtaaagctg tcaccggatg tgctttccgg
     5281 tctgatgagt ccgtgaggac gaaacagcct ctacaaataa ttttgtttaa gaactgggaa
     5341 gagcgcagat taaagaggag aaatactaga tggcttcctc cgaagacgtt atcaaagagt
     5401 tcatgcgttt caaagttcgt atggaaggtt ccgttaacgg tcacgagttc gaaatcgaag
     5461 gtgaaggtga aggtcgtccg tacgaaggta cccagaccgc taaactgaaa gttaccaaag
     5521 gtggtccgct gccgttcgct tgggacatcc tgtccccgca gttccagtac ggttccaaag
     5581 cttacgttaa acacccggct gacatcccgg actacctgaa actgtccttc ccggaaggtt
     5641 tcaaatggga acgtgttatg aacttcgaag acggtggtgt tgttaccgtt acccaggact
     5701 cctccctgca agacggtgag ttcatctaca aagttaaact gcgtggtacc aacttcccgt
     5761 ccgacggtcc ggttatgcag aaaaaaacca tgggttggga agcttccacc gaacgtatgt
     5821 acccggaaga cggtgctctg aaaggtgaaa tcaaaatgcg tctgaaactg aaagacggtg
     5881 gtcactacga cgctgaagtt aaaaccacct acatggctaa aaaaccggtt cagctgccgg
     5941 gtgcttacaa aaccgacatc aaactggaca tcacctccca caacgaagac tacaccatcg
     6001 ttgaacagta cgaacgtgct gaaggtcgtc actccaccgg tgcttaataa cgctgatagt
     6061 gctagtgtag atcgcgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     6121 ggccaacgcg cggggagagg cggtttgcgt attgggcgca tgcataaaaa ctgttgtaat
     6181 tcattaagca ttctgccgac atggaagcca tcacaaacgg catgatgaac ctgaatcgcc
     6241 agcggcatca gcaccttgtc gccttgcgta taatatttgc ccatgggggt gggcgaagaa
     6301 ctccagcatg agatccccgc gctggaggat catccagccg gcgtcccgga aaacgattcc
     6361 gaagcctcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     6421 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     6481 gacagctatt cctttgccct cggacgagtg ctggggcgtc ggtttccact atcggcgagt
     6541 acttctacac agccatcggt ccagacggcc gcgcttctgc gggcgatttg tgtacgcccg
     6601 acagtcccgg ctccggatcg gacgattgcg tcgcatcgac cctgcgccca agctgcatca
     6661 tcgaaattgc cgtcaaccaa gctctgatag agttggtcaa gaccaatgcg gagcatatac
     6721 gcccggagcc gcggcgatcc tgcaagctcc ggatgcctcc gctcgaagta gcgcgtctgc
     6781 tgctccatac aagccaacca cggcctccag aagaagatgt tggcgacctc gtattgggaa
     6841 tccccgaaca tcgcctcgct ccagtcaatg accgctgtta tgcggccatt gtccgtcagg
     6901 acattgttgg agccgaaatc cgcgtgcacg aggtgccgga cttcggggca gtcctcggcc
     6961 caaagcatca gctcatcgag agcctgcgcg acggacgcac tgacggtgtc gtccatcaca
     7021 gtttgccagt gatacacatg gggatcagca atcgcgcata tgaaatcacg ccatgtagtg
     7081 tattgaccga ttccttgcgg tccgaatggg ccgaacccgc tcgtctggct aagatcggcc
     7141 gcagcgatcg catccatggc ctccgcgacc ggctgcagaa cagcgggcag ttcggtttca
     7201 ggcaggtctt gcaacgtgac accctgtgca cggcgggaga tgcaataggt caggctctcg
     7261 ctgaattccc caatgtcaag cacttccgga atcgggagcg cggccgatgc aaagtgccga
     7321 taaacataac gatctttgta gaaaccatcg gcgcagctat ttacccgcag gacatatcca
     7381 cgccctccta catcgaagct gaaagcacga gattcttcgc cctccgagag ctgcatcagg
     7441 tcggagacgc tgtcgaactt ttcgatcaga aacttctcga cagacgtcgc ggtgagttca
     7501 ggctttttca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     7561 atgagcggat acatatttga atgaattcaa tccatttact atgttatgtt ctgaggggag
     7621 tgaaaattcc gtccagatag cccagtagct gacattcatc cca
//
