LOCUS       sequence-332864-        7455 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Broad-Host range expression vector, RK2 and pUC origin, CymRAM 
            repressor, PCymRC promoter controlling GmR, Kanamycin Resistance 
            Marker..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7455)
  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 7455)
  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: pKCyGe2
FEATURES             Location/Qualifiers
     source          1..7455
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(318..1112)
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(429..448)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     1039..1058
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     CDS             complement(1626..2774)
                     /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(3046..3417)
                     /codon_start=1
                     /gene="traJ"
                     /product="oriT-recognizing protein"
                     /label=traJ
                     /translation="MADETKPTRKGSPPIKVYCLPDERRAIEEKAAAAGMSL*AYLLAV
                     GQGYKITGVVDYEHVRELARINGDLGRLGGLLKLWLTDDPRTARFGDATILALLAKIEE
                     KQDELGKVMMGVVRPRAEP"
     oriT            3450..3559
                     /note="incP origin of transfer"
     rep_origin      3712..4343
                     /label=oriV
                     /note="incP origin of replication"
     rep_origin      4806..5394
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5295..5314
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5755..5771
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    6574..6601
                     /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    6632..6659
                     /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        6663..6712
                     /label=sTRSV HHRz
                     /note="hammerhead ribozyme from the tobacco ringspot virus 
                     satellite RNA (Khvorova et al., 2003)"
     CDS             6788..7321
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
ORIGIN
        1 ggtttgcgta ttgggcgcat gcataaaaac tgttgtaatt cattaagcat tctgccgaca
       61 tggaagccat cacaaacggc atgatgaacc tgaatcgcca gcggcatcag caccttgtcg
      121 ccttgcgtat aatatttgcc catgggggtg ggcgaagaac tccagcatga gatccccgcg
      181 ctggaggatc atccagccgg cgtcccggaa aacgattccg aagcccaacc tttcatagaa
      241 ggcggcggtg gaatcgaaat ctcgtgatgg caggttgggc gtcgcttggt cggtcatttc
      301 gaaccccaga gtcccgctca gaagaactcg tcaagaaggc gatagaaggc gatgcgctgc
      361 gaatcgggag cggcgatacc gtaaagcacg aggaagcggt cagcccattc gccgccaagc
      421 tcttcagcaa tatcacgggt agccaacgct atgtcctgat agcggtccgc cacacccagc
      481 cggccacagt cgatgaatcc agaaaagcgg ccattttcca ccatgatatt cggcaagcag
      541 gcatcgccat gggtcacgac gagatcctcg ccgtcgggca tgcgcgcctt gagcctggcg
      601 aacagttcgg ctggcgcgag cccctgatgc tcttcgtcca gatcatcctg atcgacaaga
      661 ccggcttcca tccgagtacg tgctcgctcg atgcgatgtt tcgcttggtg gtcgaatggg
      721 caggtagccg gatcaagcgt atgcagccgc cgcattgcat cagccatgat ggatactttc
      781 tcggcaggag caaggtgaga tgacaggaga tcctgccccg gcacttcgcc caatagcagc
      841 cagtcccttc ccgcttcagt gacaacgtcg agcacagctg cgcaaggaac gcccgtcgtg
      901 gccagccacg atagccgcgc tgcctcgtcc tgcagttcat tcagggcacc ggacaggtcg
      961 gtcttgacaa aaagaaccgg gcgcccctgc gctgacagcc ggaacacggc ggcatcagag
     1021 cagccgattg tctgttgtgc ccagtcatag ccgaatagcc tctccaccca agcggccgga
     1081 gaacctgcgt gcaatccatc ttgttcaatc atgcgaaacg atcctcatcc tgtctcttga
     1141 tcagatcttg atcccctgcg ccatcagatc cttggcggca agaaagccat ccagtttact
     1201 ttgcagggct tcccaacctt accagagggc gccccagctg gcaattccgg ttcgcttgct
     1261 gtccataaaa ccgcccagtc tagctatcgc catgtaagcc cactgcaagc tacctgcttt
     1321 ctctttgcgc ttgcgttttc cattgtccag atagcccagt agctgacatt catcccaggt
     1381 ggcacttttc ggggatcgtg gaaacgatag gcctatgcca tgcgggtcaa ggcgacttcc
     1441 ggcaagctat acgcgcccta gaattgtcaa ttttaatcct ctgtttatcg gcagttcgta
     1501 gagcgcgccg tgcgtcccga gcgatactga gcgaagcaag tgcgtcgagc agtgcccgct
     1561 tgttcctgaa atgccagtaa agcgctggct gctgaacccc cagccggaac tgaccccaca
     1621 aggccctagc gtttgcaatg caccaggtca tcattgaccc aggcgtgttc caccaggccg
     1681 ctgcctcgca actcttcgca ggcttcgccg acctgctcgc gccacttctt cacgcgggtg
     1741 gaatccgatc cgcacatgag gcggaaggtt tccagcttga gcgggtacgg ctcccggtgc
     1801 gagctgaaat agtcgaacat ccgtcgggcc gtcggcgaca gcttgcggta cttctcccat
     1861 atgaatttcg tgtagtggtc gccagcaaac agcacgacga tttcctcgtc gatcaggacc
     1921 tggcaacggg acgttttctt gccacggtcc aggacgcgga agcggtgcag cagcgacacc
     1981 gattccaggt gcccaacgcg gtcggacgtg aagcccatcg ccgtcgcctg taggcgcgac
     2041 aggcattcct cggccttcgt gtaataccgg ccattgatcg accagcccag gtcctggcaa
     2101 agctcgtaga acgtgaaggt gatcggctcg ccgatagggg tgcgcttcgc gtactccaac
     2161 acctgctgcc acaccagttc gtcatcgtcg gcccgcagct cgacgccggt gtaggtgatc
     2221 ttcacgtcct tgttgacgtg gaaaatgacc ttgttttgca gcgcctcgcg cgggattttc
     2281 ttgttgcgcg tggtgaacag ggcagagcgg gccgtgtcgt ttggcatcgc tcgcatcgtg
     2341 tccggccacg gcgcaatatc gaacaaggaa agctgcattt ccttgatctg ctgcttcgtg
     2401 tgtttcagca acgcggcctg cttggcctcg ctgacctgtt ttgccaggtc ctcgccggcg
     2461 gtttttcgct tcttggtcgt catagttcct cgcgtgtcga tggtcatcga cttcgccaaa
     2521 cctgccgcct cctgttcgag acgacgcgaa cgctccacgg cggccgatgg cgcgggcagg
     2581 gcagggggag ccagttgcac gctgtcgcgc tcgatcttgg ccgtagcttg ctggaccatc
     2641 gagccgacgg actggaaggt ttcgcggggc gcacgcatga cggtgcggct tgcgatggtt
     2701 tcggcatcct cggcggaaaa ccccgcgtcg atcagttctt gcctgtatgc cttccggtca
     2761 aacgtccgat tcattcaccc tccttgcggg attgccccga ctcacgccgg ggcaatgtgc
     2821 ccttattcct gatttgaccc gcctggtgcc ttggtgtcca gataatccac cttatcggca
     2881 atgaagtcgg tcccgtagac cgtctggccg tccttctcgt acttggtatt ccgaatcttg
     2941 ccctgcacga ataccagctc cgcgaagtcg ctcttcttga tggagcgcat ggggacgtgc
     3001 ttggcaatca cgcgcacccc ccggccgttt tagcggctaa aaaagtcatg gctctgccct
     3061 cgggcggacc acgcccatca tgaccttgcc aagctcgtcc tgcttctctt cgatcttcgc
     3121 cagcagggcg aggatcgtgg catcaccgaa ccgcgccgtg cgcgggtcgt cggtgagcca
     3181 gagtttcagc aggccgccca ggcggcccag gtcgccattg atgcgggcca gctcgcggac
     3241 gtgctcatag tccacgacgc ccgtgatttt gtagccctgg ccgacggcca gcaggtaggc
     3301 ctacaggctc atgccggccg ccgccgcctt ttcctcaatc gctcttcgtt cgtctggaag
     3361 gcagtacacc ttgataggtg ggctgccctt cctggttggc ttggtttcat cagccatccg
     3421 cttgccctca tctgttacgc cggcggtagc cggccagcct cgcagagcag gattcccgtt
     3481 gagcaccgcc aggtgcgaat aagggacagt gaagaaggaa cacccgctcg cgggtgggcc
     3541 tacttcacct atcctgcccg gctgacgccg ttggatacac caaggaaagt ctacacgaac
     3601 cctttggcaa aatcctgtat atcgtgcgaa aaaggatgga tataccgaaa aaatcgctat
     3661 aatgaccccg aagcagggtt atgcagcgga aaagatccgt cgaccctttc cgacgctcac
     3721 cgggctggtt gccctcgccg ctgggctggc ggccgtctat ggccctgcaa acgcgccaga
     3781 aacgccgtcg aagccgtgtg cgagacaccg cggccgccgg cgttgtggat acctcgcgga
     3841 aaacttggcc ctcactgaca gatgaggggc ggacgttgac acttgagggg ccgactcacc
     3901 cggcgcggcg ttgacagatg aggggcaggc tcgatttcgg ccggcgacgt ggagctggcc
     3961 agcctcgcaa atcggcgaaa acgcctgatt ttacgcgagt ttcccacaga tgatgtggac
     4021 aagcctgggg ataagtgccc tgcggtattg acacttgagg ggcgcgacta ctgacagatg
     4081 aggggcgcga tccttgacac ttgaggggca gagtgctgac agatgagggg cgcacatatt
     4141 gacatttgag gggctgtcca caggcagaaa atccagcatt tgcaagggtt tccgccagtt
     4201 tttcggccac cgctaacctg tcttttaacc tgcttttaaa ccaatattta taaaccttgt
     4261 ttttaaccag ggctgcgccc tgtgcgcgtg accgcgcacg ccgaaggggg gtgccccccc
     4321 ttctcgaacc ctcccggccc gctaacgcgg gcctcccatc cccccagggg ctgcgcccct
     4381 cggccgcgaa cggcctcacc ccaaaaatgg cagccaagct gaccacttct gcgctcggcc
     4441 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt
     4501 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
     4561 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
     4621 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
     4681 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     4741 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     4801 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     4861 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     4921 ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac
     4981 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     5041 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     5101 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     5161 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     5221 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     5281 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     5341 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     5401 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttcgttt aagggcacca
     5461 ataactgcct taaaaaaatt acgccccgcc ctgccactca tcgcagtacg gcctattggt
     5521 taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgctta
     5581 caatttccat tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc
     5641 ttcgctatta cgccagctgg cgaaaggggg atgtgctgca aggcgattaa gttgggtaac
     5701 gccagggttt tcccagtcac gacgttgtaa aacgacggcc actgacacga cgttgtaaaa
     5761 cgacggccag tgatcaacgt ttgaattttg cataacgttc acgtgcaatt tccagggtgc
     5821 tattacgcac acgttcaaaa cgttctttat ctttctgcca caggctacga actaccagac
     5881 cacgaacgct gttaaaaatc agccacagaa tatcttcggc atcatcacgg ctcagaccac
     5941 ggctcaccag cacacccagc cacatatctt caacaacaaa acgattacgt tcaacggtac
     6001 gctgaatacc ttcacgcagt gccggatcac gatctgctgc aacaatcaga tccaggccga
     6061 tgctaaaatc atcatccaga aaaaattctg ctgcatcatc cagcatctgc tgaataacat
     6121 catcttccgg tttcagtttt gccagacgtg cacggctacg ttcggtaatc tgctcataca
     6181 gccattcaaa ggttgccagc agcagttcca gtttggtcgg aaaatgatgg ctctgtgcac
     6241 cacggctaac accggctgca cccggaacat ctgcaatacg aaaacctgca taaccttttt
     6301 cacgcagaac acccagtgct gctgcaatca gtttaccctg ggtttccatt gcacgttctg
     6361 cctgggtacg acgtttcggg ctcataagaa tctccgtttg gtatttacta tgaatattcc
     6421 attgggcgct atcatgccat accgcgaaag gttttgcgcc attcgatggc gcgccgcccc
     6481 aattattgaa ggcctcccta acggggggcc tttttttgtt tctggtctcc cgcttaacga
     6541 tcgttggctg ggatacatat gtcaatgtac cggaacaaac agacaatctg gtctgtttgt
     6601 attatggaaa atttttctgt ataatagatt caacaaacag acaatctggt ctgtttgtaa
     6661 agctgtcacc ggatgtgctt tccggtctga tgagtccgtg aggacgaaac agcctctaca
     6721 aataattttg tttaagaact gggaagagcg cacgtgggtc gatgtttgat gttatggagc
     6781 agcaacgatg ttacgcagca gcaacgatgt tacgcagcag ggcagtcgcc ctaaaacaaa
     6841 gttaggtggc tcaagtatgg gcatcattcg cacatgtagg ctcggccctg accaagtcaa
     6901 atccatgcgg gctgctcttg atcttttcgg tcgtgagttc ggagacgtag ccacctactc
     6961 ccaacatcag ccggactccg attacctcgg gaacttgctc cgtagtaaga cattcatcgc
     7021 gcttgctgcc ttcgaccaag aagcggttgt tggcgctctc gcggcttacg ttctgcccaa
     7081 gtttgagcag ccgcgtagtg agatctatat ctatgatctc gcagtctccg gagagcaccg
     7141 gaggcagggc attgccaccg cgctcatcaa tctcctcaag catgaggcca acgcgcttgg
     7201 tgcttatgtg atctacgtgc aagcagatta cggtgacgat cccgcagtgg ctctctatac
     7261 aaagttgggc atacgggaag aagtgatgca ctttgatatc gacccaagta ccgccaccta
     7321 acaattcgtt caagccgaga tcggcttccc ggccgcggag ttgttcggta aattgtcaca
     7381 acgccgccag ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa
     7441 cgcgcgggga gaggc
//
