LOCUS       sequence-259194-        3564 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3564)
  AUTHORS   Azubuike CC, Gatehouse AMR, Howard TP
  TITLE     pCAT vectors overcome inefficient electroporation of Cupriavidus 
            necator H16.
  JOURNAL   N Biotechnol. 2021 Nov 25;65:20-30. doi: 10.1016/j.nbt.2021.07.003. 
            Epub 2021 Jul 29.
  PUBMED    34333160
REFERENCE   2  (bases 1 to 3564)
  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: "N 
            Biotechnol."; date: "2021-11-25"; volume: "65:20-30. doi"; pages: " 
            10.1016/j.nbt.2021.07.003. Epub 2021 Jul 29"
COMMENT     Sequence Label: pCAT218
FEATURES             Location/Qualifiers
     source          1..3564
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(13..18,19..34,35..41)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     CDS             80..793
                     /codon_start=1
                     /product="S65T variant of Aequorea victoria green 
                     fluorescent protein (Heim et al., 1995)"
                     /label=GFP (S65T)
                     /note="excitable with blue light"
                     /translation="MRKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(110..138)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     733..753
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      805..876
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      892..919
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             complement(989..1804)
                     /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(989..1804)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     1712..1731
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1796..1815
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2051..2820
                     /label=pBBR1 oriV
                     /note="replication origin of the broad-host-range plasmid 
                     pBBR1 from Bordetella bronchiseptica; requires the pBBR1 
                     Rep protein for replication"
     CDS             2821..3483
                     /codon_start=1
                     /product="replication protein for the broad-host-range 
                     plasmid pBBR1 from Bordetella bronchiseptica"
                     /label=pBBR1 Rep
                     /translation="MATQSREIGIQAKNKPGHWVQTERKAHEAWAGLIARKPTAAMLLH
                     HLVAQMGHQNAVVVSQKTLSKLIGRSLRTVQYAVKDLVAERWISVVKLNGPGTVSAYVV
                     NDRVAWGQPRDQLRLSVFSAAVVVDHDDQDESLLGHGDLRRIPTLYPGEQQLPTGPGEE
                     PPSQPGIPGMEPDLPALTETEEWERRGQQRLPMPDEPCFLDDGEPLEPPTRVTLPRR"
ORIGIN
        1 gaacttaatt aattgacaat taatcatcgg ctcgtataat gtgtggaatt gtgagatctt
       61 ttaagaagga gatatacata tgcgtaaagg agaagaactt ttcactggag ttgtcccaat
      121 tcttgttgaa ttagatggtg atgttaatgg gcacaaattt tctgtcagtg gagagggtga
      181 aggtgatgca acatacggaa aacttaccct taaatttatt tgcactactg gaaaactacc
      241 tgttccatgg ccaacacttg tcactacttt cggttatggt gttcaatgct ttgcgagata
      301 cccagatcat atgaaacagc atgacttttt caagagtgcc atgcccgaag gttatgtaca
      361 ggaaagaact atatttttca aagatgacgg gaactacaag acacgtgctg aagtcaagtt
      421 tgaaggtgat acccttgtta atagaatcga gttaaaaggt attgatttta aagaagatgg
      481 aaacattctt ggacacaaat tggaatacaa ctataactca cacaatgtat acatcatggc
      541 agacaaacaa aagaatggaa tcaaagttaa cttcaaaatt agacacaaca ttgaagatgg
      601 aagcgttcaa ctagcagacc attatcaaca aaatactcca attggcgatg gccctgtcct
      661 tttaccagac aaccattacc tgtccacaca atctgccctt tcgaaagatc ccaacgaaaa
      721 gagagatcac atggtccttc ttgagtttgt aacagctgct gggattacac atggcatgga
      781 tgaactatac aaataaccag gcatcaaata aaacgaaagg ctcagtcgaa agactgggcc
      841 tttcgtttta tctgttgttt gtcggtgaac gctctctact agagtcacac tggctcacct
      901 tcgggtgggc ctttctgcga agcggcgcgc cgtcccgtca agtcagcgta atgctctgcc
      961 agtgttacaa ccaattaacc aattctgatt agaaaaactc atcgagcatc aaatgaaact
     1021 gcaatttatt catatcagga ttatcaatac catatttttg aaaaagccgt ttctgtaatg
     1081 aaggagaaaa ctcaccgagg cagttccata ggatggcaag atcctggtat cggtctgcga
     1141 ttccgactcg tccaacatca atacaaccta ttaatttccc ctcgtcaaaa ataaggttat
     1201 caagtgagaa atcaccatga gtgacgactg aatccggtga gaatggcaaa agcttatgca
     1261 tttctttcca gacttgttca acaggccagc cattacgctc gtcatcaaaa tcactcgcat
     1321 caaccaaacc gttattcatt cgtgattgcg cctgagcgag acgaaatacg cgatcgctgt
     1381 taaaaggaca attacaaaca ggaatcgaat gcaaccggcg caggaacact gccagcgcat
     1441 caacaatatt ttcacctgaa tcaggatatt cttctaatac ctggaatgct gttttcccgg
     1501 ggatcgcagt ggtgagtaac catgcatcat caggagtacg gataaaatgc ttgatggtcg
     1561 gaagaggcat aaattccgtc agccagttta gtctgaccat ctcatctgta acatcattgg
     1621 caacgctacc tttgccatgt ttcagaaaca actctggcgc atcgggcttc ccatacaatc
     1681 gatagattgt cgcacctgat tgcccgacat tatcgcgagc ccatttatac ccatataaat
     1741 cagcatccat gttggaattt aatcgcggcc tcgagcaaga cgtttcccgt tgaatatggc
     1801 tcataacacc ccttgtatta ctgtttatgt aagcagacag ttttattgtt catgatgata
     1861 tatttttatc ttgtgcaatg taacatcaga gattttgaga cacaacgtgg ctttccaggt
     1921 ggccggccct tcgcaaagtc gtgaccgcct acggcggctg cggcgcccta cgggcttgct
     1981 ctccgggctt cgccctgcgc ggtcgctgcg ctcccttgcc agcccgtgga tatgtggacg
     2041 atggccgcga gcggccaccg gctggctcgc ttcgctcggc ccgtggacaa ccctgctgga
     2101 caagctgatg gacaggctgc gcctgcccac gagcttgacc acagggattg cccaccggct
     2161 acccagcctt cgaccacata cccaccggct ccaactgcgc ggcctgcggc cttgccccat
     2221 caattttttt aattttctct ggggaaaagc ctccggcctg cggcctgcgc gcttcgcttg
     2281 ccggttggac accaagtgga aggcgggtca aggctcgcgc agcgaccgcg cagcggcttg
     2341 gccttgacgc gcctggaacg acccaagcct atgcgagtgg gggcagtcga aggcgaagcc
     2401 cgcccgcctg ccccccgagc ctcacggcgg cgagtgcggg ggttccaagg gggcagcgcc
     2461 accttgggca aggccgaagg ccgcgcagtc gatcaacaag ccccggaggg gccacttttt
     2521 gccggagggg gagccgcgcc gaaggcgtgg gggaaccccg caggggtgcc cttctttggg
     2581 caccaaagaa ctagatatag ggcgaaatgc gaaagactta aaaatcaaca acttaaaaaa
     2641 ggggggtacg caacagctca ttgcggcacc ccccgcaata gctcattgcg taggttaaag
     2701 aaaatctgta attgactgcc acttttacgc aacgcataat tgttgtcgcg ctgccgaaaa
     2761 gttgcagctg attgcgcatg gtgccgcaac cgtgcggcac cctaccgcat ggagataagc
     2821 atggccacgc agtccagaga aatcggcatt caagccaaga acaagcccgg tcactgggtg
     2881 caaacggaac gcaaagcgca tgaggcgtgg gccgggctta ttgcgaggaa acccacggcg
     2941 gcaatgctgc tgcatcacct cgtggcgcag atgggccacc agaacgccgt ggtggtcagc
     3001 cagaagacac tttccaagct catcggacgt tctttgcgga cggtccaata cgcagtcaag
     3061 gacttggtgg ccgagcgctg gatctccgtc gtgaagctca acggccccgg caccgtgtcg
     3121 gcctacgtgg tcaatgaccg cgtggcgtgg ggccagcccc gcgaccagtt gcgcctgtcg
     3181 gtgttcagtg ccgccgtggt ggttgatcac gacgaccagg acgaatcgct gttggggcat
     3241 ggcgacctgc gccgcatccc gaccctgtat ccgggcgagc agcaactacc gaccggcccc
     3301 ggcgaggagc cgcccagcca gcccggcatt ccgggcatgg aaccagacct gccagccttg
     3361 accgaaacgg aggaatggga acggcgcggg cagcagcgcc tgccgatgcc cgatgagccg
     3421 tgttttctgg acgatggcga gccgttggag ccgccgacac gggtcacgct gccgcgccgg
     3481 tagcacttgg gttgcgcagc aacccgtaag tgcgctgttc cagactatcg gctgtagccg
     3541 cctcgccgcc ctataccttg tctg
//
