LOCUS       sequence-269076-        3566 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 3566)
  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 3566)
  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..3566
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(86..91,92..107,108..114)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     CDS             153..866
                     /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(183..211)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     806..826
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      878..949
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      965..992
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             complement(1062..1877)
                     /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(1062..1877)
                     /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     1785..1804
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1869..1888
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2124..2895
                     /label=pBBR1 oriV
                     /note="replication origin of the broad-host-range plasmid 
                     pBBR1 from Bordetella bronchiseptica; requires the pBBR1 
                     Rep protein for replication"
     CDS             2896..3558
                     /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 ttgcgcagca acccgtaagt gcgctgttcc agactatcgg ctgtagccgc ctcgccgccc
       61 tataccttgt ctggaactta attaattgac aattaatcat cggctcgtat aatgtgtgga
      121 attgtgagat cttttaagaa ggagatatac atatgcgtaa aggagaagaa cttttcactg
      181 gagttgtccc aattcttgtt gaattagatg gtgatgttaa tgggcacaaa ttttctgtca
      241 gtggagaggg tgaaggtgat gcaacatacg gaaaacttac ccttaaattt atttgcacta
      301 ctggaaaact acctgttcca tggccaacac ttgtcactac tttcggttat ggtgttcaat
      361 gctttgcgag atacccagat catatgaaac agcatgactt tttcaagagt gccatgcccg
      421 aaggttatgt acaggaaaga actatatttt tcaaagatga cgggaactac aagacacgtg
      481 ctgaagtcaa gtttgaaggt gatacccttg ttaatagaat cgagttaaaa ggtattgatt
      541 ttaaagaaga tggaaacatt cttggacaca aattggaata caactataac tcacataatg
      601 tatacatcat ggcagacaaa caaaagaatg gaatcaaagt taacttcaaa attagacaca
      661 acattgaaga tggaagcgtt caactagcag accattatca acaaaatact ccaattggcg
      721 atggccctgt ccttttacca gacaaccatt acctgtccac acaatctgcc ctttcgaaag
      781 atcccaacga aaagagagat cacatggtcc ttcttgagtt tgtaacagct gctgggatta
      841 cacatggcat ggatgaacta tacaaataac caggcatcaa ataaaacgaa aggctcagtc
      901 gaaagactgg gcctttcgtt ttatctgttg tttgtcggtg aacgctctct actagagtca
      961 cactggctca ccttcgggtg ggcctttctg cgaagcggcg cgccgtcccg tcaagtcagc
     1021 gtaatgctct gccagtgtta caaccaatta accaattctg attagaaaaa ctcatcgagc
     1081 atcaaatgaa actgcaattt attcatatca ggattatcaa taccatattt ttgaaaaagc
     1141 cgtttctgta atgaaggaga aaactcaccg aggcagttcc ataggatggc aagatcctgg
     1201 tatcggtctg cgattccgac tcgtccaaca tcaatacaac ctattaattt cccctcgtca
     1261 aaaataaggt tatcaagtga gaaatcacca tgagtgacga ctgaatccgg tgagaatggc
     1321 aaaagcttat gcatttcttt ccagacttgt tcaacaggcc agccattacg ctcgtcatca
     1381 aaatcactcg catcaaccaa accgttattc attcgtgatt gcgcctgagc gagacgaaat
     1441 acgcgatcgc tgttaaaagg acaattacaa acaggaatcg aatgcaaccg gcgcaggaac
     1501 actgccagcg catcaacaat attttcacct gaatcaggat attcttctaa tacctggaat
     1561 gctgttttcc cggggatcgc agtggtgagt aaccatgcat catcaggagt acggataaaa
     1621 tgcttgatgg tcggaagagg cataaattcc gtcagccagt ttagtctgac catctcatct
     1681 gtaacatcat tggcaacgct acctttgcca tgtttcagaa acaactctgg cgcatcgggc
     1741 ttcccataca atcgatagat tgtcgcacct gattgcccga cattatcgcg agcccattta
     1801 tacccatata aatcagcatc catgttggaa tttaatcgcg gcctcgagca agacgtttcc
     1861 cgttgaatat ggctcataac accccttgta ttactgttta tgtaagcaga cagttttatt
     1921 gttcatgatg atatattttt atcttgtgca atgtaacatc agagattttg agacacaacg
     1981 tggatttcca ggtggccggc ccttcgcaaa gtcgtgaccg cctacggcgg ctgcggcgcc
     2041 ctacgggctt gctctccggg cttcgccctg cgcggtcgct gcgctccctt gccagcccgt
     2101 ggatatgtgg acgatggccg cgagcggcca ccggctggct cgcttcgctc ggcccgtgga
     2161 caaccctgct ggacaagctg atggacaggc tgcgcctgcc cacgagcttg accacaggga
     2221 ttgcccaccg gctacccagc cttcgaccac atacccaccg gctccaactg cgcggcctgc
     2281 ggccttgccc catcaatttt tttaattttc tctggggaaa agcctccggc ctgcggcctg
     2341 cgcgcttcgc ttgccggttg gacaccaagt ggaaggcggg tcaaggctcg cgcagcgacc
     2401 gcgcagcggc ttggccttga cgcgcctgga acgacccaag cctatgcgag tgggggcagt
     2461 cgaagggcga agcccgcccg cctgcccccc gagcctcacg gcggcgagtg cgggggttcc
     2521 aagggggcag cgccaccttg ggcaaggccg aaggccgcgc agtcgatcaa caagccccgg
     2581 aggggccact ttttgccgga gggggagccg cgccgaaggc gtgggggaac cccgcagggg
     2641 tgcccttctt tgggcaccaa agaactagat atagggcgaa atgcgaaaga cttaaaaatc
     2701 aacaacttaa aaaagggggg tacgcaacag ctcattgcgg caccccccgc aatagctcat
     2761 tgcgtaggtt aaagaaaatc tgtaattgac tgccactttt acgcaacgca taattgttgt
     2821 cgcgctgccg aaaagttgca gctgattgcg catggtgccg caaccgtgcg gcacccctac
     2881 cgcatggaga taagcatggc cacgcagtcc agagaaatcg gcattcaagc caagaacaag
     2941 cccggtcact gggtgcaaac ggaacgcaaa gcgcatgagg cgtgggccgg gcttattgcg
     3001 aggaaaccca cggcggcaat gctgctgcat cacctcgtgg cgcagatggg ccaccagaac
     3061 gccgtggtgg tcagccagaa gacactttcc aagctcatcg gacgttcttt gcggacggtc
     3121 caatacgcag tcaaggactt ggtggccgag cgctggatct ccgtcgtgaa gctcaacggc
     3181 cccggcaccg tgtcggccta cgtggtcaat gaccgcgtgg cgtggggcca gccccgcgac
     3241 cagttgcgcc tgtcggtgtt cagtgccgcc gtggtggttg atcacgacga ccaggacgaa
     3301 tcgctgttgg ggcatggcga cctgcgccgc atcccgaccc tgtatccggg cgagcagcaa
     3361 ctaccgaccg gccccggcga ggagccgccc agccagcccg gcattccggg catggaacca
     3421 gacctgccag ccttgaccga aacggaggaa tgggaacggc gcgggcagca gcgcctgccg
     3481 atgcccgatg agccgtgttt tctggacgat ggcgagccgt tggagccgcc gacacgggtc
     3541 acgctgccgc gccggtagca cttggg
//
