LOCUS       sequence-269101-        3600 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 3600)
  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 3600)
  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: pCAT221
FEATURES             Location/Qualifiers
     source          1..3600
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             81..794
                     /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(111..139)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     734..754
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      806..877
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      893..920
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             complement(990..1805)
                     /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(990..1805)
                     /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     1713..1732
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1797..1816
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2052..2823
                     /label=pBBR1 oriV
                     /note="replication origin of the broad-host-range plasmid 
                     pBBR1 from Bordetella bronchiseptica; requires the pBBR1 
                     Rep protein for replication"
     CDS             2824..3486
                     /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 tgacacaggt ggaaatttag aataacttgt tagtaaacct aatggatcga ccttagatct
       61 tttaagaagg agatatacat atgcgtaaag gagaagaact tttcactgga gttgtcccaa
      121 ttcttgttga attagatggt gatgttaatg ggcacaaatt ttctgtcagt ggagagggtg
      181 aaggtgatgc aacatacgga aaacttaccc ttaaatttat ttgcactact ggaaaactac
      241 ctgttccatg gccaacactt gtcactactt tcggttatgg tgttcaatgc tttgcgagat
      301 acccagatca tatgaaacag catgactttt tcaagagtgc catgcccgaa ggttatgtac
      361 aggaaagaac tatatttttc aaagatgacg ggaactacaa gacacgtgct gaagtcaagt
      421 ttgaaggtga tacccttgtt aatagaatcg agttaaaagg tattgatttt aaagaagatg
      481 gaaacattct tggacacaaa ttggaataca actataactc acacaatgta tacatcatgg
      541 cagacaaaca aaagaatgga atcaaagtta acttcaaaat tagacacaac attgaagatg
      601 gaagcgttca actagctgac cattatcaac aaaatactcc aattggcgat ggccctgtcc
      661 ttttaccaga caaccattac ctgtccacac aatctgccct ttcgaaagat cccaacgaaa
      721 agagagatca catggtcctt cttgagtttg taacagctgc tgggattaca catggcatgg
      781 atgaactata caaataacca ggcatcaaat aaaacgaaag gctcagtcga aagactgggc
      841 ctttcgtttt atctgttgtt tgtcggtgaa cgctctctac tagagtcaca ctggctcacc
      901 ttcgggtggg cctttctgcg aagcggcgcg ccgtcccgtc aagtcagcgt aatgctctgc
      961 cagtgttaca accaattaac caattctgat tagaaaaact catcgagcat caaatgaaac
     1021 tgcaatttat tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat
     1081 gaaggagaaa actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg
     1141 attccgactc gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta
     1201 tcaagtgaga aatcaccatg agtgacgact gaatccggtg agaatggcaa aagcttatgc
     1261 atttctttcc agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca
     1321 tcaaccaaac cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg
     1381 ttaaaaggac aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca
     1441 tcaacaatat tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg
     1501 gggatcgcag tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc
     1561 ggaagaggca taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg
     1621 gcaacgctac ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat
     1681 cgatagattg tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa
     1741 tcagcatcca tgttggaatt taatcgcggc ctcgagcaag acgtttcccg ttgaatatgg
     1801 ctcataacac cccttgtatt actgtttatg taagcagaca gttttattgt tcatgatgat
     1861 atatttttat cttgtgcaat gtaacatcag agattttgag acacaacgtg gctttccagg
     1921 tggccggccc ttcgcaaagt cgtgaccgcc tacggcggct gcggcgccct acgggcttgc
     1981 tctccgggct tcgccctgcg cggtcgctgc gctcccttgc cagcccgtgg atatgtggac
     2041 gatggccgcg agcggccacc ggctggctcg cttcgctcgg cccgtggaca accctgctgg
     2101 acaagctgat ggacaggctg cgcctgccca cgagcttgac cacagggatt gcccaccggc
     2161 tacccagcct tcgaccacat acccaccggc tccaactgcg cggcctgcgg ccttgcccca
     2221 tcaatttttt taattttctc tggggaaaag cctccggcct gcggcctgcg cgcttcgctt
     2281 gccggttgga caccaagtgg aaggcgggtc aaggctcgcg cagcgaccgc gcagcggctt
     2341 ggccttgacg cgcctggaac gacccaagcc tatgcgagtg ggggcagtcg aagggcgaag
     2401 cccgcccgcc tgccccccga gcctcacggc ggcgagtgcg ggggttccaa gggggcagcg
     2461 ccaccttggg caaggccgaa ggccgcgcag tcgatcaaca agccccggag gggccacttt
     2521 ttgccggagg gggagccgcg ccgaaggcgt gggggaaccc cgcaggggtg cccttctttg
     2581 ggcaccaaag aactagatat agggcgaaat gcgaaagact taaaaatcaa caacttaaaa
     2641 aaggggggta cgcaacagct cattgcggca ccccccgcaa tagctcattg cgtaggttaa
     2701 agaaaatctg taattgactg ccacttttac gcaacgcata attgttgtcg cgctgccgaa
     2761 aagttgcagc tgattgcgca tggtgccgca accgtgcggc acccctaccg catggagata
     2821 agcatggcca cgcagtccag agaaatcggc attcaagcca agaacaagcc cggtcactgg
     2881 gtgcaaacgg aacgcaaagc gcatgaggcg tgggccgggc ttattgcgag gaaacccacg
     2941 gcggcaatgc tgctgcatca cctcgtggcg cagatgggcc accagaacgc cgtggtggtc
     3001 agccagaaga cactttccaa gctcatcgga cgttctttgc ggacggtcca atacgcagtc
     3061 aaggacttgg tggccgagcg ctggatctcc gtcgtgaagc tcaacggccc cggcaccgtg
     3121 tcggcctacg tggtcaatga ccgcgtggcg tggggccagc cccgcgacca gttgcgcctg
     3181 tcggtgttca gtgccgccgt ggtggttgat cacgacgacc aggacgaatc gctgttgggg
     3241 catggcgacc tgcgccgcat cccgaccctg tatccgggcg agcagcaact accgaccggc
     3301 cccggcgagg agccgcccag ccagcccggc attccgggca tggaaccaga cctgccagcc
     3361 ttgaccgaaa cggaggaatg ggaacggcgc gggcagcagc gcctgccgat gcccgatgag
     3421 ccgtgttttc tggacgatgg cgagccgttg gagccgccga cacgggtcac gctgccgcgc
     3481 cggtagcact tgggttgcgc agcaacccgt aagtgcgctg ttccagacta tcggctgtag
     3541 ccgcctcgcc gccctatacc ttgtctggaa cttaattaaa gcggatataa aaaccgttat
//
