LOCUS       sequence-269099-        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 13 to 47)
  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: pCAT220
FEATURES             Location/Qualifiers
     source          1..3600
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        13..47
                     /label=﻿BBa_J23100
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 1.0
                     "
                     /citation=[1]
     CDS             78..791
                     /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(108..136)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     731..751
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      807..878
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      894..921
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             complement(995..1810)
                     /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(995..1810)
                     /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     1718..1737
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1802..1821
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2057..2828
                     /label=pBBR1 oriV
                     /note="replication origin of the broad-host-range plasmid 
                     pBBR1 from Bordetella bronchiseptica; requires the pBBR1 
                     Rep protein for replication"
     CDS             2829..3491
                     /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 ctcaatctct atttgacggc tagctcagtc ctaggtacag tgctagcgta ctttaacttt
       61 aagaaggaga tatacatatg cgtaaaggag aagaactttt cactggagtt gtcccaattc
      121 ttgttgaatt agatggtgat gttaatgggc acaaattttc tgtcagtgga gagggtgaag
      181 gtgatgcaac atacggaaaa cttaccctta aatttatttg cactactgga aaactacctg
      241 ttccatggcc aacacttgtc actactttcg gttatggtgt tcaatgcttt gcgagatacc
      301 cagatcatat gaaacagcat gactttttca agagtgccat gcccgaaggt tatgtacagg
      361 aaagaactat atttttcaaa gatgacggga actacaagac acgtgctgaa gtcaagtttg
      421 aaggtgatac ccttgttaat agaatcgagt taaaaggtat tgattttaaa gaagatggaa
      481 acattcttgg acacaaattg gaatacaact ataactcaca caatgtatac atcatggcag
      541 acaaacaaaa gaatggaatc aaagttaact tcaaaattag acacaacatt gaagatggaa
      601 gcgttcaact agcagaccat tatcaacaaa atactccaat tggcgatggc cctgtccttt
      661 taccagacaa ccattacctg tccacacaat ctgccctttc gaaagatccc aacgaaaaga
      721 gagatcacat ggtccttctt gagtttgtaa cagctgctgg gattacacat ggcatggatg
      781 aactatacaa ataatcgacc aggcatcaaa taaaacgaaa ggctcagtcg aaagactggg
      841 cctttcgttt tatctgttgt ttgtcggtga acgctctcta ctagagtcac actggctcac
      901 cttcgggtgg gcctttctgc gtttaaagcg gcgcgccgtc ccgtcaagtc agcgtaatgc
      961 tctgccagtg ttacaaccaa ttaaccaatt ctgattagaa aaactcatcg agcatcaaat
     1021 gaaactgcaa tttattcata tcaggattat caataccata tttttgaaaa agccgtttct
     1081 gtaatgaagg agaaaactca ccgaggcagt tccataggat ggcaagatcc tggtatcggt
     1141 ctgcgattcc gactcgtcca acatcaatac aacctattaa tttcccctcg tcaaaaataa
     1201 ggttatcaag tgagaaatca ccatgagtga cgactgaatc cggtgagaat ggcaaaagct
     1261 tatgcatttc tttccagact tgttcaacag gccagccatt acgctcgtca tcaaaatcac
     1321 tcgcatcaac caaaccgtta ttcattcgtg attgcgcctg agcgagacga aatacgcgat
     1381 cgctgttaaa aggacaatta caaacaggaa tcgaatgcaa ccggcgcagg aacactgcca
     1441 gcgcatcaac aatattttca cctgaatcag gatattcttc taatacctgg aatgctgttt
     1501 tcccggggat cgcagtggtg agtaaccatg catcatcagg agtacggata aaatgcttga
     1561 tggtcggaag aggcataaat tccgtcagcc agtttagtct gaccatctca tctgtaacat
     1621 cattggcaac gctacctttg ccatgtttca gaaacaactc tggcgcatcg ggcttcccat
     1681 acaatcgata gattgtcgca cctgattgcc cgacattatc gcgagcccat ttatacccat
     1741 ataaatcagc atccatgttg gaatttaatc gcggcctcga gcaagacgtt tcccgttgaa
     1801 tatggctcat aacacccctt gtattactgt ttatgtaagc agacagtttt attgttcatg
     1861 atgatatatt tttatcttgt gcaatgtaac atcagagatt ttgagacaca acgtggcttt
     1921 ccaggtggcc ggcccttcgc aaagtcgtga ccgcctacgg cggctgcggc gccctacggg
     1981 cttgctctcc gggcttcgcc ctgcgcggtc gctgcgctcc cttgccagcc cgtggatatg
     2041 tggacgatgg ccgcgagcgg ccaccggctg gctcgcttcg ctcggcccgt ggacaaccct
     2101 gctggacaag ctgatggaca ggctgcgcct gcccacgagc ttgaccacag ggattgccca
     2161 ccggctaccc agccttcgac cacataccca ccggctccaa ctgcgcggcc tgcggccttg
     2221 ccccatcaat ttttttaatt ttctctgggg aaaagcctcc ggcctgcggc ctgcgcgctt
     2281 cgcttgccgg ttggacacca agtggaaggc gggtcaaggc tcgcgcagcg accgcgcagc
     2341 ggcttggcct tgacgcgcct ggaacgaccc aagcctatgc gagtgggggc agtcgaaggg
     2401 cgaagcccgc ccgcctgccc cccgagcctc acggcggcga gtgcgggggt tccaaggggg
     2461 cagcgccacc ttgggcaagg ccgaaggccg cgcagtcgat caacaagccc cggaggggcc
     2521 actttttgcc ggagggggag ccgcgccgaa ggcgtggggg aaccccgcag gggtgccctt
     2581 ctttgggcac caaagaacta gatatagggc gaaatgcgaa agacttaaaa atcaacaact
     2641 taaaaaaggg gggtacgcaa cagctcattg cggcaccccc cgcaatagct cattgcgtag
     2701 gttaaagaaa atctgtaatt gactgccact tttacgcaac gcataattgt tgtcgcgctg
     2761 ccgaaaagtt gcagctgatt gcgcatggtg ccgcaaccgt gcggcacccc taccgcatgg
     2821 agataagcat ggccacgcag tccagagaaa tcggcattca agccaagaac aagcccggtc
     2881 actgggtgca aacggaacgc aaagcgcatg aggcgtgggc cgggcttatt gcgaggaaac
     2941 ccacggcggc aatgctgctg catcacctcg tggcgcagat gggccaccag aacgccgtgg
     3001 tggtcagcca gaagacactt tccaagctca tcggacgttc tttgcggacg gtccaatacg
     3061 cagtcaagga cttggtggcc gagcgctgga tctccgtcgt gaagctcaac ggccccggca
     3121 ccgtgtcggc ctacgtggtc aatgaccgcg tggcgtgggg ccagccccgc gaccagttgc
     3181 gcctgtcggt gttcagtgcc gccgtggtgg ttgatcacga cgaccaggac gaatcgctgt
     3241 tggggcatgg cgacctgcgc cgcatcccga ccctgtatcc gggcgagcag caactaccga
     3301 ccggccccgg cgaggagccg cccagccagc ccggcattcc gggcatggaa ccagacctgc
     3361 cagccttgac cgaaacggag gaatgggaac ggcgcgggca gcagcgcctg ccgatgcccg
     3421 atgagccgtg ttttctggac gatggcgagc cgttggagcc gccgacacgg gtcacgctgc
     3481 cgcgccggta gcacttgggt tgcgcagcaa cccgtaagtg cgctgttcca gactatcggc
     3541 tgtagccgcc tcgccgccct ataccttgtc tggaacttaa ttaagccgct tctagaacgt
//
