LOCUS       sequence-259200-        3891 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 3891)
  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 3891)
  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: pCAT224
FEATURES             Location/Qualifiers
     source          1..3891
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(93..458)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWVPGPPSRSTVSISLISNSCSPGDPLV
                     LERPPPRWSSNSPYSESYYARSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEAR
                     TDRPSQQLRSLNGEWKL"
     primer_bind     227..249
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     241..258
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     242..258
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     268..287
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        268..286
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    295..402
                     /label=MCS
                     /note="pBluescript multiple cloning site"
     primer_bind     319..335
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     319..335
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(369..385)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(370..386)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     complement(415..435)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(415..433)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(454..470)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(454..470)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(467..489)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    478..494
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(502..508,509..526,527..532))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     CDS             complement(623..1438)
                     /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(623..1438)
                     /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     1346..1365
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1430..1449
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1685..2454
                     /label=pBBR1 oriV
                     /note="replication origin of the broad-host-range plasmid 
                     pBBR1 from Bordetella bronchiseptica; requires the pBBR1 
                     Rep protein for replication"
     CDS             2455..3117
                     /codon_start=1
                     /product="replication protein for the broad-host-range 
                     plasmid pBBR1 from Bordetella bronchiseptica"
                     /label=pBBR1 Rep
                     /translation="MATQSREIGIQAKNKPGHWVQTERKAHEAWAGLIARKPTAAMLLH
                     HLVAQMGHQNAVVVSQKTLSKLIGRSLRTVQYAVKDLVAERWISVVKLNGPGTVSAYVV
                     NDRVAWGQPRDQLRLSVFSAAVVVDHDDQDESLLGHGDLRRIPTLYPGEQQLPTGPGEE
                     PPSQPGIPGMEPDLPALTETEEWERRGQQRLPMPDEPCFLDDGEPLEPPTRVTLPRR"
     rep_origin      complement(3268..3856)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3348..3367)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gaacttaatt aactcatcgc agtcggccta ttggttaaaa aatgagctga tttaacaaaa
       61 atttaacgcg aattttaaca aaatattaac gcttacaatt tccattcgcc attcaggctg
      121 cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc tattacgcca gctggcgaaa
      181 gggggatgtg ctgcaaggcg attaagttgg gtaacgccag ggttttccca gtcacgacgt
      241 tgtaaaacga cggccagtga gcgcgcgtaa tacgactcac tatagggcga attggagctc
      301 caccgcggtg gcggccgctc tagaactagt ggatcccccg ggctgcagga attcgatatc
      361 aagcttatcg ataccgtcga cctcgagggg gggcccggta cccagctttt gttcccttta
      421 gtgagggtta attgcgcgct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg
      481 ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg
      541 tgcctaatga gtgaagcggc gcgccgtccc gtcaagtcag cgtaatgctc tgccagtgtt
      601 acaaccaatt aaccaattct gattagaaaa actcatcgag catcaaatga aactgcaatt
      661 tattcatatc aggattatca ataccatatt tttgaaaaag ccgtttctgt aatgaaggag
      721 aaaactcacc gaggcagttc cataggatgg caagatcctg gtatcggtct gcgattccga
      781 ctcgtccaac atcaatacaa cctattaatt tcccctcgtc aaaaataagg ttatcaagtg
      841 agaaatcacc atgagtgacg actgaatccg gtgagaatgg caaaagctta tgcatttctt
      901 tccagacttg ttcaacaggc cagccattac gctcgtcatc aaaatcactc gcatcaacca
      961 aaccgttatt cattcgtgat tgcgcctgag cgagacgaaa tacgcgatcg ctgttaaaag
     1021 gacaattaca aacaggaatc gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa
     1081 tattttcacc tgaatcagga tattcttcta atacctggaa tgctgttttc ccggggatcg
     1141 cagtggtgag taaccatgca tcatcaggag tacggataaa atgcttgatg gtcggaagag
     1201 gcataaattc cgtcagccag tttagtctga ccatctcatc tgtaacatca ttggcaacgc
     1261 tacctttgcc atgtttcaga aacaactctg gcgcatcggg cttcccatac aatcgataga
     1321 ttgtcgcacc tgattgcccg acattatcgc gagcccattt atacccatat aaatcagcat
     1381 ccatgttgga atttaatcgc ggcctcgagc aagacgtttc ccgttgaata tggctcataa
     1441 caccccttgt attactgttt atgtaagcag acagttttat tgttcatgat gatatatttt
     1501 tatcttgtgc aatgtaacat cagagatttt gagacacaac gtggctttcc aggtggccgg
     1561 cccttcgcaa agtcgtgacc gcctacggcg gctgcggcgc cctacgggct tgctctccgg
     1621 gcttcgccct gcgcggtcgc tgcgctccct tgccagcccg tggatatgtg gacgatggcc
     1681 gcgagcggcc accggctggc tcgcttcgct cggcccgtgg acaaccctgc tggacaagct
     1741 gatggacagg ctgcgcctgc ccacgagctt gaccacaggg attgcccacc ggctacccag
     1801 ccttcgacca catacccacc ggctccaact gcgcggcctg cggccttgcc ccatcaattt
     1861 ttttaatttt ctctggggaa aagcctccgg cctgcggcct gcgcgcttcg cttgccggtt
     1921 ggacaccaag tggaaggcgg gtcaaggctc gcgcagcgac cgcgcagcgg cttggccttg
     1981 acgcgcctgg aacgacccaa gcctatgcga gtgggggcag tcgaaggcga agcccgcccg
     2041 cctgcccccc gagcctcacg gcggcgagtg cgggggttcc aagggggcag cgccaccttg
     2101 ggcaaggccg aaggccgcgc agtcgatcaa caagccccgg aggggccact ttttgccgga
     2161 gggggagccg cgccgaaggc gtgggggaac cccgcagggg tgcccttctt tgggcaccaa
     2221 agaactagat atagggcgaa atgcgaaaga cttaaaaatc aacaacttaa aaaagggggg
     2281 tacgcaacag ctcattgcgg caccccccgc aatagctcat tgcgtaggtt aaagaaaatc
     2341 tgtaattgac tgccactttt acgcaacgca taattgttgt cgcgctgccg aaaagttgca
     2401 gctgattgcg catggtgccg caaccgtgcg gcaccctacc gcatggagat aagcatggcc
     2461 acgcagtcca gagaaatcgg cattcaagcc aagaacaagc ccggtcactg ggtgcaaacg
     2521 gaacgcaaag cgcatgaggc gtgggccggg cttattgcga ggaaacccac ggcggcaatg
     2581 ctgctgcatc acctcgtggc gcagatgggc caccagaacg ccgtggtggt cagccagaag
     2641 acactttcca agctcatcgg acgttctttg cggacggtcc aatacgcagt caaggacttg
     2701 gtggccgagc gctggatctc cgtcgtgaag ctcaacggcc ccggcaccgt gtcggcctac
     2761 gtggtcaatg accgcgtggc gtggggccag ccccgcgacc agttgcgcct gtcggtgttc
     2821 agtgccgccg tggtggttga tcacgacgac caggacgaat cgctgttggg gcatggcgac
     2881 ctgcgccgca tcccgaccct gtatccgggc gagcagcaac taccgaccgg ccccggcgag
     2941 gagccgccca gccagcccgg cattccgggc atggaaccag acctgccagc cttgaccgaa
     3001 acggaggaat gggaacggcg cgggcagcag cgcctgccga tgcccgatga gccgtgtttt
     3061 ctggacgatg gcgagccgtt ggagccgccg acacgggtca cgctgccgcg ccggtagcac
     3121 ttgggttgcg cagcaacccg taagtgcgct gttccagact atcggctgta gccgcctcgc
     3181 cgccctatac cttgtctggg tagtttaaac gtgagcaaaa ggccagcaaa aggccaggaa
     3241 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     3301 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     3361 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     3421 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     3481 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     3541 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     3601 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     3661 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg
     3721 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     3781 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     3841 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg c
//
