LOCUS       sequence-269507-        4162 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 4162)
  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 4162)
  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: pCAT222
FEATURES             Location/Qualifiers
     source          1..4162
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(60..65,66..83,84..90)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    98..114
                     /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)."
     primer_bind     103..125
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     122..138
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     122..138
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             134..499
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWVPGPPSRSTVSISLISNSCSPGDPLV
                     LERPPPRWSSNSPYSESYYARSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEAR
                     TDRPSQQLRSLNGEWKL"
     primer_bind     157..177
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        159..177
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     misc_feature    190..297
                     /label=MCS
                     /note="pBluescript multiple cloning site"
     primer_bind     206..222
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     207..223
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(257..273)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(257..273)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(305..324)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(306..324)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(334..351)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(334..350)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(343..365)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     promoter        648..750
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             751..1410
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(817..836)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             complement(1635..2297)
                     /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      2298..3069
                     /label=pBBR1 oriV
                     /note="replication origin of the broad-host-range plasmid 
                     pBBR1 from Bordetella bronchiseptica; requires the pBBR1 
                     Rep protein for replication"
     primer_bind     complement(3305..3324)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             3316..4131
                     /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             3316..4131
                     /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     complement(3389..3408)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
ORIGIN
        1 agagcattac gctgacttga cgggacggcg cgccgcttca ctcattaggc accccaggct
       61 ttacacttta tgcttccggc tcgtatgttg tgtggaattg tgagcggata acaatttcac
      121 acaggaaaca gctatgacca tgattacgcc aagcgcgcaa ttaaccctca ctaaagggaa
      181 caaaagctgg gtaccgggcc ccccctcgag gtcgacggta tcgataagct tgatatcgaa
      241 ttcctgcagc ccgggggatc cactagttct agagcggccg ccaccgcggt ggagctccaa
      301 ttcgccctat agtgagtcgt attacgcgcg ctcactggcc gtcgttttac aacgtcgtga
      361 ctgggaaaac cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag
      421 ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa
      481 tggcgaatgg aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa
      541 atcagctcat tttttaacca ataggccgac tgcgatgagt taattaagtt cggtgtccct
      601 gttgataccg ggaagccctg ggccaacttt tggcgaaaat gagacgttga tcggcacgta
      661 agaggttcca actttcacca taatgaaata agatcactac cgggcgtatt ttttgagtta
      721 tcgagatttt caggagctaa ggaagctaaa atggagaaaa aaatcactgg atataccacc
      781 gttgatatat cccaatggca tcgtaaagaa cattttgagg catttcagtc agttgctcaa
      841 tgtacctata accagaccgt tcagctggat attacggcct ttttaaagac cgtaaagaaa
      901 aataagcaca agttttatcc ggcctttatt cacattcttg cccgcctgat gaatgctcat
      961 ccggaattcc gtatggcaat gaaagacggt gagctggtga tatgggatag tgttcaccct
     1021 tgttacaccg ttttccatga gcaaactgaa acgttttcat cgctctggag tgaataccac
     1081 gacgatttcc ggcagtttct acacatatat tcgcaagatg tggcgtgtta cggtgaaaac
     1141 ctggcctatt tccctaaagg gtttattgag aatatgtttt tcgtctcagc caatccctgg
     1201 gtgagtttca ccagttttga tttaaacgtg gccaatatgg acaacttctt cgcccccgtt
     1261 ttcaccatgg gcaaatatta tacgcaaggc gacaaggtgc tgatgccgct ggcgattcag
     1321 gttcatcatg ccgtttgtga tggcttccat gtcggcagaa tgcttaatga attacaacag
     1381 tactgcgatg agtggcaggg cggggcgtaa tttttttaag gcagttattg gtgcccttaa
     1441 acgcctggtt gctacgcctg aataagtgat aataagcgga tgaatggcag aaattcgaaa
     1501 gcaaattcga cccggtcgtc ggttcagggc agggtcgtta agtttaaact acccagacaa
     1561 ggtatagggc ggcgaggcgg ctacagccga tagtctggaa cagcgcactt acgggttgct
     1621 gcgcaaccca agtgctaccg gcgcggcagc gtgacccgtg tcggcggctc caacggctcg
     1681 ccatcgtcca gaaaacacgg ctcatcgggc atcggcaggc gctgctgccc gcgccgttcc
     1741 cattcctccg tttcggtcaa ggctggcagg tctggttcca tgcccggaat gccgggctgg
     1801 ctgggcggct cctcgccggg gccggtcggt agttgctgct cgcccggata cagggtcggg
     1861 atgcggcgca ggtcgccatg ccccaacagc gattcgtcct ggtcgtcgtg atcaaccacc
     1921 acggcggcac tgaacaccga caggcgcaac tggtcgcggg gctggcccca cgccacgcgg
     1981 tcattgacca cgtaggccga cacggtgccg gggccgttga gcttcacgac ggagatccag
     2041 cgctcggcca ccaagtcctt gactgcgtat tggaccgtcc gcaaagaacg tccgatgagc
     2101 ttggaaagtg tcttctggct gaccaccacg gcgttctggt ggcccatctg cgccacgagg
     2161 tgatgcagca gcattgccgc cgtgggtttc ctcgcaataa gcccggccca cgcctcatgc
     2221 gctttgcgtt ccgtttgcac ccagtgaccg ggcttgttct tggcttgaat gccgatttct
     2281 ctggactgcg tggccatgct tatctccatg cggtaggggt gccgcacggt tgcggcacca
     2341 tgcgcaatca gctgcaactt ttcggcagcg cgacaacaat tatgcgttgc gtaaaagtgg
     2401 cagtcaatta cagattttct ttaacctacg caatgagcta ttgcgggggg tgccgcaatg
     2461 agctgttgcg tacccccctt ttttaagttg ttgattttta agtctttcgc atttcgccct
     2521 atatctagtt ctttggtgcc caaagaaggg cacccctgcg gggttccccc acgccttcgg
     2581 cgcggctccc cctccggcaa aaagtggccc ctccggggct tgttgatcga ctgcgcggcc
     2641 ttcggccttg cccaaggtgg cgctgccccc ttggaacccc cgcactcgcc gccgtgaggc
     2701 tcggggggca ggcgggcggg cttcgccctt cgactgcccc cactcgcata ggcttgggtc
     2761 gttccaggcg cgtcaaggcc aagccgctgc gcggtcgctg cgcgagcctt gacccgcctt
     2821 ccacttggtg tccaaccggc aagcgaagcg cgcaggccgc aggccggagg cttttcccca
     2881 gagaaaatta aaaaaattga tggggcaagg ccgcaggccg cgcagttgga gccggtgggt
     2941 atgtggtcga aggctgggta gccggtgggc aatccctgtg gtcaagctcg tgggcaggcg
     3001 cagcctgtcc atcagcttgt ccagcagggt tgtccacggg ccgagcgaag cgagccagcc
     3061 ggtggccgct cgcggccatc gtccacatat ccacgggctg gcaagggagc gcagcgaccg
     3121 cgcagggcga agcccggaga gcaagcccgt agggcgccgc agccgccgta ggcggtcacg
     3181 actttgcgaa gggccggcca cctggaaagc cacgttgtgt ctcaaaatct ctgatgttac
     3241 attgcacaag ataaaaatat atcatcatga acaataaaac tgtctgctta cataaacagt
     3301 aatacaaggg gtgttatgag ccatattcaa cgggaaacgt cttgctcgag gccgcgatta
     3361 aattccaaca tggatgctga tttatatggg tataaatggg ctcgcgataa tgtcgggcaa
     3421 tcaggtgcga caatctatcg attgtatggg aagcccgatg cgccagagtt gtttctgaaa
     3481 catggcaaag gtagcgttgc caatgatgtt acagatgaga tggtcagact aaactggctg
     3541 acggaattta tgcctcttcc gaccatcaag cattttatcc gtactcctga tgatgcatgg
     3601 ttactcacca ctgcgatccc cgggaaaaca gcattccagg tattagaaga atatcctgat
     3661 tcaggtgaaa atattgttga tgcgctggca gtgttcctgc gccggttgca ttcgattcct
     3721 gtttgtaatt gtccttttaa cagcgatcgc gtatttcgtc tcgctcaggc gcaatcacga
     3781 atgaataacg gtttggttga tgcgagtgat tttgatgacg agcgtaatgg ctggcctgtt
     3841 gaacaagtct ggaaagaaat gcataagctt ttgccattct caccggattc agtcgtcact
     3901 catggtgatt tctcacttga taaccttatt tttgacgagg ggaaattaat aggttgtatt
     3961 gatgttggac gagtcggaat cgcagaccga taccaggatc ttgccatcct atggaactgc
     4021 ctcggtgagt tttctccttc attacagaaa cggctttttc aaaaatatgg tattgataat
     4081 cctgatatga ataaattgca gtttcatttg atgctcgatg agtttttcta atcagaattg
     4141 gttaattggt tgtaacactg gc
//
