LOCUS       sequence-141453-        5708 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Small IncP-based broad host range replicating vector.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5708)
  TITLE     broad host range pcm66t
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5708)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pCM66T: trimmed pCM66 backbone
FEATURES             Location/Qualifiers
     source          1..5708
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      11..642
                     /label=oriV
                     /note="incP origin of replication"
     rep_origin      1105..1693
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1594..1613
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1847..1864
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    1981..2002
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2017..2022,2023..2040,2041..2047)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2055..2071
                     /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     2060..2082
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2079..2095
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2079..2095
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             2091..2414
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSLHACRSTLEDPRVPSSNSLAVVLQRRDWENPGVTQLNR
                     LAAHPPFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCTLSTICS
                     DAA"
     misc_feature    2108..2164
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(2165..2182)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2165..2181)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2174..2196)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2390..2409)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2509..2531
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2532..3347)
                     /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(2532..3347)
                     /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     3255..3274
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3339..3358
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             complement(3633..4781)
                     /codon_start=1
                     /product="trans-acting replication protein that binds to 
                     and activates oriV"
                     /label=trfA
                     /translation="MNRTFDRKAYRQELIDAGFSAEDAETIASRTVMRAPRETFQSVGS
                     MVQQATAKIERDSVQLAPPALPAPSAAVERSRRLEQEAAGLAKSMTIDTRGTMTTKKRK
                     TAGEDLAKQVSEAKQAALLKHTKQQIKEMQLSLFDIAPWPDTMRAMPNDTARSALFTTR
                     NKKIPREALQNKVIFHVNKDVKITYTGVELRADDDELVWQQVLEYAKRTPIGEPITFTF
                     YELCQDLGWSINGRYYTKAEECLSRLQATAMGFTSDRVGHLESVSLLHRFRVLDRGKKT
                     SRCQVLIDEEIVVLFAGDHYTKFIWEKYRKLSPTARRMFDYFSSHREPYPLKLETFRLM
                     CGSDSTRVKKWREQVGEACEELRGSGLVEHAWVNDDLVHCKR"
     CDS             complement(5053..5424)
                     /codon_start=1
                     /gene="traJ"
                     /product="oriT-recognizing protein"
                     /label=traJ
                     /translation="MADETKPTRKGSPPIKVYCLPDERRAIEEKAAAAGMSL*AYLLAV
                     GQGYKITGVVDYEHVRELARINGDLGRLGGLLKLWLTDDPRTARFGDATILALLAKIEE
                     KQDELGKVMMGVVRPRAEP"
     oriT            5457..5566
                     /note="incP origin of transfer"
ORIGIN
        1 gaccctttcc gacgctcacc gggctggttg ccctcgccgc tgggctggcg gccgtctatg
       61 gccctgcaaa cgcgccagaa acgccgtcga agccgtgtgc gagacaccgc ggccgccggc
      121 gttgtggata cctcgcggaa aacttggccc tcactgacag atgaggggcg gacgttgaca
      181 cttgaggggc cgactcaccc ggcgcggcgt tgacagatga ggggcaggct cgatttcggc
      241 cggcgacgtg gagctggcca gcctcgcaaa tcggcgaaaa cgcctgattt tacgcgagtt
      301 tcccacagat gatgtggaca agcctgggga taagtgccct gcggtattga cacttgaggg
      361 gcgcgactac tgacagatga ggggcgcgat ccttgacact tgaggggcag agtgctgaca
      421 gatgaggggc gcacctattg acatttgagg ggctgtccac aggcagaaaa tccagcattt
      481 gcaagggttt ccgcccgttt ttcggccacc gctaacctgt cttttaacct gcttttaaac
      541 caatatttat aaaccttgtt tttaaccagg gctgcgccct gtgcgcgtga ccgcgcacgc
      601 cgaagggggg tgccccccct tctcgaaccc tcccggcccg ctaacgcggg cctcccatcc
      661 ccccaggggc tgcgcccctc ggccgcgaac ggcctcaccc caaaaatggc agccaagctg
      721 accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg
      781 tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat
      841 cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc
      901 tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat
      961 actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt
     1021 tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
     1081 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     1141 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     1201 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt
     1261 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     1321 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     1381 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     1441 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     1501 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
     1561 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
     1621 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
     1681 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
     1741 ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc
     1801 ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag
     1861 cgaggaagcg gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca
     1921 ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat
     1981 taatgtgagt tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg
     2041 tatgttgtgt ggaattgtga gcggataaca atttcacaca ggaaacagct atgaccatga
     2101 ttacgccaag cttgcatgcc tgcaggtcga ctctagagga tccccgggta ccgagctcga
     2161 attcactggc cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta
     2221 atcgccttgc agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg
     2281 atcgcccttc ccaacagttg cgcagcctga atggcgaatg gcgcctgatg cggtattttc
     2341 tccttacgca tctgtgcggt atttcacacc gcatatggtg cactctcagt acaatctgct
     2401 ctgatgccgc atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac
     2461 gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca
     2521 tgtgtcagag gttagaaaaa ctcatcgagc atcaaatgaa actgcaattt attcatatca
     2581 ggattatcaa taccatattt ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg
     2641 aggcagttcc ataggatggc aagatcctgg tatcggtctg cgattccgac tcgtccaaca
     2701 tcaatacaac ctattaattt cccctcgtca aaaataaggt tatcaagtga gaaatcacca
     2761 tgagtgacga ctgaatccgg tgagaatggc aaaagcttat gcatttcttt ccagacttgt
     2821 tcaacaggcc agccattacg ctcgtcatca aaatcactcg catcaaccaa accgttattc
     2881 attcgtgatt gcgcctgagc gagacgaaat acgcgatcgc tgttaaaagg acaattacaa
     2941 acaggaatcg aatgcaaccg gcgcaggaac actgccagcg catcaacaat attttcacct
     3001 gaatcaggat attcttctaa tacctggaat gctgttttcc cggggatcgc agtggtgagt
     3061 aaccatgcat catcaggagt acggataaaa tgcttgatgg tcggaagagg cataaattcc
     3121 gtcagccagt ttagtctgac catctcatct gtaacatcat tggcaacgct acctttgcca
     3181 tgtttcagaa acaactctgg cgcatcgggc ttcccataca atcgatagat tgtcgcacct
     3241 gattgcccga cattatcgcg agcccattta tacccatata aatcagcatc catgttggaa
     3301 tttaatcgcg gcctcgagca agacgtttcc cgttgaatat ggctcataac accccttgta
     3361 ttactgttta tgtaagcaga cagttttatt gttcatgatg atatattttt atcttgtgca
     3421 atgtaacatc agagattttg agacacaacg tggctttccc ccccccccct gcaggtccga
     3481 cacggggatg gatggcgttc ccgatcatgg tcctgcttgc ttcgggagcg atactgagcg
     3541 aagcaagtgc gtcgagcagt gcccgcttgt tcctgaaatg ccagtaaagc gctggctgct
     3601 gaacccccag ccggaactga ccccacaagg ccctagcgtt tgcaatgcac caggtcatca
     3661 ttgacccagg cgtgttccac caggccgctg cctcgcaact cttcgcaggc ttcgccgacc
     3721 tgctcgcgcc acttcttcac gcgggtggaa tccgatccgc acatgaggcg gaaggtttcc
     3781 agcttgagcg ggtacggctc ccggtgcgag ctgaaatagt cgaacatccg tcgggccgtc
     3841 ggcgacagct tgcggtactt ctcccatatg aatttcgtgt agtggtcgcc agcaaacagc
     3901 acgacgattt cctcgtcgat caggacctgg caacgggacg ttttcttgcc acggtccagg
     3961 acgcggaagc ggtgcagcag cgacaccgat tccaggtgcc caacgcggtc ggacgtgaag
     4021 cccatcgccg tcgcctgtag gcgcgacagg cattcctcgg ccttcgtgta ataccggcca
     4081 ttgatcgacc agcccaggtc ctggcaaagc tcgtagaacg tgaaggtgat cggctcgccg
     4141 ataggggtgc gcttcgcgta ctccaacacc tgctgccaca ccagttcgtc atcgtcggcc
     4201 cgcagctcga cgccggtgta ggtgatcttc acgtccttgt tgacgtggaa aatgaccttg
     4261 ttttgcagcg cctcgcgcgg gattttcttg ttgcgcgtgg tgaacagggc agagcgggcc
     4321 gtgtcgtttg gcatcgctcg catcgtgtcc ggccacggcg caatatcgaa caaggaaagc
     4381 tgcatttcct tgatctgctg cttcgtgtgt ttcagcaacg cggcctgctt ggcctcgctg
     4441 acctgttttg ccaggtcctc gccggcggtt tttcgcttct tggtcgtcat agttcctcgc
     4501 gtgtcgatgg tcatcgactt cgccaaacct gccgcctcct gttcgagacg acgcgaacgc
     4561 tccacggcgg ccgatggcgc gggcagggca gggggagcca gttgcacgct gtcgcgctcg
     4621 atcttggccg tagcttgctg gaccatcgag ccgacggact ggaaggtttc gcggggcgca
     4681 cgcatgacgg tgcggcttgc gatggtttcg gcatcctcgg cggaaaaccc cgcgtcgatc
     4741 agttcttgcc tgtatgcctt ccggtcaaac gtccgattca ttcaccctcc ttgcgggatt
     4801 gccccgactc acgccggggc aatgtgccct tattcctgat ttgacccgcc tggtgccttg
     4861 gtgtccagat aatccacctt atcggcaatg aagtcggtcc cgtagaccgt ctggccgtcc
     4921 ttctcgtact tggtattccg aatcttgccc tgcacgaata ccagctccgc gaagtcgctc
     4981 ttcttgatgg agcgcatggg gacgtgcttg gcaatcacgc gcaccccccg gccgttttag
     5041 cggctaaaaa agtcatggct ctgccctcgg gcggaccacg cccatcatga ccttgccaag
     5101 ctcgtcctgc ttctcttcga tcttcgccag cagggcgagg atcgtggcat caccgaaccg
     5161 cgccgtgcgc gggtcgtcgg tgagccagag tttcagcagg ccgcccaggc ggcccaggtc
     5221 gccattgatg cgggccagct cgcggacgtg ctcatagtcc acgacgcccg tgattttgta
     5281 gccctggccg acggccagca ggtaggccta caggctcatg ccggccgccg ccgccttttc
     5341 ctcaatcgct cttcgttcgt ctggaaggca gtacaccttg ataggtgggc tgcccttcct
     5401 ggttggcttg gtttcatcag ccatccgctt gccctcatct gttacgccgg cggtagccgg
     5461 ccagcctcgc agagcaggat tcccgttgag caccgccagg tgcgaataag ggacagtgaa
     5521 gaaggaacac ccgctcgcgg gtgggcctac ttcacctatc ctgcccggct gacgccgttg
     5581 gatacaccaa ggaaagtcta cacgaaccct ttggcaaaat cctgtatatc gtgcgaaaaa
     5641 ggatggatat accgaaaaaa tcgctataat gaccccgaag cagggttatg cagcggaaaa
     5701 gatccgtc
//
