LOCUS       sequence-64399-e        8120 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8120)
  AUTHORS   Marx CJ, Lidstrom ME
  TITLE     Development of improved versatile broad-host-range vectors for use 
            in methylotrophs and other Gram-negative bacteria.
  JOURNAL   Microbiology. 2001 Aug;147(Pt 8):2065-75.
  PUBMED    11495985
REFERENCE   2  (bases 1 to 8120)
  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: 
            "Microbiology."; date: "2001-08"; volume: "147(Pt 8)"; pages: 
            "2065-75"
COMMENT     Sequence Label: pCM66
FEATURES             Location/Qualifiers
     source          1..8120
                     /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"
     promoter        2115..2133
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     2115..2132
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     misc_feature    2140..2196
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(2198..2217)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2199..2217)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2224..2241)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2224..2240)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2233..2255)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2381..2836
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2468..2487)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2678..2699
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2903..2922)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3022..3044
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3082..3100)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     CDS             complement(4558..5373)
                     /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(4558..5373)
                     /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     5281..5300
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5365..5384
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             complement(6045..7193)
                     /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(7465..7836)
                     /codon_start=1
                     /gene="traJ"
                     /product="oriT-recognizing protein"
                     /label=traJ
                     /translation="MADETKPTRKGSPPIKVYCLPDERRAIEEKAAAAGMSL*AYLLAV
                     GQGYKITGVVDYEHVRELARINGDLGRLGGLLKLWLTDDPRTARFGDATILALLAKIEE
                     KQDELGKVMMGVVRPRAEP"
     oriT            7869..7978
                     /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 tccttctagt
     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 ttacgcgcca agctatttag gtgacactat agaatactca agcttgcatg cctgcaggtc
     2161 gactctagag gatccccggg taccgagctc gaattcgccc tatagtgagt cgtattacaa
     2221 ttcactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa
     2281 tcgccttgca gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga
     2341 tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg acgcgccctg tagcggcgca
     2401 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta
     2461 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
     2521 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
     2581 cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt
     2641 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga
     2701 acaacactca accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg
     2761 gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata
     2821 ttaacgctta caatttcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac
     2881 accgcatatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagcccc
     2941 gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt
     3001 acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac
     3061 cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga
     3121 taataatggt ttcttagcac cctttctcgg tccttcaacg ttcctgacaa cgagcctcct
     3181 tttcgccaat ccatcgacaa tcaccgcgag tccctgctcg aacgctgcgt ccggaccggc
     3241 ttcgtcgaag gcgtctatcg cggcccgcaa cagcggcgag agcggagcct gttcaacggt
     3301 gccgccgcgc tcgccggcat cgctgtcgcc ggcctgctcc tcaagcacgg ccccaacagt
     3361 gaagtagctg attgtcatca gcgcattgac ggcgtccccg gccgaaaaac ccgcctcgca
     3421 gaggaagcga agctgcgcgt cggccgtttc catctgcggt gcgcccggtc gcgtgccggc
     3481 atggatgcgc gcgccatcgc ggtaggcgag cagcgcctgc ctgaagctgc gggcattccc
     3541 gatcagaaat gagcgccagt cgtcgtcggc tctcggcacc gaatgcgtat gattctccgc
     3601 cagcatggct tcggccagtg cgtcgagcag cgcccgcttg ttcctgaagt gccagtaaag
     3661 cgccggctgc tgaaccccca accgttccgc cagtttgcgt gtcgtcagac cgtctacgcc
     3721 gacctcgttc aacaggtcca gggcggcacg gatcactgta ttcggctgca actttgtcat
     3781 gattgacact ttatcactga taaacataat atgtccacca acttatcagt gataaagaat
     3841 ccgcgcgttc aatcggacca gcggaggctg gtccggaggc cagacgtgaa acccaacata
     3901 cccctgatcg taattctgag cactgtcgcg ctcgacgctg tcggcatcgg cctgattatg
     3961 ccggtgctgc cgggcctcct gcgcgatctg gttcactcga acgacgtcac cgcccactat
     4021 ggcattctgc tggcgctgta tgcgttggtg caatttgcct gcgcacctgt gctgggcgcg
     4081 ctgtcggatc gtttcgggcg gcggccaatc ttgctcgtct cgctggccgg cgccactgtc
     4141 gactacgcca tcatggcgac agcgcctttc ctttgggttc tctatatcgg gcggatcgtg
     4201 gccggcatca ccggggcgac tggggcggta gccggcgctt atattgccga tgacctgcag
     4261 gggggggggg gcgctgaggt ctgcctcgtg aagaaggtgt tgctgactca taccaggcct
     4321 gaatcgcccc atcatccagc cagaaagtga gggagccacg gttgatgaga gctttgttgt
     4381 aggtggacca gttggtgatt ttgaactttt gctttgccac ggaacggtct gcgttgtcgg
     4441 gaagatgcgt gatctgatcc ttcaactcag caaaagttcg atttattcaa caaagccgcc
     4501 gtcccgtcaa gtcagcgtaa tgctctgcca gtgttacaac caattaacca attctgatta
     4561 gaaaaactca tcgagcatca aatgaaactg caatttattc atatcaggat tatcaatacc
     4621 atatttttga aaaagccgtt tctgtaatga aggagaaaac tcaccgaggc agttccatag
     4681 gatggcaaga tcctggtatc ggtctgcgat tccgactcgt ccaacatcaa tacaacctat
     4741 taatttcccc tcgtcaaaaa taaggttatc aagtgagaaa tcaccatgag tgacgactga
     4801 atccggtgag aatggcaaaa gcttatgcat ttctttccag acttgttcaa caggccagcc
     4861 attacgctcg tcatcaaaat cactcgcatc aaccaaaccg ttattcattc gtgattgcgc
     4921 ctgagcgaga cgaaatacgc gatcgctgtt aaaaggacaa ttacaaacag gaatcgaatg
     4981 caaccggcgc aggaacactg ccagcgcatc aacaatattt tcacctgaat caggatattc
     5041 ttctaatacc tggaatgctg ttttcccggg gatcgcagtg gtgagtaacc atgcatcatc
     5101 aggagtacgg ataaaatgct tgatggtcgg aagaggcata aattccgtca gccagtttag
     5161 tctgaccatc tcatctgtaa catcattggc aacgctacct ttgccatgtt tcagaaacaa
     5221 ctctggcgca tcgggcttcc catacaatcg atagattgtc gcacctgatt gcccgacatt
     5281 atcgcgagcc catttatacc catataaatc agcatccatg ttggaattta atcgcggcct
     5341 cgagcaagac gtttcccgtt gaatatggct cataacaccc cttgtattac tgtttatgta
     5401 agcagacagt tttattgttc atgatgatat atttttatct tgtgcaatgt aacatcagag
     5461 attttgagac acaacgtggc tttccccccc ccccctgcag gtccgacacg gggatggatg
     5521 gcgttcccga tcatggtcct gcttgcttcg ggtggcatcg gaatgccggc gctgcaagca
     5581 atgttgtcca ggcaggtgga tgaggaacgt caggggcagc tgcaaggctc actggcggcg
     5641 ctcaccagcc tgacctcgat cgtcggaccc ctcctcttca cggcgatcta tgcggcttct
     5701 ataacaacgt ggaacgggtg ggcatggatt gcaggcgctg ccctctactt gctctgcctg
     5761 ccggcgctgc gtcgcgggct ttggagcggc gcagggcaac gagccgatcg ctgatcgtgg
     5821 aaacgatagg cctatgccat gcgggtcaag gcgacttccg gcaagctata cgcgccctag
     5881 aattgtcaat tttaatcctc tgtttatcgg cagttcgtag agcgcgccgt gcgtcccgag
     5941 cgatactgag cgaagcaagt gcgtcgagca gtgcccgctt gttcctgaaa tgccagtaaa
     6001 gcgctggctg ctgaaccccc agccggaact gaccccacaa ggccctagcg tttgcaatgc
     6061 accaggtcat cattgaccca ggcgtgttcc accaggccgc tgcctcgcaa ctcttcgcag
     6121 gcttcgccga cctgctcgcg ccacttcttc acgcgggtgg aatccgatcc gcacatgagg
     6181 cggaaggttt ccagcttgag cgggtacggc tcccggtgcg agctgaaata gtcgaacatc
     6241 cgtcgggccg tcggcgacag cttgcggtac ttctcccata tgaatttcgt gtagtggtcg
     6301 ccagcaaaca gcacgacgat ttcctcgtcg atcaggacct ggcaacggga cgttttcttg
     6361 ccacggtcca ggacgcggaa gcggtgcagc agcgacaccg attccaggtg cccaacgcgg
     6421 tcggacgtga agcccatcgc cgtcgcctgt aggcgcgaca ggcattcctc ggccttcgtg
     6481 taataccggc cattgatcga ccagcccagg tcctggcaaa gctcgtagaa cgtgaaggtg
     6541 atcggctcgc cgataggggt gcgcttcgcg tactccaaca cctgctgcca caccagttcg
     6601 tcatcgtcgg cccgcagctc gacgccggtg taggtgatct tcacgtcctt gttgacgtgg
     6661 aaaatgacct tgttttgcag cgcctcgcgc gggattttct tgttgcgcgt ggtgaacagg
     6721 gcagagcggg ccgtgtcgtt tggcatcgct cgcatcgtgt ccggccacgg cgcaatatcg
     6781 aacaaggaaa gctgcatttc cttgatctgc tgcttcgtgt gtttcagcaa cgcggcctgc
     6841 ttggcctcgc tgacctgttt tgccaggtcc tcgccggcgg tttttcgctt cttggtcgtc
     6901 atagttcctc gcgtgtcgat ggtcatcgac ttcgccaaac ctgccgcctc ctgttcgaga
     6961 cgacgcgaac gctccacggc ggccgatggc gcgggcaggg cagggggagc cagttgcacg
     7021 ctgtcgcgct cgatcttggc cgtagcttgc tggaccatcg agccgacgga ctggaaggtt
     7081 tcgcggggcg cacgcatgac ggtgcggctt gcgatggttt cggcatcctc ggcggaaaac
     7141 cccgcgtcga tcagttcttg cctgtatgcc ttccggtcaa acgtccgatt cattcaccct
     7201 ccttgcggga ttgccccgac tcacgccggg gcaatgtgcc cttattcctg atttgacccg
     7261 cctggtgcct tggtgtccag ataatccacc ttatcggcaa tgaagtcggt cccgtagacc
     7321 gtctggccgt ccttctcgta cttggtattc cgaatcttgc cctgcacgaa taccagctcc
     7381 gcgaagtcgc tcttcttgat ggagcgcatg gggacgtgct tggcaatcac gcgcaccccc
     7441 cggccgtttt agcggctaaa aaagtcatgg ctctgccctc gggcggacca cgcccatcat
     7501 gaccttgcca agctcgtcct gcttctcttc gatcttcgcc agcagggcga ggatcgtggc
     7561 atcaccgaac cgcgccgtgc gcgggtcgtc ggtgagccag agtttcagca ggccgcccag
     7621 gcggcccagg tcgccattga tgcgggccag ctcgcggacg tgctcatagt ccacgacgcc
     7681 cgtgattttg tagccctggc cgacggccag caggtaggcc tacaggctca tgccggccgc
     7741 cgccgccttt tcctcaatcg ctcttcgttc gtctggaagg cagtacacct tgataggtgg
     7801 gctgcccttc ctggttggct tggtttcatc agccatccgc ttgccctcat ctgttacgcc
     7861 ggcggtagcc ggccagcctc gcagagcagg attcccgttg agcaccgcca ggtgcgaata
     7921 agggacagtg aagaaggaac acccgctcgc gggtgggcct acttcaccta tcctgcccgg
     7981 ctgacgccgt tggatacacc aaggaaagtc tacacgaacc ctttggcaaa atcctgtata
     8041 tcgtgcgaaa aaggatggat ataccgaaaa aatcgctata atgaccccga agcagggtta
     8101 tgcagcggaa aagatccgtc
//
