LOCUS       sequence-64398-e        6878 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 6878)
  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 6878)
  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: pCM62
FEATURES             Location/Qualifiers
     source          1..6878
                     /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"
     primer_bind     complement(2569..2587)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     CDS             3373..4572
                     /codon_start=1
                     /gene="tetA"
                     /product="tetracycline efflux protein"
                     /label=TcR
                     /note="confers resistance to tetracycline"
                     /translation="MKPNIPLIVILSTVALDAVGIGLIMPVLPGLLRDLVHSNDVTAHY
                     GILLALYALVQFACAPVLGALSDRFGRRPILLVSLAGATVDYAIMATAPFLWVLYIGRI
                     VAGITGATGAVAGAYIADITDGDERARHFGFMSACFGFGMVAGPVLGGLMGGFSPHAPF
                     FAAAALNGLNFLTGCFLLPESHKGERRPLRREALNPLASFRWARGMTVVAALMAVFFIM
                     QLVGQVPAALWVIFGEDRFHWDATTIGISLAAFGILHSLAQAMITGPVAARLGERRALM
                     LGMIADGTGYILLAFATRGWMAFPIMVLLASGGIGMPALQAMLSRQVDEERQGQLQGSL
                     AALTSLTSIVGPLLFTAIYAASITTWNGWAWIAGAALYLLCLPALRRGLWSGAGQRADR
                     "
     CDS             complement(4803..5951)
                     /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(6223..6594)
                     /codon_start=1
                     /gene="traJ"
                     /product="oriT-recognizing protein"
                     /label=traJ
                     /translation="MADETKPTRKGSPPIKVYCLPDERRAIEEKAAAAGMSL*AYLLAV
                     GQGYKITGVVDYEHVRELARINGDLGRLGGLLKLWLTDDPRTARFGDATILALLAKIEE
                     KQDELGKVMMGVVRPRAEP"
     oriT            6627..6736
                     /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 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 gttttcaccg tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac
     2581 gcctattttt ataggttaat gtcatgataa taatggtttc ttagcaccct ttctcggtcc
     2641 ttcaacgttc ctgacaacga gcctcctttt cgccaatcca tcgacaatca ccgcgagtcc
     2701 ctgctcgaac gctgcgtccg gaccggcttc gtcgaaggcg tctatcgcgg cccgcaacag
     2761 cggcgagagc ggagcctgtt caacggtgcc gccgcgctcg ccggcatcgc tgtcgccggc
     2821 ctgctcctca agcacggccc caacagtgaa gtagctgatt gtcatcagcg cattgacggc
     2881 gtccccggcc gaaaaacccg cctcgcagag gaagcgaagc tgcgcgtcgg ccgtttccat
     2941 ctgcggtgcg cccggtcgcg tgccggcatg gatgcgcgcg ccatcgcggt aggcgagcag
     3001 cgcctgcctg aagctgcggg cattcccgat cagaaatgag cgccagtcgt cgtcggctct
     3061 cggcaccgaa tgcgtatgat tctccgccag catggcttcg gccagtgcgt cgagcagcgc
     3121 ccgcttgttc ctgaagtgcc agtaaagcgc cggctgctga acccccaacc gttccgccag
     3181 tttgcgtgtc gtcagaccgt ctacgccgac ctcgttcaac aggtccaggg cggcacggat
     3241 cactgtattc ggctgcaact ttgtcatgat tgacacttta tcactgataa acataatatg
     3301 tccaccaact tatcagtgat aaagaatccg cgcgttcaat cggaccagcg gaggctggtc
     3361 cggaggccag acgtgaaacc caacataccc ctgatcgtaa ttctgagcac tgtcgcgctc
     3421 gacgctgtcg gcatcggcct gattatgccg gtgctgccgg gcctcctgcg cgatctggtt
     3481 cactcgaacg acgtcaccgc ccactatggc attctgctgg cgctgtatgc gttggtgcaa
     3541 tttgcctgcg cacctgtgct gggcgcgctg tcggatcgtt tcgggcggcg gccaatcttg
     3601 ctcgtctcgc tggccggcgc cactgtcgac tacgccatca tggcgacagc gcctttcctt
     3661 tgggttctct atatcgggcg gatcgtggcc ggcatcaccg gggcgactgg ggcggtagcc
     3721 ggcgcttata ttgccgatat cactgatggc gatgagcgcg cgcggcactt cggcttcatg
     3781 agcgcctgtt tcgggttcgg gatggtcgcg ggacctgtgc tcggtgggct gatgggcggt
     3841 ttctcccccc acgctccgtt cttcgccgcg gcagccttga acggcctcaa tttcctgacg
     3901 ggctgtttcc ttttgccgga gtcgcacaaa ggcgaacgcc ggccgttacg ccgggaggct
     3961 ctcaacccgc tcgcttcgtt ccggtgggcc cggggcatga ccgtcgtcgc cgccctgatg
     4021 gcggtcttct tcatcatgca acttgtcgga caggtgccgg ccgcgctttg ggtcattttc
     4081 ggcgaggatc gctttcactg ggacgcgacc acgatcggca tttcgcttgc cgcatttggc
     4141 attctgcatt cactcgccca ggcaatgatc accggccctg tagccgcccg gctcggcgaa
     4201 aggcgggcac tcatgctcgg aatgattgcc gacggcacag gctacatcct gcttgccttc
     4261 gcgacacggg gatggatggc gttcccgatc atggtcctgc ttgcttcggg tggcatcgga
     4321 atgccggcgc tgcaagcaat gttgtccagg caggtggatg aggaacgtca ggggcagctg
     4381 caaggctcac tggcggcgct caccagcctg acctcgatcg tcggacccct cctcttcacg
     4441 gcgatctatg cggcttctat aacaacgtgg aacgggtggg catggattgc aggcgctgcc
     4501 ctctacttgc tctgcctgcc ggcgctgcgt cgcgggcttt ggagcggcgc agggcaacga
     4561 gccgatcgct gatcgtggaa acgataggcc tatgccatgc gggtcaaggc gacttccggc
     4621 aagctatacg cgccctagaa ttgtcaattt taatcctctg tttatcggca gttcgtagag
     4681 cgcgccgtgc gtcccgagcg atactgagcg aagcaagtgc gtcgagcagt gcccgcttgt
     4741 tcctgaaatg ccagtaaagc gctggctgct gaacccccag ccggaactga ccccacaagg
     4801 ccctagcgtt tgcaatgcac caggtcatca ttgacccagg cgtgttccac caggccgctg
     4861 cctcgcaact cttcgcaggc ttcgccgacc tgctcgcgcc acttcttcac gcgggtggaa
     4921 tccgatccgc acatgaggcg gaaggtttcc agcttgagcg ggtacggctc ccggtgcgag
     4981 ctgaaatagt cgaacatccg tcgggccgtc ggcgacagct tgcggtactt ctcccatatg
     5041 aatttcgtgt agtggtcgcc agcaaacagc acgacgattt cctcgtcgat caggacctgg
     5101 caacgggacg ttttcttgcc acggtccagg acgcggaagc ggtgcagcag cgacaccgat
     5161 tccaggtgcc caacgcggtc ggacgtgaag cccatcgccg tcgcctgtag gcgcgacagg
     5221 cattcctcgg ccttcgtgta ataccggcca ttgatcgacc agcccaggtc ctggcaaagc
     5281 tcgtagaacg tgaaggtgat cggctcgccg ataggggtgc gcttcgcgta ctccaacacc
     5341 tgctgccaca ccagttcgtc atcgtcggcc cgcagctcga cgccggtgta ggtgatcttc
     5401 acgtccttgt tgacgtggaa aatgaccttg ttttgcagcg cctcgcgcgg gattttcttg
     5461 ttgcgcgtgg tgaacagggc agagcgggcc gtgtcgtttg gcatcgctcg catcgtgtcc
     5521 ggccacggcg caatatcgaa caaggaaagc tgcatttcct tgatctgctg cttcgtgtgt
     5581 ttcagcaacg cggcctgctt ggcctcgctg acctgttttg ccaggtcctc gccggcggtt
     5641 tttcgcttct tggtcgtcat agttcctcgc gtgtcgatgg tcatcgactt cgccaaacct
     5701 gccgcctcct gttcgagacg acgcgaacgc tccacggcgg ccgatggcgc gggcagggca
     5761 gggggagcca gttgcacgct gtcgcgctcg atcttggccg tagcttgctg gaccatcgag
     5821 ccgacggact ggaaggtttc gcggggcgca cgcatgacgg tgcggcttgc gatggtttcg
     5881 gcatcctcgg cggaaaaccc cgcgtcgatc agttcttgcc tgtatgcctt ccggtcaaac
     5941 gtccgattca ttcaccctcc ttgcgggatt gccccgactc acgccggggc aatgtgccct
     6001 tattcctgat ttgacccgcc tggtgccttg gtgtccagat aatccacctt atcggcaatg
     6061 aagtcggtcc cgtagaccgt ctggccgtcc ttctcgtact tggtattccg aatcttgccc
     6121 tgcacgaata ccagctccgc gaagtcgctc ttcttgatgg agcgcatggg gacgtgcttg
     6181 gcaatcacgc gcaccccccg gccgttttag cggctaaaaa agtcatggct ctgccctcgg
     6241 gcggaccacg cccatcatga ccttgccaag ctcgtcctgc ttctcttcga tcttcgccag
     6301 cagggcgagg atcgtggcat caccgaaccg cgccgtgcgc gggtcgtcgg tgagccagag
     6361 tttcagcagg ccgcccaggc ggcccaggtc gccattgatg cgggccagct cgcggacgtg
     6421 ctcatagtcc acgacgcccg tgattttgta gccctggccg acggccagca ggtaggccta
     6481 caggctcatg ccggccgccg ccgccttttc ctcaatcgct cttcgttcgt ctggaaggca
     6541 gtacaccttg ataggtgggc tgcccttcct ggttggcttg gtttcatcag ccatccgctt
     6601 gccctcatct gttacgccgg cggtagccgg ccagcctcgc agagcaggat tcccgttgag
     6661 caccgccagg tgcgaataag ggacagtgaa gaaggaacac ccgctcgcgg gtgggcctac
     6721 ttcacctatc ctgcccggct gacgccgttg gatacaccaa ggaaagtcta cacgaaccct
     6781 ttggcaaaat cctgtatatc gtgcgaaaaa ggatggatat accgaaaaaa tcgctataat
     6841 gaccccgaag cagggttatg cagcggaaaa gatccgtc
//
