LOCUS       40924_11381        7607 bp DNA     circular SYN 17-DEC-2018
DEFINITION  Cloning vector pCM66, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7607)
  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 147 (Pt 8), 2065-2075 (2001)
  PUBMED    11495985
REFERENCE   2  (bases 1 to 7607)
  AUTHORS   Marx CJ, Lidstrom ME.
  TITLE     Direct Submission
  JOURNAL   Submitted (12-DEC-2000) Microbiology, University of Washington, Box 
            357242, Seattle, WA 98195, USA
REFERENCE   3  (bases 1 to 7607)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 7607)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Microbiology 147 (Pt 8), 2065-2075 (2001)"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (12-DEC-2000) Microbiology, University of Washington, Box 357242, 
            Seattle, WA 98195, USA"
COMMENT     SGRef: number: 3; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..7607
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      3..712
                     /label=oriV
                     /note="incP origin of replication"
     rep_origin      1105..1693
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     protein_bind    1981..2002
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     promoter        2017..2047
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2055..2071
                     /label=lac operator
                     /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     2079..2095
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    complement(2108..2164)
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(2165..2181)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             complement(2626..3267)
                     /codon_start=1
                     /label=TetR
                     /note="tetracycline resistance regulatory protein"
                     /translation="MTKLQPNTVIRAALDLLNEVGVDGLTTRKLAERLGVQQPALYWHF
                     RNKRALLDALAEAMLAENHTHSVPRADDDWRSFLIGNARSFRQALLAYRDGARIHAGTR
                     PGAPQMETADAQLRFLCEAGFSAGDAVNALMTISYFTVGAVLEEQAGDSDAGERGGTVE
                     QAPLSPLLRAAIDAFDEAGPDAAFEQGLAVIVDGLAKRRLVVRNVEGPRKG"
     CDS             complement(4048..4860)
                     /codon_start=1
                     /label=KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(5535..6680)
                     /codon_start=1
                     /label=trfA
                     /note="trans-acting replication protein that binds to and 
                     activates oriV"
                     /translation="MNRTFDRKAYRQELIDAGFSAEDAETIASRTVMRAPRETFQSVGS
                     MVQQATAKIERDSVQLAPPALPAPSAAVERSRRLEQEAAGLAKSMTIDTRGTMTTKKRK
                     TAGEDLAKQVSEAKQAALLKHTKQQIKEMQLSLFDIAPWPDTMRAMPNDTARSALFTTR
                     NKKIPREALQNKVIFHVNKDVKITYTGVELRADDDELVWQQVLEYAKRTPIGEPITFTF
                     YELCQDLGWSINGRYYTKAEECLSRLQATAMGFTSDRVGHLESVSLLHRFRVLDRGKKT
                     SRCQVLIDEEIVVLFAGDHYTKFIWEKYRKLSPTARRMFDYFSSHREPYPLKLETFRLM
                     CGSDSTRVKKWREQVGEACEELRGSGLVEHAWVNDDLVHCKR"
     CDS             complement(6952..7323)
                     /codon_start=1
                     /gene="traJ"
                     /product="oriT-recognizing protein"
                     /label=traJ
                     /translation="MADETKPTRKGSPPIKVYCLPDERRAIEEKAAAAGMSL*AYLLAV
                     GQGYKITGVVDYEHVRELARINGDLGRLGGLLKLWLTDDPRTARFGDATILALLAKIEE
                     KQDELGKVMMGVVRPRAEP"
     CDS             complement(7207..7323)
                     /codon_start=1
                     /gene="traJ'"
                     /product="TraJ'"
                     /label=traJ'
                     /note="truncated oriT-recognizing protein"
                     /protein_id="AAK73404.1"
                     /translation="MADETKPTRKGSPPIKVYCLPDERRAIEEKAAAAGMSL"
     gene            complement(7207..7323)
                     /gene="traJ'"
                     /label=traJ'
     oriT            complement(7356..7465)
                     /direction=LEFT
                     /label=oriT
                     /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 ttgccgatga cctgcagggg ggggggggcg ctgaggtctg cctcgtgaag
     3781 aaggtgttgc tgactcatac caggcctgaa tcgccccatc atccagccag aaagtgaggg
     3841 agccacggtt gatgagagct ttgttgtagg tggaccagtt ggtgattttg aacttttgct
     3901 ttgccacgga acggtctgcg ttgtcgggaa gatgcgtgat ctgatccttc aactcagcaa
     3961 aagttcgatt tattcaacaa agccgccgtc ccgtcaagtc agcgtaatgc tctgccagtg
     4021 ttacaaccaa ttaaccaatt ctgattagaa aaactcatcg agcatcaaat gaaactgcaa
     4081 tttattcata tcaggattat caataccata tttttgaaaa agccgtttct gtaatgaagg
     4141 agaaaactca ccgaggcagt tccataggat ggcaagatcc tggtatcggt ctgcgattcc
     4201 gactcgtcca acatcaatac aacctattaa tttcccctcg tcaaaaataa ggttatcaag
     4261 tgagaaatca ccatgagtga cgactgaatc cggtgagaat ggcaaaagct tatgcatttc
     4321 tttccagact tgttcaacag gccagccatt acgctcgtca tcaaaatcac tcgcatcaac
     4381 caaaccgtta ttcattcgtg attgcgcctg agcgagacga aatacgcgat cgctgttaaa
     4441 aggacaatta caaacaggaa tcgaatgcaa ccggcgcagg aacactgcca gcgcatcaac
     4501 aatattttca cctgaatcag gatattcttc taatacctgg aatgctgttt tcccggggat
     4561 cgcagtggtg agtaaccatg catcatcagg agtacggata aaatgcttga tggtcggaag
     4621 aggcataaat tccgtcagcc agtttagtct gaccatctca tctgtaacat cattggcaac
     4681 gctacctttg ccatgtttca gaaacaactc tggcgcatcg ggcttcccat acaatcgata
     4741 gattgtcgca cctgattgcc cgacattatc gcgagcccat ttatacccat ataaatcagc
     4801 atccatgttg gaatttaatc gcggcctcga gcaagacgtt tcccgttgaa tatggctcat
     4861 aacacccctt gtattactgt ttatgtaagc agacagtttt attgttcatg atgatatatt
     4921 tttatcttgt gcaatgtaac atcagagatt ttgagacaca acgtggcttt cccccccccc
     4981 cctgcaggtc cgacacgggg atggatggcg ttcccgatca tggtcctgct tgcttcgggt
     5041 ggcatcggaa tgccggcgct gcaagcaatg ttgtccaggc aggtggatga ggaacgtcag
     5101 gggcagctgc aaggctcact ggcggcgctc accagcctga cctcgatcgt cggacccctc
     5161 ctcttcacgg cgatctatgc ggcttctata acaacgtgga acgggtgggc atggattgca
     5221 ggcgctgccc tctacttgct ctgcctgccg gcgctgcgtc gcgggctttg gagcggcgca
     5281 gggcaacgag ccgatcgctg atcgtggaaa cgataggcct atgccatgcg ggtcaaggcg
     5341 acttccggca agctatacgc gccctagaat tgtcaatttt aatcctctgt ttatcggcag
     5401 ttcgtagagc gcgccgtgcg tcccgagcga tactgagcga agcaagtgcg tcgagcagtg
     5461 cccgcttgtt cctgaaatgc cagtaaagcg ctggctgctg aacccccagc cggaactgac
     5521 cccacaaggc cctagcgttt gcaatgcacc aggtcatcat tgacccaggc gtgttccacc
     5581 aggccgctgc ctcgcaactc ttcgcaggct tcgccgacct gctcgcgcca cttcttcacg
     5641 cgggtggaat ccgatccgca catgaggcgg aaggtttcca gcttgagcgg gtacggctcc
     5701 cggtgcgagc tgaaatagtc gaacatccgt cgggccgtcg gcgacagctt gcggtacttc
     5761 tcccatatga atttcgtgta gtggtcgcca gcaaacagca cgacgatttc ctcgtcgatc
     5821 aggacctggc aacgggacgt tttcttgcca cggtccagga cgcggaagcg gtgcagcagc
     5881 gacaccgatt ccaggtgccc aacgcggtcg gacgtgaagc ccatcgccgt cgcctgtagg
     5941 cgcgacaggc attcctcggc cttcgtgtaa taccggccat tgatcgacca gcccaggtcc
     6001 tggcaaagct cgtagaacgt gaaggtgatc ggctcgccga taggggtgcg cttcgcgtac
     6061 tccaacacct gctgccacac cagttcgtca tcgtcggccc gcagctcgac gccggtgtag
     6121 gtgatcttca cgtccttgtt gacgtggaaa atgaccttgt tttgcagcgc ctcgcgcggg
     6181 attttcttgt tgcgcgtggt gaacagggca gagcgggccg tgtcgtttgg catcgctcgc
     6241 atcgtgtccg gccacggcgc aatatcgaac aaggaaagct gcatttcctt gatctgctgc
     6301 ttcgtgtgtt tcagcaacgc ggcctgcttg gcctcgctga cctgttttgc caggtcctcg
     6361 ccggcggttt ttcgcttctt ggtcgtcata gttcctcgcg tgtcgatggt catcgacttc
     6421 gccaaacctg ccgcctcctg ttcgagacga cgcgaacgct ccacggcggc cgatggcgcg
     6481 ggcagggcag ggggagccag ttgcacgctg tcgcgctcga tcttggccgt agcttgctgg
     6541 accatcgagc cgacggactg gaaggtttcg cggggcgcac gcatgacggt gcggcttgcg
     6601 atggtttcgg catcctcggc ggaaaacccc gcgtcgatca gttcttgcct gtatgccttc
     6661 cggtcaaacg tccgattcat tcaccctcct tgcgggattg ccccgactca cgccggggca
     6721 atgtgccctt attcctgatt tgacccgcct ggtgccttgg tgtccagata atccacctta
     6781 tcggcaatga agtcggtccc gtagaccgtc tggccgtcct tctcgtactt ggtattccga
     6841 atcttgccct gcacgaatac cagctccgcg aagtcgctct tcttgatgga gcgcatgggg
     6901 acgtgcttgg caatcacgcg caccccccgg ccgttttagc ggctaaaaaa gtcatggctc
     6961 tgccctcggg cggaccacgc ccatcatgac cttgccaagc tcgtcctgct tctcttcgat
     7021 cttcgccagc agggcgagga tcgtggcatc accgaaccgc gccgtgcgcg ggtcgtcggt
     7081 gagccagagt ttcagcaggc cgcccaggcg gcccaggtcg ccattgatgc gggccagctc
     7141 gcggacgtgc tcatagtcca cgacgcccgt gattttgtag ccctggccga cggccagcag
     7201 gtaggcctac aggctcatgc cggccgccgc cgccttttcc tcaatcgctc ttcgttcgtc
     7261 tggaaggcag tacaccttga taggtgggct gcccttcctg gttggcttgg tttcatcagc
     7321 catccgcttg ccctcatctg ttacgccggc ggtagccggc cagcctcgca gagcaggatt
     7381 cccgttgagc accgccaggt gcgaataagg gacagtgaag aaggaacacc cgctcgcggg
     7441 tgggcctact tcacctatcc tgcccggctg acgccgttgg atacaccaag gaaagtctac
     7501 acgaaccctt tggcaaaatc ctgtatatcg tgcgaaaaag gatggatata ccgaaaaaat
     7561 cgctataatg accccgaagc agggttatgc agcggaaaag atccgtc
//
