LOCUS       Exported                7607 bp ds-DNA     circular SYN 17-DEC-2018
DEFINITION  Cloning vector pCM66, complete sequence.
ACCESSION   AF327715
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)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..7607
                     /organism="Cloning vector pCM66"
                     /mol_type="other DNA"
                     /note="derived from pCM62 by inserting the 1.3 kb HincII 
                     fragment of pUC4K between the EcoRV and NruI sites"
                     /db_xref="taxon:163914"
     rep_origin      4..712
                     /note="oriV; IncP origin of replication"
     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"
     rep_origin      1180..1735
                     /label=colE1
                     /note="colE1"
     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."
     regulatory      2017..2072
                     /regulatory_class="promoter"
                     /note="Plac; lac operon promoter/operator"
     promoter        2017..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     2079..2095
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             2091..2414
                     /codon_start=1
                     /gene="lacZa"
                     /product="LacZ"
                     /label=lacZa
                     /note="beta-galactosidase alpha peptide"
                     /protein_id="AAK73401.1"
                     /translation="MTMITPSLHACRSTLEDPRVPSSNSLAVVLQRRDWENPGVTQLNR
                     LAAHPPFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCTLSTICS
                     DAA"
     gene            2091..2414
                     /gene="lacZa"
                     /label=lacZa
     misc_feature    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(4045..4860)
                     /codon_start=1
                     /gene="kan"
                     /product="Kan"
                     /function="kanamycin resistance"
                     /label=kan
                     /protein_id="AAK73402.1"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     gene            complement(4045..4860)
                     /gene="kan"
                     /label=kan
     CDS             complement(5532..6680)
                     /codon_start=1
                     /gene="trfA"
                     /product="TrfA"
                     /function="trans-acting replication"
                     /label=trfA
                     /protein_id="AAK73403.1"
                     /translation="MNRTFDRKAYRQELIDAGFSAEDAETIASRTVMRAPRETFQSVGS
                     MVQQATAKIERDSVQLAPPALPAPSAAVERSRRLEQEAAGLAKSMTIDTRGTMTTKKRK
                     TAGEDLAKQVSEAKQAALLKHTKQQIKEMQLSLFDIAPWPDTMRAMPNDTARSALFTTR
                     NKKIPREALQNKVIFHVNKDVKITYTGVELRADDDELVWQQVLEYAKRTPIGEPITFTF
                     YELCQDLGWSINGRYYTKAEECLSRLQATAMGFTSDRVGHLESVSLLHRFRVLDRGKKT
                     SRCQVLIDEEIVVLFAGDHYTKFIWEKYRKLSPTARRMFDYFSSHREPYPLKLETFRLM
                     CGSDSTRVKKWREQVGEACEELRGSGLVEHAWVNDDLVHCKR"
     gene            complement(5532..6680)
                     /gene="trfA"
                     /label=trfA
     oriT            6862..7600
                     /label=IncP origin of transfer
                     /note="IncP origin of transfer"
     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            7356..7465
                     /note="incP origin of transfer"