LOCUS       Exported                6878 bp ds-DNA     circular SYN 17-DEC-2018
DEFINITION  Cloning vector pCM62, complete sequence.
ACCESSION   AF327714
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 147 (Pt 8), 2065-2075 (2001)
  PUBMED    11495985
REFERENCE   2  (bases 1 to 6878)
  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 6878)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..6878
                     /organism="Cloning vector pCM62"
                     /mol_type="other DNA"
                     /note="created by ligating together the blunted 5.3 kb 
                     HindIII-XmnI region of pCM50 (which comprises the minimal 
                     replicon pCM51) with the blunted 1.9 kb AatII-AvaII region 
                     of pUC19. During this step, an additional 356 bp inward 
                     from the site of XmnI cleavage of pCM50 was lost, 
                     presumably caused by either non-specific cleavage or the 
                     exonuclease activity of T4 DNA polymerase"
                     /db_xref="taxon:163913"
     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="AAK73396.1"
                     /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(2602..3267)
                     /codon_start=1
                     /gene="tetR"
                     /product="TetR"
                     /function="tetracycline resistance regulation"
                     /label=tetR
                     /note="loss of region reported following ligation leads to 
                     a small alteration of the C-terminus, from -GDD to 
                     -GAKKPLLS, which does not adversely affect tetracycline 
                     resistance conferred in either Escherichia coli or 
                     Methylobacterium extorquens AM1."
                     /protein_id="AAK73397.1"
                     /translation="MTKLQPNTVIRAALDLLNEVGVDGLTTRKLAERLGVQQPALYWHF
                     RNKRALLDALAEAMLAENHTHSVPRADDDWRSFLIGNARSFRQALLAYRDGARIHAGTR
                     PGAPQMETADAQLRFLCEAGFSAGDAVNALMTISYFTVGAVLEEQAGDSDAGERGGTVE
                     QAPLSPLLRAAIDAFDEAGPDAAFEQGLAVIVDGLAKRRLVVRNVEGPRKGAKKPLLS"
     gene            complement(2602..3267)
                     /gene="tetR"
                     /label=tetR
     CDS             3373..4572
                     /codon_start=1
                     /gene="tetA"
                     /product="TetA"
                     /function="tetracycline resistance"
                     /label=tetA
                     /protein_id="AAK73398.1"
                     /translation="MKPNIPLIVILSTVALDAVGIGLIMPVLPGLLRDLVHSNDVTAHY
                     GILLALYALVQFACAPVLGALSDRFGRRPILLVSLAGATVDYAIMATAPFLWVLYIGRI
                     VAGITGATGAVAGAYIADITDGDERARHFGFMSACFGFGMVAGPVLGGLMGGFSPHAPF
                     FAAAALNGLNFLTGCFLLPESHKGERRPLRREALNPLASFRWARGMTVVAALMAVFFIM
                     QLVGQVPAALWVIFGEDRFHWDATTIGISLAAFGILHSLAQAMITGPVAARLGERRALM
                     LGMIADGTGYILLAFATRGWMAFPIMVLLASGGIGMPALQAMLSRQVDEERQGQLQGSL
                     AALTSLTSIVGPLLFTAIYAASITTWNGWAWIAGAALYLLCLPALRRGLWSGAGQRADR
                     "
     gene            3373..4572
                     /gene="tetA"
                     /label=tetA
     CDS             complement(4803..5951)
                     /codon_start=1
                     /gene="trfA"
                     /product="TrfA"
                     /function="trans-acting replication"
                     /label=trfA
                     /protein_id="AAK73399.1"
                     /translation="MNRTFDRKAYRQELIDAGFSAEDAETIASRTVMRAPRETFQSVGS
                     MVQQATAKIERDSVQLAPPALPAPSAAVERSRRLEQEAAGLAKSMTIDTRGTMTTKKRK
                     TAGEDLAKQVSEAKQAALLKHTKQQIKEMQLSLFDIAPWPDTMRAMPNDTARSALFTTR
                     NKKIPREALQNKVIFHVNKDVKITYTGVELRADDDELVWQQVLEYAKRTPIGEPITFTF
                     YELCQDLGWSINGRYYTKAEECLSRLQATAMGFTSDRVGHLESVSLLHRFRVLDRGKKT
                     SRCQVLIDEEIVVLFAGDHYTKFIWEKYRKLSPTARRMFDYFSSHREPYPLKLETFRLM
                     CGSDSTRVKKWREQVGEACEELRGSGLVEHAWVNDDLVHCKR"
     gene            complement(4803..5951)
                     /gene="trfA"
                     /label=trfA
     oriT            6133..6871
                     /label=IncP origin of transfer
                     /note="IncP origin of transfer"
     CDS             complement(6223..6594)
                     /codon_start=1
                     /gene="traJ"
                     /product="oriT-recognizing protein"
                     /label=traJ
                     /translation="MADETKPTRKGSPPIKVYCLPDERRAIEEKAAAAGMSL*AYLLAV
                     GQGYKITGVVDYEHVRELARINGDLGRLGGLLKLWLTDDPRTARFGDATILALLAKIEE
                     KQDELGKVMMGVVRPRAEP"
     CDS             complement(6478..6594)
                     /codon_start=1
                     /gene="traJ"
                     /product="TraJ"
                     /label=traJ
                     /note="truncated oriT-recognizing protein"
                     /protein_id="AAK73400.1"
                     /translation="MADETKPTRKGSPPIKVYCLPDERRAIEEKAAAAGMSL"
     gene            complement(6478..6594)
                     /gene="traJ"
                     /label=traJ
     oriT            6627..6736
                     /note="incP origin of transfer"