pCM110 vector (V008445)

Basic Vector Information

The plasmid pCM110 is an expression vector specifically developed for use in Methylobacterium extorquens AM1. It is constructed based on an IncP plasmid that lacks both the ColE1 ori and Plac. The construction of pCM110 involves inserting the 0.4 kb HindIII - NsiI fragment of pCM27 containing Pmax into pCM60 (C. J. Marx & M. E. Lidstrom, unpublished results) which had been cut with HindIII and NsiI.
This expression vector provides minimal expression in E. coli, which is advantageous for the introduction of toxic genes into M. extorquens AM1. For example, a construct containing xylE cloned into pCM110 (pCM111) was created to compare its XylE expression level to that of another vector (pCM81) in both organisms. The results showed that pCM111 provided a high level of expression in M. extorquens AM1, 1.5 - 2-fold higher than that achieved with pCM81; however, extracts from E. coli JM109 bearing pCM111 had a basal level of XylE activity, nearly at the background. These results indicate that the Pmax is expressed at very low levels in E. coli. Therefore, while other expression vectors like pCM80 are useful for the expression of most cloned genes, pCM110 may be required to express genes that are toxic in E. coli.

Vector Name:: pCM110

Antibiotic Resistance:: Tetracycline

Length:: 5834 bp

Type:: Cloning vector

Replication origin:: oriV

Source/Author:: Marx CJ, Lidstrom ME.

pCM110 vector Map

Copy Sequence View Map

References

Marx CJ, Lidstrom ME. Development of improved versatile broad-host-range vectors for use in methylotrophs and other Gram-negative bacteria. Microbiology (Reading). 2001 Aug;147(Pt 8):2065-2075. doi: 10.1099/00221287-147-8-2065. PMID: 11495985.

pCM110 vector Sequence

LOCUS       40924_11316        5834 bp DNA     circular SYN 17-DEC-2018
DEFINITION  Cloning vector pCM110, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5834)
  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 5834)
  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 5834)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 5834)
  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..5834
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      3..712
                     /label=oriV
                     /note="incP origin of replication"
     RBS             820..828
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     regulatory      856..1195
                     /note="PmxaF; Methylobacterium extorquens mxa operon
                     promoter"
                     /regulatory_class="promoter"
     CDS             complement(1576..2223)
                     /label=TetR
                     /note="tetracycline resistance regulatory protein"
     CDS             2329..3525
                     /label=TcR
                     /note="tetracycline efflux protein"
     CDS             complement(3762..4907)
                     /label=trfA
                     /note="trans-acting replication protein that binds to and 
                     activates oriV"
     CDS             complement(5179..5550)
                     /codon_start=1
                     /gene="traJ"
                     /product="oriT-recognizing protein"
                     /label=traJ
                     /translation="MADETKPTRKGSPPIKVYCLPDERRAIEEKAAAAGMSL*AYLLAV
                     GQGYKITGVVDYEHVRELARINGDLGRLGGLLKLWLTDDPRTARFGDATILALLAKIEE
                     KQDELGKVMMGVVRPRAEP"
     CDS             complement(5434..5550)
                     /codon_start=1
                     /gene="traJ'"
                     /product="TraJ'"
                     /label=traJ'
                     /note="truncated oriT-recognizing protein"
                     /protein_id="AAK73417.1"
                     /translation="MADETKPTRKGSPPIKVYCLPDERRAIEEKAAAAGMSL"
     gene            complement(5434..5550)
                     /gene="traJ'"
                     /label=traJ'
     oriT            complement(5583..5692)
                     /direction=LEFT
                     /label=oriT
                     /note="incP origin of transfer"

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