pKLCF-c Vector

  • This product was discontinued on January 05, 2015

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N3745
  • Discontinued
    Discontinued
  • Product Information
    The vector pKLCF-c permits secreted expression of a recombinant protein having a chitin-binding domain (CBD) affinity tag fused to its carboxy-terminus in the yeast Kluyveromyces lactis. It is compatible with the K. lactis Protein Expression Kit (NEB #E1000). CBD fusion proteins expressed from pKLCFc can be affinity purified directly from untreated culture medium using Chitin Beads (NEB #S6651) or Chitin Magnetic Beads (NEB #E8036).

    Vector pKLCF-c contains the strong K. lactis PLAC4-PBI promoter (1), DNA encoding the K. lactis Cts1p chitin-binding domain (2), a universal multiple cloning site (MCS), the K. lactis LAC4 transcription terminator (TT), and a fungal acetamidase selectable marker gene (amdS) expressed from the yeast ADH1 promoter (PADH1). An E. coli replication origin (ori) and ampicillin resistance gene (ApR) is present for propagation of pKLCF-c in E. coli. SacII or BstXI linearized pKLCF-c integrates into the LAC4 locus of the K. lactis genome upon transformation of K. lactis competent cells.

    The sequence of the pKLCF-c vector (GenBank HQ236722) and additional pKLCF-c information are available here.
    N3745
    N3745
    Figure 1:  pKLCF-c multiple cloning site (MCS). The K. lactis α-mating factor is shown with a blue background and the chitin-binding domain is shown with a purple background. Only unique restriction sites are shown.

    DNASU is a central repository for plasmid clones and collections that may also be helpful.

    Highlights

    • PLAC4-PBI promoter does not express in E. coli allowing toxic genes to be cloned prior to their expression in yeast.
    • Universal MCS lies downstream of DNA encoding CBD and PLAC4-PBI promoter.
    • Acetamidase expression for non-antibiotic selection in K. lactis.
    • Ampicillin resistance for propagation in E. coli.
    • Permits expression of CBD-tagged fusion proteins and their one-step purification directly from growth medium.
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