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  • pMCP-tag(m) Vector

    Description

    pMCP-tag(m) Vector is a mammalian expression plasmid encoding MCP, an MCP-tag protein, which is expressed under control of the CMV promoter. This plasmid is intended for the cloning and transient expression of MCP-tag protein fusions in mammalian cells. pMCP-tag(m) Vector contains two multiple cloning sites to allow cloning of the fusion partner as a fusion to the C-terminus of the MCP-tag and an appropriate signal peptide to the N-terminus of the MCP-tag.

    The MCP-tag is a small protein tag (8 kDa) based on the acyl carrier protein (ACP) containing two mutations (D36-T36 and D39-G39), for labeling cell membrane proteins. It allows the specific, covalent attachment of virtually any molecule to a protein of interest. MCP-tag substrates are derivatives of coenzyme A (CoA). In the labeling reaction, the substituted phosphopantetheine group of CoA is covalently attached to a conserved serine residue of the MCP-tag by SFP Synthase. Having no cysteines, the MCP-tag is particularly suited for specifically labeling cell-surface proteins, and should be useful for labeling secreted proteins with disulfide bridges such as antibodies.

    There are two steps to using this system: sub cloning and expression of the protein of interest as a MCP-tag fusion, and labeling of the fusion protein using SFP Synthase with the substrate of choice. The cloning and expression of MCP-tag protein fusions is described in this document. The labeling of fusion proteins with CoA substrates is described in the documentation supplied with CoA substrates and SFP Synthase.

    N9317
    Cloning Region of pMCP-tag(m) Vector: Unique restriction sites in the regions flanking the MCP gene are displayed above the coding strand.

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

    Advantages and Features

    Features


    BACKGROUND:

    This plasmid encodes the gene MCP which is a mutant carrier protein. In the plasmid sequence, the MCP gene is encoded from bp 690 to 923. The MCP gene was cloned via HindIII and PstI including additional flanking restriction sites.

    This plasmid is intended as a transient expression vector for MCP-tag gene fusions in mammalian cell lines. It is not recommended for the production of stable cell lines. The plasmid contains the b-lactamase (Ampicillin resistance) gene for maintenance in bacteria. The plasmid also contains a CMV promoter and SV40 polyadenylation signals for correct mRNA processing. The gene of interest should be cloned downstream of the MCP-tag coding sequence, as a fusion to the C-terminus of the MCP-tag. An appropriate cell surface signal peptide should be cloned upstream of the MCP-tag as an N-terminal fusion. A Kozak sequence is located upstream of the MCP gene to increase the translation efficiency of the fusion protein. The MCP-tag gene can be isolated from the plasmid using PCR or direct cloning in order to subclone it into a different vector system of choice.

    Properties and Usage

    Materials Required but not Supplied

    • Mammalian cell lines 
    • Transfection reagents 
    • CoA substrates

    Storage Temperature

    -20°C

    Notes

    1. Storage: pMCP-tag(m) Vector is supplied in TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA) at a concentration of 0.5 µg/µl. Plasmid solutions can be stored at 4°C for up to one week. For long-term storage -20°C is recommended.
    2. NEB 10-beta Competent E. coli (High Efficiency) (NEB #C3019) is recommended for propagating and subcloning this vector. 

    Supporting Documents

    Material Safety Datasheets

    The following is a list of Material Safety Data Sheets (MSDS) that apply to this product to help you use it safely. The following file naming structure is used to name these document files: [Product Name] MSDS. For international versions please contact us at info@neb.com.

    Datacards

    The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at info@neb.com or fill out the Technical Support Form for appropriate document.
    1. What is the ACP-tag?
    2. How does it work?
    3. How specific is the binding of substrate to the ACP-tag?
    4. What linker type and length would you recommend?
    5. Can I clone my protein as fusion to the N- or C-terminus of the ACP-tag?
    6. Are ACP-tag substrates stable to fixation?
    7. Can ACP-tag be multiplexed with other protein labeling systems (GFP, Antibody)?
    8. Can you use ACP-tag for in vivo FRET?
    9. Does the ACP-tag labeling reaction work in Yeast?
    1. Expression of MCP Fusions (N9317)
    2. Cloning of MCP-tag Fusions in pMCP-tag(m) Vector (N9317)

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