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  • pSNAPf Vector

    Description

    pSNAPf Vector is a mammalian expression plasmid intended for the cloning and stable or transient expression of SNAP-tag® protein fusions in mammalian cells. This plasmid encodes SNAPf, a SNAP-tag protein, which is expressed under control of the CMV promoter. The expression vector has an IRES (internal ribosome entry site) and a neomycin resistance gene downstream of the SNAPf for the efficient selection of stable transfectants. pSNAPf Vector contains two multiple cloning sites to allow cloning of the fusion partner as a fusion to the N- or C-terminus of the SNAPf.

    The SNAP-tag is a novel tool for protein research, allowing the specific, covalent attachment of virtually any molecule to a protein of interest. The SNAP-tag is a small protein based on mammalian O6-alkylguanine-DNA-alkyltransferase (AGT). SNAP-tag substrates are derivatives of benzyl purines and benzyl pyrimidines. In the labeling reaction, the substituted benzyl group of the substrate is covalently attached to the SNAP-tag.

    pSNAPf contains an improved version of SNAP-tag, termed SNAPf. SNAPf displays faster kinetics in in vitro labeling and fast, specific and efficient labeling in live and fixed cell applications, thereby rendering it a desired research tool for analysis of protein dynamics.

    There are two steps to using this system: sub cloning and expression of the protein of interest as a SNAPf fusion, and labeling of the fusion with the SNAP-tag substrate of choice. Cloning and expression of SNAPf fusion proteins are described in this document. The labeling of the fusion proteins with SNAP-tag substrates is described in the instructions supplied with the SNAP-tag substrates.

    N9183

    Cloning Region of pSNAPf
    Unique restriction sites in the regions flanking the SNAPf gene are displayed above the coding strand. The complete sequence for pSNAPf and the control plasmids can be downloaded.


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

    Advantages and Features

    Features


    BACKGROUND:

    A plasmid map and the sequence of the cloning region can be found with these instructions. The complete plasmid sequence can be downloaded. This plasmid encodes the gene SNAPf which is a mutant form of the human gene for O6-alkylguanine-DNA-alkyltransferase (hAGT). The codon usage of the gene is optimized for expression in mammalian cells. In the plasmid sequence, the SNAPf gene is encoded from 969 bp to 1514 bp.

    This plasmid is intended for the cloning and stable or transient expression of SNAP-tag protein fusions in mammalian cells. It is suitable for the efficient production of stable cell lines expressing SNAPf gene fusions. The plasmid contains the CMV promoter followed by the genes for SNAPf and neomycin resistance separated by the IRES of the encephalomyocarditis virus (ECMV), which permits the translation of two open reading frames from one messenger RNA; therefore after selection of stable mammalian cells for neomycin resistance, nearly all surviving colonies should stably express the SNAPf fusion protein. Unless your expression experiments require a pure population of cells, you can simply use the pool of resistant cells, otherwise cell clones can be isolated and characterized using standard procedures.

    The plasmid contains the β-lactamase (Ampicillin resistance) gene for maintenance in bacteria. The gene of interest can be cloned upstream or downstream of the SNAPf coding sequence, as a fusion to the N- or C-terminus of the SNAP-tag. pSNAPf Vector can also be used as an expression control plasmid, expressing SNAPf alone, in which case the SNAP-tag protein is distributed throughout the cell. The SNAPf gene can be isolated from the plasmid using PCR or direct cloning in order to subclone it into a different vector of choice.

    Properties and Usage

    Materials Required but not Supplied

    Tissue culture reagents and media
    Mammalian cell line(s)
    Transfection reagents

    Storage Temperature

    -20°C

    Notes

    1. Storage: pSNAPf 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.

    References

    1. Keppler, A. et al. (2003). Nat. Biotechnol. 21, 86.
    2. Gautier, A. et al. (2008). Chem. Biol. 15, 128.
    3. Keppler, A. et al. (2004). Proc. Natl. Acad. Sci USA. 101, 9955.
    4. Maurel, D. et al. (2008). Nat. Methods. 5, 561.
    5. Jansen, L.E. et al. (2007). J. of Cell Biol. 176, 795.
    6. Krayl, M., Guiard, B. Paal, K. and Vous, W. (2006). Anal. Biol. Chem. 355, 81-89.
    7. Banala, S., Arnold, A. and Johnsson, K. (2008). ChemBio Chem. 9, 38-41.

    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. Cellular Imaging and Analysis FAQs
    1. Expression of SNAPf Fusions (N9183)
    2. Cloning of SNAP-tag Fusions in pSNAPf (N9183)

    Selection Tools

    Troubleshooting Guides

    Interactive Tools

    Application Notes

    If you generate more plasmid DNA by bacterial transformation, we recommend isolating the plasmid DNA using an endotoxin-free plasmid prep kit prior to transfection into mammalian cells.