Product Class: Other

pSNAPf Vector

Product Introduction

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.

  • View sequence details
  • 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
  • pSNAPf contains an improved version of SNAP-tag
  • SNAPf displays faster kinetics in in vitro labeling than SNAP-tag, and fast, specific and efficient labeling in live and fixed cell applications

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Catalog # Size Concentration
N9183S 20 µg 0.5 mg/ml

Product Information

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.

Cloning Region of pSNAP Cloning Region of pSNAP


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.
Product Categories:
Cellular Analysis Vectors Products,
DNA Plasmids & Substrates Products,
Protein Labeling Products
Applications:
Snap Surface Products,
In vivo Imaging,
Pulse Chase
,
Receptor Internalization
,
Protein Localization
,
Protein Labeling Snap Clip

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 & Usage

Materials Required but not Supplied

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

Lac Repressor on Plasmid

0

Storage Temperature

-20°C

Sequence Files

Fasta GenBank

Product Notes

  1. Storage: pSNAPf Vector issupplied in TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA) at a concentration of0.5 µg/µl. Plasmid solutions can be stored at 4°C for up to one week. Forlong-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.

Protocols, Manuals & Usage

Protocols

  1. Expression of SNAPf Fusions (N9183)
  2. Cloning of SNAP-tag Fusions in pSNAPf (N9183)

Application Notes

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 [email protected] or fill out the Technical Support Form for appropriate document.)

Tools & Resources

Selection Charts

Web Tools

FAQs & Troubleshooting

FAQs

  1. Cellular Imaging and Analysis FAQs
  2. What is the DNA sequence of this product?

Tech Tips

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.

Quality, Safety & Legal

Quality Assurance Statement

Quality Control tests are performed on each new lot of NEB product to meet the specifications designated for it. Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. Further information regarding NEB product quality can be found here.

Specifications

The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]

Certificate Of Analysis

The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality controls for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]

Safety DataSheets

The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.

Legal and Disclaimers

This product is covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB).

While NEB develops and validates its products for various applications, the use of this product may require the buyer to obtain additional third party intellectual property rights for certain applications.

For more information about commercial rights, please contact NEB's Global Business Development team at [email protected].

This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.

New England Biolabs (NEB) is committed to practicing ethical science – we believe it is our job as researchers to ask the important questions that when answered will help preserve our quality of life and the world that we live in. However, this research should always be done in safe and ethical manner. Learn more.

Licenses

Notice To Buyer/User: The Buyer/User has a non-exclusive license to use this system or any component thereof for RESEARCH AND DEVELOPMENT PURPOSES ONLY. Commercial use of this system or any components thereof requires a license from New England Biolabs, Inc., 240 County Road, Ipswich, MA 01938.

Cellular Imaging and Analysis (i.e., SNAP and CLIP products)

Notice to Buyer/User: The Buyer/User has a non-exclusive license to use this system or any component thereof for RESEARCH AND DEVELOPMENT ONLY. Commercial use of this system or any components thereof requires a license from New England Biolabs, Inc., 240 County Road, Ipswich, MA 01938.

These patents and patent applications are owned by Covalys, or owned by the Ecole Polytechnique Fédérale de Lausanne (EPFL) and exclusively licensed to Covalys and NEB.

The CMV promoter is covered under U.S. Patent No. 5,385,839 and its use is permitted for research purposes only. Any other use of the CMV promoter requires a license from the University of Iowa Research Foundation, 214 Technology Innovation Center, Iowa City, IA 52242.

The products and/or their use may be covered by one or more of the following patents and patent applications:
Methods for Using O6-Alkylguanine-DNA-Alkyltransferases: US 7,939,284; US 8,367,361; EP 1 410 023; EP 2 037 271; EP 1 696 234; EP 2 211 177; JP 4195815; JP 4226053; CN 1295510; CN 1975422; and SG 100125.

Mutants of O6-Alkylguanine-DNA-Alkyltransferases: US 7,888,090. EP 1720981 (pending).