pMCP-tag(m) Vector

Catalog # N9317 was discontinued on January 05, 2015

Product Information
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.

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.

Product Categories:

Discontinued Products
- Advantages and Features
  
  Features
  
  BACKGROUND:
  
  This plasmidencodes the gene MCP which is a mutant carrierprotein. In the plasmid sequence, the MCP gene isencoded from bp 690 to 923. The MCP gene wascloned via HindIII and PstI including additionalflanking restriction sites.
  
  This plasmid is intended as a transient expressionvector for MCP-tag gene fusions in mammalian celllines. It is not recommended for the productionof stable cell lines. The plasmid contains theb-lactamase (Ampicillin resistance) gene formaintenance in bacteria. The plasmid also containsa CMV promoter and SV40 polyadenylation signalsfor correct mRNA processing. The gene of interestshould be cloned downstream of the MCP-tagcoding sequence, as a fusion to the C-terminusof the MCP-tag. An appropriate cell surface signalpeptide should be cloned upstream of the MCP-tagas an N-terminal fusion. A Kozak sequence islocated upstream of the MCP gene to increase thetranslation efficiency of the fusion protein. TheMCP-tag gene can be isolated from the plasmidusing PCR or direct cloning in order to subclone itinto a different vector system of choice.
- Properties & Usage
  Materials Required but not Supplied
  
  Mammalian cell lines
  
  Transfection reagents
  
  CoA substrates
  
  Sequence Files
  Fasta GenBank
- Related Products
  Companion Products
  
  NEB® 10-beta Competent E. coli (High Efficiency)
- Product Notes
  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.
  
  NEB 10-beta Competent E. coli (High Efficiency) (NEB #C3019) is recommended for propagating and subcloning this vector.
Protocols, Manuals & Usage
- Protocols
  Expression of MCP Fusions (N9317)
  
  Cloning of MCP-tag Fusions in pMCP-tag(m) Vector (N9317)
- 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.)
  
  N9317Datasheet-Lot0011206
  
  N9317Datasheet-Lot0011012
  
  N9317Datasheet-Lot0011310
Tools & Resources
- Selection Charts
  SNAP-tag®/CLIP-tag® Cloning Vector Selection Chart
- Web Tools
  DNA Sequences and Maps Tool
FAQs & Troubleshooting
- FAQs
  What is the ACP-tag?
  
  How does it work?
  
  How specific is the binding of substrate to the ACP-tag?
  
  What linker type and length would you recommend?
  
  Can I clone my protein as fusion to the N- or C-terminus of the ACP-tag?
  
  Are ACP-tag substrates stable to fixation?
  
  Can ACP-tag be multiplexed with other protein labeling systems (GFP, Antibody)?
  
  Can you use ACP-tag for in vivo FRET?
  
  Does the ACP-tag labeling reaction work in Yeast?
Quality, Safety & Legal
- Quality Control Assays
  
  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.
- Safety Data Sheets
  The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.
  
  pMCP-tag(m) Vector
- 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.)
  
  N9317Datasheet-Lot0011206
  
  N9317Datasheet-Lot0011012
  
  N9317Datasheet-Lot0011310
- 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 gbd@neb.com.
  
  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
  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. For detailed information see our Terms of Use .
  
  The products and/or their use may be covered by one or more of the following patents and patent applications:
  7,939,284 (Methods for Using O6-Alkylguanine-DNA-Alkyltransferases)
  7,888,090 (Mutants of O6-Alkylguanine-DNA-Alkyltransferases)
  8,163,479 Specific Substrates for O6-Alkylguanine-DNA-Alkyltransferases)
  8,178,314 (Pyrimidines Reacting With O6-Alkylguanine-DNA-Alkyltransferases)
  PCT/EP2007/057597 (Labeling of Fusion Proteins with Synthetic Probes)
  EP07117800 (Drug Delivery)
  EP07117802 (Drug Delivery)
  EP07120288 (GTPase-Transient Protein Protein Interactions)
  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.