Features

BACKGROUND:

A plasmid map and the sequence of the cloning region can be found above. The complete plasmid sequence can be downloaded. This plasmid encodes the gene ACPwt which is the wild type Acyl Carrier Protein from E. coli. In the plasmid sequence, the ACPwt gene is encoded from bp 975 to 1208. The ACPwt gene was cloned via EcoRI and SbfI including additional flanking restriction sites.

This plasmid is intended for the cloning and stable or transient expression of ACP-tag protein fusions in mammalian cells. It is suitable for the efficient production of stable cell lines expressing ACP-tag gene fusions. The plasmid contains the CMV promoter followed by the genes for the ACP-tag 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 ACP-tag fusion protein. Unless the expression experiments require a pure population of cells, the pool of resistant cells can simply be used, 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 should be cloned downstream of the ACP-tag coding sequence, as a fusion to the C-terminus of the ACP-tag. An appropriate cell surface signal peptide should be cloned upstream of the ACP-tag as an N-terminal fusion. A Kozak sequence is located upstream of the ACPwt gene to increase the translation efficiency of the fusion protein. The ACPwt gene can be isolated from the plasmid using PCR or direct cloning in order to subclone it into a different vector of choice.

Materials Required but not Supplied

• Mammalian cell lines
• Transfection reagents
• CoA substrates
• ACP or SFP Synthase
• Tissue culture reagents and media

Storage Temperature

-20°C

1. Storage: pACP-tag(m)-2 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.
   
   

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. Cloning of ACP-tag Fusions in pACP-tag(m)-2 (N9322)
2. Expression of ACP-tag Fusions (N9322)

• Comparison of SNAP-tag®/CLIP-tag™ Technologies to GFP
• SNAP-tag®/CLIP-tag® Cloning Vector and Plasmid Selection Chart

• Labeling with SNAP-tag® Technology Troubleshooting Guide

• DNA Sequences and Maps Tool

• NEBioCalculator

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

• pACP-tag(m)-2 Vector

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.

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.

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