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Home Discontinued Products pACP-tag(m)-2 Vector

pACP-tag(m)-2 Vector

  • This product was discontinued on January 05, 2015

Ordering Information

N9322
  • Discontinued
    Discontinued
  • Product Information
    pACP-tag(m)-2 Vector is a mammalian expression plasmid encoding ACPwt, an ACP-tag protein, which is expressed under control of the CMV promoter. The expression plasmid has an IRES (Internal Ribosome Entry Site) and a neomycin resistance gene downstream of the ACP-tag for the efficient selection of stable transfectants. This plasmid is intended for the cloning and transient or stable expression of ACP-tag protein fusions in mammalian cells. pACP-tag(m)-2 contains two multiple cloning sites to allow cloning of the fusion partner as a fusion to the C-terminus of the ACP-tag and an appropriate signal peptide to the N-terminus of the ACP-tag.

    The ACP-tag is a small protein tag (8 kDa) based on the acyl carrier protein (ACP). It allows the specific, covalent attachment of virtually any molecule to a protein of interest. ACP-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 ACP-tag by a phosphopantetheine transferase (ACP or SFP Synthase). Having no cysteines, the ACP-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: subcloning and expression of the protein of interest as an ACP-tag fusion, and labeling of the fusion protein with the CoA substrate of choice. In this document, the cloning and expression of ACP-tag protein fusions is described. The labeling of fusion proteins with CoA substrates is described in the documentation supplied with CoA substrates and ACP or SFP Synthase.



    Cloning Region of pACP-tag(m)-2
    Unique restriction sites in the regions flanking the ACP-tag gene are displayed above the coding strand. The complete sequence for pACP-tag(m)-2 and pACP-ADRβ2 can be downloaded here 

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

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    Discontinued
    • Advantages and Features

      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.
    • Properties and Usage

      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
    • Notes
      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.

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  • Quality & Safety
    • 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.
    • 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.)
  • Legal Information
    • 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.

      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.


      FuGENE® is a registered trademark of Roche.

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