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CKAR
(Plasmid #14860)

Ordering

Item Catalog # Description Quantity Price (USD)
Plasmid 14860 Standard format: Plasmid sent in bacteria as agar stab 1 $65

This material is available to academics and nonprofits only.

Backbone

  • Vector backbone
    pcDNA3
  • Backbone manufacturer
    Invitrogen
  • Backbone size w/o insert (bp) 5400
  • Vector type
    Mammalian Expression
  • Selectable markers
    Neomycin (select with G418)

Growth in Bacteria

  • Bacterial Resistance(s)
    Ampicillin
  • Growth Temperature
    37°C
  • Growth Strain(s)
    DH5alpha
  • Copy number
    High Copy

Gene/Insert

  • Gene/Insert name
    C kinase activity reporter
  • Alt name
    CFP-FHA2-peptide-YFP
  • Tags / Fusion Proteins
    • CFP (N terminal on insert)
    • YFP (C terminal on insert)

Cloning Information

  • Cloning method Restriction Enzyme
  • 5′ cloning site HindIII (not destroyed)
  • 3′ cloning site XbaI (not destroyed)
  • 5′ sequencing primer T7
  • (Common Sequencing Primers)

Resource Information

Depositor Comments

PKC substrate sequence flanked by flexible linker sequence: GGSGG RFRRFQTLKIKAKA GGSGG. A206K mutation present in both CFP (mCFP) and citrine (mYFP) to reduce the intrinsic homoaffinity of all GFPs and preclude intermolecular FRET by CFP-YFP dimerization. CFP was amplified by PCR from a plasmid template to encode a HindIII restriction site followed by a consensus initiation site for translation (CGCCACC) before the initiating ATG of CFP, and a KpnI restriction site at the 3' end instead of a terminating codon. FHA2, a gift from Michael Yaffe (MIT, Cambridge, MA) was amplified by PCR to include KpnI and BamHI restriction sites at the 5' and 3' ends, respectively. Citrine was amplified by PCR to include a 5' BamHI followed by the PKC substrate sequence and a 3' XbaI following the terminating codon.

How to cite this plasmid ( Back to top)

These plasmids were created by your colleagues. Please acknowledge the Principal Investigator, cite the article in which the plasmids were described, and include Addgene in the Materials and Methods of your future publications.

  • For your Materials & Methods section:

    CKAR was a gift from Alexandra Newton (Addgene plasmid # 14860 ; http://n2t.net/addgene:14860 ; RRID:Addgene_14860)
  • For your References section:

    A genetically encoded fluorescent reporter reveals oscillatory phosphorylation by protein kinase C. Violin JD, Zhang J, Tsien RY, Newton AC. J Cell Biol. 2003 Jun 9. 161(5):899-909. 10.1083/jcb.200302125 PubMed 12782683