CKAR
(Plasmid #14860)

• Depositing Lab: Alexandra Newton
• Publication: Violin et al J Cell Biol. 2003 Jun 9. 161(5):899-909.

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, 100 μg/mL
• 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

Resource Information

• Articles Citing this Plasmid:
  • 10 References

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
• Industry Terms:
  • Not Available to Industry

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.