pCAGFP
(Plasmid #32748)

• Depositing Lab: Jeanne Hardy
• Publication: Nicholls et al J Biol Chem. 2011 Jul 15;286(28):24977-86. Epub 2011 May 10.

Backbone

• Vector backbone: pmKate2-C
• Backbone manufacturer: Clontech
• Backbone size w/o insert (bp): 3970
• Vector type: Mammalian Expression
• Selectable markers: Neomycin (select with G418)

Growth in Bacteria

• Bacterial Resistance(s): Kanamycin, 50 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH5alpha
• Copy number: High Copy

Gene/Insert

• Gene/Insert name: GFP
• Insert Size (bp): 854
• Mutation: S65T
• Promoter: CMV
• Tag / Fusion Protein:
  • DEVDFQGPCNDSSDPLVVAASIIGILHLILWILDRL (C terminal on insert)

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ cloning site: NheI (not destroyed)
• 3′ cloning site: EcoRI (not destroyed)
• 5′ sequencing primer: CMV-F
• 3′ sequencing primer: SV40pA-R

Resource Information

• Articles Citing this Plasmid:
  • 3 References

Terms and Licenses

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

Depositor Comments

The gene for dark GFP (S65T) (referred to as GFP from this point forward) was created by PCR amplifying the GFP gene using a reverse primer that also encoded the 27 amino acid quenching peptide derived from the transmembrane region of influenza M2. A sequence encoding the linker (LEVLFQGP) was then inserted between the EGFP and M2 genes using the QuikChange mutagenesis (Stratagene) approach. The newly inserted linker sequence was then mutated to encode the caspase-7 cleavage recognition site (DEVDFQGP). The final sequence of CA-GFP protein is GFP with the fusion of DEVDFQGPCNDSSDPLVVAASIIGILHLILWILDRL at the C terminus. This construct was used for expression and purification of CA-GFP.