pcDNA-NES-ZapCY2
(Plasmid #59016)

• Purpose: Cytosolic zinc-binding FRET biosensor
• Depositing Lab: Amy Palmer
• Publication: Qin et al ACS Chem Biol. 2013 Nov 15;8(11):2366-71. doi: 10.1021/cb4003859. Epub 2013 Sep 3.

Backbone

• Vector backbone: pcDNA3.1(+)
• Backbone manufacturer: Invitrogen
• Backbone size w/o insert (bp): 5500
• Total vector size (bp): 7200
• Modifications to backbone: Leu-rich NES sequence added upstream of BamHI site
• 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): DH10B
• Copy number: High Copy

Gene/Insert

• Gene/Insert name: NES-ZapCY2 genetically encoded cytosolic Zn(II) sensor
• Species: Synthetic
• Insert Size (bp): 1671
• Mutation: See comment
• Promoter: CMV
• Tag / Fusion Protein:
  • Nuclear Exclusion Signal (N terminal on backbone)

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ cloning site: BamHI (not destroyed)
• 3′ cloning site: EcoRI (not destroyed)
• 5′ sequencing primer: T7 Forward
• 3′ sequencing primer: BGH Reverse

Terms and Licenses

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

Depositor Comments

Genetically encoded, ratiometric, fluorescent biosensor. Contains nuclear exclusion signal sequence at N-terminus. Zn(II)-binding domain derived from the first 2 zinc fingers of Zap 1 from Saccharomyces cerevisiae. Contains truncated CFP and Citrine fluorescent proteins.

The Zn(II) binding domain of ZapCY2 is derived from ZapCY1. The first cysteine in each zinc finger is mutated to histidine.