pcDNA-NES-ZapCmR2
(Plasmid #59015)

• Purpose: Cytosolic zinc-binding FRET biosensor
• Depositing Lab: Amy Palmer
• Publication: Miranda et al PLoS One. 2012;7(11):e49371. doi: 10.1371/journal.pone.0049371. Epub 2012 Nov 16.

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-ZapCmR2 genetically encoded cytosolic Zn(II) sensor
• Species: Synthetic
• Insert Size (bp): 1689
• 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 Clover and mRuby fluorescent proteins.

The Zn(II) binding domain of ZapCmR2 is derived from ZapCmR1.1. The first cysteine in the second zinc finger is mutated to histidine.