pb-NES-ZapCV5 (cpV143)
(Plasmid #112061)

• Purpose: Genetically-encoded Cyan-Yellow Zinc FRET sensor. Localizes to the cytosol. Low affinity to free zinc (Kd ~ 300nM, n = 0.55) due to mutation of the zinc binding Cys residues to His.
• Depositing Lab: Amy Palmer
• Publication: Fiedler et al Anal Chem. 2017 Jan 3;89(1):711-719. doi: 10.1021/acs.analchem.6b03235. Epub 2016 Dec 13.

Backbone

• Vector backbone: pPBbsr2-3594NLS
• Vector type: Mammalian Expression
• Selectable markers: Blasticidin

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH5alpha
• Growth instructions: Depositing lab uses Omnimax cells.
• Copy number: High Copy

Gene/Insert

• Gene/Insert name: NES-ZapCV5
• Species: Synthetic
• Insert Size (bp): 1737
• Mutation: ECFP gene is truncated 36 bp at 3' end. Venus gene is circurlarly permuted at residue 173. Zap5 zinc binding domain derived from S. cerevisiae Zap1 zinc finger domains 1 & 2 with C581H, C586H, C618H and C623H mutations .
• Promoter: CBA
• Tag / Fusion Protein:
  • Nuclear Export Signal (NES) derived from HIV-1 (N terminal on insert)

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ cloning site: EcoRI (not destroyed)
• 3′ cloning site: SalI (not destroyed)
• 5′ sequencing primer: Quintara Bio: pCX-cMyc-5 (5'-GCGAGCCGCAGCCATTGCCTTTTA-3')
• 3′ sequencing primer: Quintara Bio: IRES-Rev (5'-CCTCACATTGCCAAAAGACG-3')

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
  • piggyBac Limited Use Label License
• Industry Terms:
  • Not Available to Industry