pET-(-30)dGFP-9xGGS-Cath-NLS-Cas9-6xHis
(Plasmid #62373)

• Purpose: Expression of (-30)dGFP-9xGGS-Cath-NLS-Cas9-6xHis in bacterial cells
• Depositing Lab: David Liu
• Publication: Zuris et al Nat Biotechnol. 2015 Jan;33(1):73-80. doi: 10.1038/nbt.3081. Epub 2014 Oct 30.

Backbone

• Vector backbone: pET29
• Backbone manufacturer: Novagen
• Backbone size w/o insert (bp): 5161
• Total vector size (bp): 10102
• Vector type: Bacterial Expression

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): Mach1
• Growth instructions: For protein expression, transform into BL21 Star (DE3) cells. Induce at 0.6 OD with 0.5 mM IPTG final concentration. Growing overnight (16 hours) at 20-25 C.
• Copy number: High Copy

Gene/Insert

• Gene/Insert name: (-30)dGFP-9xGGS-Cath-NLS-Cas9-6xHis
• Species: S. pyogenes
• Insert Size (bp): 4941
• Mutation: Y67S amino acid substitution in (-30)GFP renders GFP non-fluorescent
• Promoter: T7
• Tags / Fusion Proteins:
  • 6xHis tag (C terminal on backbone)
  • (-30)dGFP (N terminal on insert)
  • Cathepsin cleavage site (N terminal on insert)
  • NLS (N terminal on insert)

Cloning Information

• Cloning method: Gibson Cloning
• 5′ sequencing primer: T7 promoter
• 3′ sequencing primer: T7 terminator

Resource Information

• Supplemental Documents:
  • pET-(-30)dGFP-9xGGS-Cath-NLS-Cas9-6xHis.gb
• A portion of this plasmid was derived from a plasmid made by: pMJ806 served as template for Cas9 gene. This was reported in Pattanayak et. al. Nature Biotechnology 2013.

Terms and Licenses

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

Depositor Comments

Alternative plasmid name: pJAZ0003