AP70_pU.CAG.Cas9-D10A-K848A-R1060A.rBGpA
(Plasmid #199254)

• Purpose: Expression construct encoding the high specificity SpCas9-KARA-D10A nickase
• Depositing Lab: Manuel Goncalves
• Publication: Wang et al Nucleic Acids Res. 2021 Jan 25;49(2):1173-1198. doi: 10.1093/nar/gkaa1236.

Backbone

• Vector backbone: pBR322
• Backbone size w/o insert (bp): 4965
• Total vector size (bp): 9360
• Vector type: Mammalian Expression, CRISPR

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH5alpha
• Copy number: High Copy

Gene/Insert

• Gene/Insert name: High specificity S. pyogenes Cas9-D10A-KARA nickase
• Alt name: SpCas9-KARA-D10A
• Species: Streptococcus pyogenes
• Insert Size (bp): 4266
• Mutation: D10A K848A R1060A
• Promoter: CAG promoter
• Tags / Fusion Proteins:
  • 3XFLAG epitope (N terminal on insert)
  • SV40 NLS (N terminal on insert)
  • Nucleoplasmin NLS (C terminal on insert)

Cloning Information

• Cloning method: Unknown
• 5′ sequencing primer: M13 rev
• 3′ sequencing primer: M13 fwd

Resource Information

• Supplemental Documents:
  • AP70_pU.CAG.Cas9-D10A-K848A-R1060A.rBGpA.gbk
• A portion of this plasmid was derived from a plasmid made by: The SpCas9 endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system, mutated to K848A and R1060A to improve targeting specificity (Slaymaker et al., Science 2016 351:84-88) was modified via the introduction of the RuvC nuclease domain-disabling mutation D10A and placed in a pCAG.rBGpA expression plasmid backbone.

Terms and Licenses

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