MSP712
(Plasmid #65768)

• Purpose: Bacterial expression plasmid for Sp-dCas9 & sgRNA (need to clone in spacer into BsaI sites): T7-humanSpdCas9(D10A/H840A)-T7-BsaIcassette-Sp-sgRNA
• Depositing Lab: Keith Joung
• Publication: Kleinstiver et al Nature. 2015 Jun 22. doi: 10.1038/nature14592.

Backbone

• Vector backbone: pACYCDuet-1
• Backbone manufacturer: EMD Millipore
• Vector type: Bacterial Expression, CRISPR

Growth in Bacteria

• Bacterial Resistance(s): Chloramphenicol, 25 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): XL1 Blue
• Copy number: Low Copy

Gene/Insert

• Gene/Insert name: mammalian codon-optimized Streptococcus pyogenes dCas9 (D10A/H840A)-NLS-3XFlag, and SpCas9 gRNA
• Mutation: D10A and H840A mutations in Cas9
• Promoter: T7 (x2)
• Tags / Fusion Proteins:
  • NLS (C terminal on insert)
  • 3x FLAG (C terminal on insert)

Cloning Information

• Cloning method: Gibson Cloning
• 5′ sequencing primer: ACYC-DuetUP1
• 3′ sequencing primer: oBK390 (5'-GCAAATTCGACCCGGTCGTCG-3')

Resource Information

• Supplemental Documents:
  • Annotated Genbank file
  • Joung Lab gRNA Cloning Protocol
• A portion of this plasmid was derived from a plasmid made by: The pACYCDuet-1 plasmid backbone was provided by The University of Western Ontario
• Article Citing this Plasmid:
  • 1 Reference

Terms and Licenses

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

Depositor Comments

Use BsaI sites to insert sgRNA spacer sequence into the vector.