MSP2292
(Plasmid #70707)

• Purpose: Bacterial expression plasmid for KKH SaCas9 & sgRNA targeted to site 2: T7-humanSaCas9(E782K/N968K/R1015H)-NLS-3xFLAG-T7-Sa-sgRNA(84) #2
• Depositing Lab: Keith Joung
• Publication: Kleinstiver et al Nat Biotechnol. 2015 Nov 2. doi: 10.1038/nbt.3404.

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): DH5alpha
• Copy number: Low Copy

Gene/Insert 1

• Gene/Insert name: mammalian codon-optimized KKH variant Staphylococcus aureus Cas9
• Alt name: SauCas9
• Species: S. aureus
• Mutation: E782K, N968K and R1015H in SaCas9
• Promoter: T7
• Tag / Fusion Protein:
  • NLS-3xFLAG (C terminal on insert)

Cloning Information for Gene/Insert 1

• Cloning method: Unknown
• 5′ sequencing primer: ACYCDuetUP1
• 3′ sequencing primer: oBK390 (5'-GCAAATTCGACCCGGTCGTCG-3')

Gene/Insert 2

• Gene/Insert name: SaCas9 sgRNA (+84)
• Promoter: T7

Cloning Information for Gene/Insert 2

• Cloning method: Unknown
• 5′ sequencing primer: DuetDOWN1
• 3′ sequencing primer: T7-term

Resource Information

• Supplemental Documents:
  • Annotated GenBank file

Terms and Licenses

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

Depositor Comments

For this plasmid, the sequence of the sgRNA spacer is GTCGCCCTCGAACTTCACCT. The length of the sgRNA (not including the spacer sequence) is 84 nucleotides, where this truncated repeat/anti-repeat sgRNA is similar to that described previously by Ran et al., Nature 2015 (PubMed ID: 25830891) and conveys higher activity than the canonical full length sgRNA.