BPK1520
(Plasmid #65777)

• Purpose: Human expression plasmid for SpCas9 sgRNA (need to clone in spacer into BsmBI sites): U6-BsmBIcassette-Sp-sgRNA
• Depositing Lab: Keith Joung
• Publication: Kleinstiver et al Nature. 2015 Jun 22. doi: 10.1038/nature14592.

Backbone

• Vector backbone: MLM3636
• Backbone manufacturer: Joung lab (Addgene Plasmid # 43860)
• Vector type: Mammalian Expression, CRISPR

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): XL1 Blue
• Copy number: High Copy

Gene/Insert

• Gene/Insert name: SpCas9 gRNA backbone, without spacer sequence
• Species: Synthetic
• Promoter: U6

Cloning Information

• Cloning method: Gibson Cloning
• 5′ sequencing primer: OS280 (5'-CAGGGTTATTGTCTCATGAGCGG-3')

Resource Information

• Supplemental Documents:
  • Annotated Genbank file
  • Joung Lab gRNA Cloning Protocol
• Addgene Notes:
  • Addgene Diagnostic Digest
• Articles Citing this Plasmid:
  • 38 References

Terms and Licenses

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

Depositor Comments

Use BsmBI sites to insert sgRNA spacer sequence into the vector. See the supplemental document "Joung Lab gRNA Cloning Protocol" for more details.

This plasmid has been found to be somewhat unstable and prone to concatenation. Concatenation often does not impact plasmid function, but may reduce transformation efficiencies. You may need to screen multiple colonies to isolate the monomeric version of this plasmid. If you still have trouble isolating the monomeric version, you might consider linearizing, gel extracting, re-ligating, and transforming the plasmid.