SpCas9_sgRNA_expression_in_pBluescript
(Plasmid #122089)

• Purpose: U6 driven SpCas9 sgRNA expression vector for cloning own guides
• Depositing Lab: Scot Wolfe
• Publication: Bolukbasi et al Nat Commun. 2018 Nov 19;9(1):4856. doi: 10.1038/s41467-018-07310-x.

Backbone

• Vector backbone: pBS
• Backbone size w/o insert (bp): 4280
• Total vector size (bp): 4356
• 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: SpCas9 tracr
• Species: Synthetic; S. pyogenes
• Insert Size (bp): 76
• Entrez Gene: NEWENTRY
• Promoter: U6

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ cloning site: BfuAI (destroyed during cloning)
• 3′ cloning site: BfuAI (destroyed during cloning)
• 5′ sequencing primer: T3 Promoter
• 3′ sequencing primer: T7 Promoter

Resource Information

• Supplemental Documents:
  • SpCas9_sgRNA_expression in pBluescript.gb
• Articles Citing this Plasmid:
  • 2 References

Terms and Licenses

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

Depositor Comments

To design oligos for cloning: 5'-ACCG-Spacer_Sequence-3' and 5'-AAAC-Rev_Comp_Spacer-3'
For sgRNA cloning: Digest with BfuAI and ligate in phosphorylated and annealed oligos