lentiCas9n(D10A)-Blast
(Plasmid #63593)

• Purpose: Expresses human codon-optimized S. pyogenes Cas9n (D10A nickase) protein and blasticidin resistance from EFS promoter. Lentiviral backbone.
• Depositing Lab: Feng Zhang
• Publication: Sanjana et al Nat Methods. 2014 Aug;11(8):783-4. doi: 10.1038/nmeth.3047.

Backbone

• Vector backbone: pFUGW
• Total vector size (bp): 12860
• Vector type: Mammalian Expression, Lentiviral, CRISPR
• Selectable markers: Blasticidin

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): NEB Stable
• Growth instructions: Use SapI digest to check for unwanted recombination of lentiviral plasmid. Only amplify in RecA- bacteria (eg. Stbl3).
• Copy number: High Copy

Gene/Insert 1

• Gene/Insert name: Cas9
• Alt name: S. pyogenes CRISPR-Cas9
• Species: Synthetic
• Insert Size (bp): 4200
• Mutation: D10A, nickase mutant
• Promoter: EFS-NS
• Tag / Fusion Protein:
  • FLAG (C terminal on insert)

Cloning Information for Gene/Insert 1

• Cloning method: Restriction Enzyme
• 5′ cloning site: AgeI, XbaI, AfeI (not destroyed)
• 3′ cloning site: BamHI (not destroyed)
• 5′ sequencing primer: pLLU-F
• 3′ sequencing primer: blast-R

Gene/Insert 2

• Gene/Insert name: Blasticidin resistance
• Alt name: blasticidin S deaminase
• Alt name: bsd
• Insert Size (bp): 399
• Promoter: EFS-NS

Cloning Information for Gene/Insert 2

• Cloning method: Restriction Enzyme
• 5′ cloning site: BamHI (not destroyed)
• 3′ cloning site: EcoRI (not destroyed)
• 5′ sequencing primer: needs custom
• 3′ sequencing primer: WPRE-R

Resource Information

• Supplemental Documents:
  • lentiCas9n Blast genbank file
• Articles Citing this Plasmid:
  • 3 References

Terms and Licenses

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

Depositor Comments

Special note from the Zhang lab: We are constantly improving our CRISPR reagents. Please check https://zlab.bio/ for the most up-to-date information.