pLSC-5
(Plasmid #62889)

• Purpose: Lenti vector for expression of inducible split Cas9
• Depositing Lab: Feng Zhang
• Publication: Zetsche et al Nat Biotechnol. 2015 Feb 2. doi: 10.1038/nbt.3149.

Backbone

• Vector backbone: lentiCRISPR
• Backbone manufacturer: Zhang lab
• Backbone size w/o insert (bp): 8809
• Total vector size (bp): 13792
• Vector type: Mammalian Expression, Lentiviral, CRISPR
• Selectable markers: Puromycin

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): Stbl3
• Copy number: Unknown

Gene/Insert 1

• Gene/Insert name: SpCas9(574-1368)
• Insert Size (bp): 2916
• Mutation: *see comment below
• Promoter: EFS
• Tags / Fusion Proteins:
  • NLS SV40 (N terminal on insert)
  • FFBP12 (N terminal on insert)
  • P2A (C terminal on insert)

Cloning Information for Gene/Insert 1

• Cloning method: Restriction Enzyme
• 5′ cloning site: Age1 (not destroyed)
• 3′ cloning site: BamH1 (not destroyed)
• 5′ sequencing primer: NA

Gene/Insert 2

• Gene/Insert name: SpCas9(2-573)
• Insert Size (bp): 2121
• Promoter: EFS
• Tags / Fusion Proteins:
  • NES PTK2 (N terminal on insert)
  • FRB (C terminal on insert)
  • P2A (C terminal on insert)

Cloning Information for Gene/Insert 2

• Cloning method: Restriction Enzyme
• 5′ cloning site: BamH1 (not destroyed)
• 3′ cloning site: BamH1 (not destroyed)
• 5′ sequencing primer: NA

Resource Information

• Supplemental Documents:
  • pLSC-5 annotated sequence
• A portion of this plasmid was derived from a plasmid made by: SpCas9 pieces were PCR amplified of PX330.
• Articles Citing this Plasmid:
  • 4 References

Terms and Licenses

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

Depositor Comments

*Note: The C-terminal fragment of Cas9 has a T593A amino acid residue substitution, but this residue change does not affect protein function. (The mutation is located at the base of a small alpha helix, which does not interact with the guide RNA or target DNA.)