lentiCRISPR v2
(Plasmid #52961)

• Purpose: Replaces original lentiCRISPRv1 (Addgene Plasmid 49535) and produces ~10-fold higher titer virus. 3rd generation 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: Custom
• Backbone size w/o insert (bp): 10000
• Total vector size (bp): 14873
• 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
• 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
• 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: hEF1aprom-F
• 3′ sequencing primer: WPRE-R

Gene/Insert 2

• Gene/Insert name: Puromycin resistance
• Alt name: puromycin N-acetyl-transferase
• Alt name: PAC
• Insert Size (bp): 600
• Promoter: EFS-NS

Cloning Information for Gene/Insert 2

• Cloning method: Restriction Enzyme
• 5′ cloning site: BamHI (not destroyed)
• 3′ cloning site: MluI (not destroyed)
• 5′ sequencing primer: Puro-F2
• 3′ sequencing primer: WPRE-R

Resource Information

• Supplemental Documents:
  • GenBank file
  • lentiCRISPRv2 and lentiGuide oligo cloning protocol
• Addgene Notes:
  • Addgene Diagnostic Digest
  • Addgene Diagnostic Digest
• Articles Citing this Plasmid:
  • 2473 References

Terms and Licenses

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

Depositor Comments

This plasmid is an updated version of the original lentiCRISPR (Addgene plasmid #49535)

IMPORTANT: The primers suggestions listed above are for gene inserts that exist in the untouched vector. After you have inserted your gRNA, you should use hU6-F (5'-GAGGGCCTATTTCCCATGATT-3') or LKO.1 5’(5'- GACTATCATATGCTTACCGT-3') to sequence that region.

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.

Information for Cloning Grade DNA (Catalog # 52961-DNA.cg) ( Back to top)

Item is discontinued.

Addgene no longer distributes this item. Contact for more information.

2 µg of cloning grade DNA in Tris buffer

Purpose

Cloning grade DNA is suitable for use in PCR, cloning reactions, or transformation into E. coli. The purity and amount is not suitable for direct transfections.

Delivery

• Amount: 2 µg
• Guaranteed Concentration: 100 ng/µl +/- 5 ng/µl
• Pricing: $105 USD
• Storage: DNA can be stored at 4℃ (short term) or -20℃ (long term).

Terms and Licenses

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

Quality Control

Addgene has verified this plasmid using Next Generation Sequencing. Results are available here