lentiGuide-Puro
(Plasmid #52963)

• Purpose: Expresses S. pyogenes CRISPR chimeric RNA element with customizable sgRNA from U6 promoter and puromycin resistance from EF-1a promoter. 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
• Total vector size (bp): 10183
• 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): NEB Stable
• Growth instructions: Use SapI digest to check for unwanted recombination of lentiviral plasmid. Only amplify in RecA- bacteria (eg. Stbl3 or NEB stable).
• Copy number: High Copy

Gene/Insert 1

• Gene/Insert name: S. pyogenes sgRNA cassette
• Alt name: S. pyogenes CRISPR customizable RNA element
• Species: Synthetic
• Insert Size (bp): 100
• Promoter: hU6

Cloning Information for Gene/Insert 1

• Cloning method: Restriction Enzyme
• 5′ cloning site: BsmBI (not destroyed)
• 3′ cloning site: BsmBI (not destroyed)
• 5′ sequencing primer: hU6-F
• 3′ sequencing primer: hGata4-rev (5'-ATTGTGGATGAATACTGCC-3')

Gene/Insert 2

• Gene/Insert name: Puromycin resistance
• Alt name: puromycin N-acetyl-transferase
• Alt name: PAC
• Insert Size (bp): 600
• Promoter: Ef1-a

Cloning Information for Gene/Insert 2

• Cloning method: Restriction Enzyme
• 5′ cloning site: BsiWI (not destroyed)
• 3′ cloning site: MluI (not destroyed)
• 5′ sequencing primer: EF-1a-F
• 3′ sequencing primer: WPRE-R

Resource Information

• Supplemental Documents:
  • GenBank file
  • lentiCRISPRv2 and lentiGuide oligo cloning protocol
• Articles Citing this Plasmid:
  • 560 References

Terms and Licenses

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

Depositor Comments

Note that this plasmid does NOT contain Cas9. It should be used in conjunction with lentiCas9-Blast (Addgene #52962) or otherwise with cell lines already expressing Cas9.

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 # 52963-DNA.cg) ( Back to top)

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