pLCKO
(Plasmid #73311)

• Purpose: (Empty Backbone) Lentiviral backbone for cloning and expressing U6 driven sgRNAs with BfuAI cloning sites and puromycin selection.
• Depositing Lab: Jason Moffat
• Publication: Hart et al Cell. 2015 Dec 3;163(6):1515-26. doi: 10.1016/j.cell.2015.11.015. Epub 2015 Nov 25.

Backbone

• Vector backbone: pLKO.1
• Backbone manufacturer: Broad Institute, Cambridge, MA
• Backbone size (bp): 9436
• Modifications to backbone: The invariant gRNA scaffold was amplified from pX330-U6-Chimeric_BB-CBh-hSpCas9 (Addgene 42230) with 5’ BspAI/BfuAI sites and cloned into the pLKO.TRC005 lentiviral vector (Broad Institute, Cambridge, MA) between the AgeI and EcoRI sites. BspAI/ BfuAI sites were used to clone individual 20 bp gRNA sequences.
• Vector type: Mammalian Expression, Lentiviral, CRISPR
• Promoter: U6
• Selectable markers: Puromycin

Growth in Bacteria

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

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ sequencing primer: U6 fwd

Resource Information

• Supplemental Documents:
  • Protocol for cloning individual sgRNAs into pLCKO lentiviral vector
• A portion of this plasmid was derived from a plasmid made by: pX330-U6-Chimeric_BB-CBh-hSpCas9 (Addgene 42230)
• Articles Citing this Plasmid:
  • 15 References

Terms and Licenses

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