pLentiGuide
(Plasmid #117986)

• Purpose: pLentiGuide is a gRNA insert lentiviral vector, with a stuffer sequence for BsmBI cloning of annealed oligos corresponding to sgRNA sequence. It has Puromycin resistance and Ampicilin resistance.
• Depositing Lab: Paul Khavari
• Publication: Rubin et al Cell. 2018 Dec 11. pii: S0092-8674(18)31514-9. doi: 10.1016/j.cell.2018.11.022.

Backbone

• Vector backbone: pCESAx (Addgene 59986)
• Modifications to backbone: For sgRNA expression, we modified the sgRNA F+E scaffold (Chen et al., 2013; https://www.addgene.org/59986/ Addgene: U6>sgRNA(F+E) www.addgene.org U6 promoter based on Nishiyama, A. and Fujiwara, S. (2008). RNA interference by expressing short hairpin RNA in the Ciona intestinalis embryo. ) in two ways. First, we exchanged the murine U6 promoter and telomerase-targeting sgRNA with the human U6 promoter, stuffer region, and associated BsmBI cloning sites from lentiCRISPRv2. Additionally, we removed a BsmBI restriction site in the puromycin resistance gene by introducing a non-synonymous mutation.
• Vector type: Lentiviral
• Selectable markers: Puromycin

Growth in Bacteria

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

Gene/Insert

• Gene/Insert name: sgRNA backbone (F+E)
• gRNA/shRNA sequence: Empty

Resource Information

• Articles Citing this Plasmid:
  • 9 References

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
  • Takara Bio Limited Use Label License (formerly Clontech)
• Industry Terms:
  • Not Available to Industry

Depositor Comments

Users should follow the oligo cloning protocol for plasmid lentiCRISPR v2 (Addgene #52961) to 1) remove stuffer fragment and 2) insert annealed oligos to complete gRNA.