pdCas9+sgRNA expression vector precursor
(Plasmid #171798)

• Purpose: (Empty Backbone) PICASSO: Library expression vector precursor for paired dCas9+sgRNA expression
• Depositing Lab: Stephen Elledge
• Publication: Barber et al Mol Cell. 2021 Aug 4. pii: S1097-2765(21)00596-7. doi: 10.1016/j.molcel.2021.07.027.

Backbone

• Vector backbone: pgRNA (Qi et al 2013)
• Modifications to backbone: Combined elements of pdCas9 (Qi et al 2013) with pgRNA (Qi et al 2013) to create dCas9-fusion+sgRNA co-expression precursor vector
• Vector type: Bacterial Expression, CRISPR
• Promoter: pLtetO

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH5alpha
• Copy number: High Copy

Resource Information

• A portion of this plasmid was derived from a plasmid made by: This plasmid was derived by combining elements from two preexisting plasmids: Addgene #44251 + Addgene #44249
• Article Citing this Plasmid:
  • 1 Reference

Terms and Licenses

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

Depositor Comments

C-terminal dCas9 fusion peptide or protein + sgRNA spacer sequence should be inserted via PvuI/BsaI sites in this vector (restriction digestion/ligation or Gibson is possible; resulting RFP excision from this vector should yield white colonies when plated).

A second subcloning step to insert the necessary expression scaffold for proper dCas9-fusion and sgRNA expression should then be introduced via SalI/SpeI digestion/ligation. SalI/SpeI sites should be encoded immediately 3' to dCas9-fusion and 5' to sgRNA spacer introduced in first subcloning step.