Expression scaffold for dCas9+sgRNA
(Plasmid #171799)

• Purpose: PICASSO: Expression scaffold for second subcloning step to properly co-express paired dCas9-fusion+sgRNA
• 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: TOPO
• Vector type: CRISPR

Growth in Bacteria

• Bacterial Resistance(s): Kanamycin, 50 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH5alpha
• Copy number: High Copy

Gene/Insert

• Gene/Insert name: PICASSO: expression scaffold for proper dCas9-fusion + sgRNA expression
• Insert Size (bp): 1191

Resource Information

• Article Citing this Plasmid:
  • 1 Reference

Terms and Licenses

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

Depositor Comments

Encodes expression scaffold for proper dCas9-fusion and sgRNA co-expression. To be introduced in second subcloning step for PICASSO library preparation.

Insert encodes C-terminal dCas9-fusion epitope tag HA, stop codon, translational termination, chloramphenicol resistance marker, and sgRNA promoter.

Recommended primers for amplification (introduce HA+6His C-terminal epitope tags and T7 promoter for sgRNA expression):
5'-agtcagtcGTCGACTACCCGTACGACGTTCCGGACTACGCGggtggtcaccatcatcaccatcattaataagg-3'
5'-CAACCAACACTAGTCTATAGTGAGTCGTATTAGATCTTTAGGACGTCGATATCTG-3'

After amplification, the amplicon should be digested with SalI/SpeI for insertion into the dCas9+sgRNA paired expression vector (second subcloning step after peptide+sgRNA spacer insertion with internal SalI/SpeI restriction sites).