EF1a-dCas9-KRAB-GFP backbone
(Plasmid #194281)

• Purpose: Vector for CRISPRi-GFP expression ready for gateway cloning of sgRNA
• Depositing Lab: Elizabeth Kirby
• Publication: Rieskamp et al iScience. 2023 Jun 8;26(7):107068. doi: 10.1016/j.isci.2023.107068. eCollection 2023 Jul 21.

Backbone

• Vector backbone: FUGW; Addgene 71237
• Backbone size w/o insert (bp): 7000
• Total vector size (bp): 14927
• Modifications to backbone: Added gateway cloning (Esp3I) downstream of U6 promoter; replaced hUbC promoter with EF1a
• Vector type: Lentiviral, CRISPR

Growth in Bacteria

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

Gene/Insert

• Gene/Insert name: dCas9-KRAB-GFP
• Alt name: CRISPRi
• gRNA/shRNA sequence: gateway cloning site plus gRNA scaffold
• Species: M. musculus (mouse)
• Promoter: EF1a
• Tag / Fusion Protein:
  • GFP

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ cloning site: Pacl (unknown if destroyed)
• 3′ cloning site: Acc65I (unknown if destroyed)
• 5′ sequencing primer: EF1a-F; U6-F
• 3′ sequencing primer: GFP-R

Resource Information

• Supplemental Documents:
  • p046.ape

Terms and Licenses

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

Depositor Comments

Use Esp3I (type IIS restriction enzyme) for scarless insert of desired sgRNA downstream of U6 promoter.
5’ ggacG(N)20 3’
5’ aaac(N’)20c 3’
Where N = 20 nt sgRNA sequence for your target gene
ggac and aaac are the overhanging ends to complement the Esp3I cut p031. The extra g in the plus strand (and the complement c in the minus strand) is to promote RNA pol III transcriptional initiation.

Please visit https://doi.org/10.1101/2022.10.31.514562 for bioRxiv preprint.