lentiCRISPR v2-dCas9
(Plasmid #112233)

• Purpose: Lentiviral plasmid for expression of gRNA and dCas9 in mammalian cells; derivative of lentiCRISPR v2
• Depositing Lab: Thomas Gilmore
• Publication: Babaei et al PLoS One. 2019 Sep 25;14(9):e0222588. doi: 10.1371/journal.pone.0222588. eCollection 2019.

Backbone

• Vector backbone: Custom
• Backbone size w/o insert (bp): 10000
• Total vector size (bp): 14873
• Vector type: Mammalian Expression, Lentiviral, CRISPR
• Selectable markers: Puromycin

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 30°C
• Growth Strain(s): NEB Stable
• Growth instructions: Only amplify in RecA- bacteria
• Copy number: High Copy

Gene/Insert 1

• Gene/Insert name: dCas9
• Species: Synthetic
• Insert Size (bp): 4200
• Mutation: Changed: Aspartic acid 10 to Glycine, Histidine 840 to Alanine
• Promoter: EFS-NS
• Tag / Fusion Protein:
  • FLAG (C terminal on insert)

Cloning Information for Gene/Insert 1

• Cloning method: Restriction Enzyme
• 5′ cloning site: AgeI, XbaI, AfeI (not destroyed)
• 3′ cloning site: BamHI (not destroyed)
• 5′ sequencing primer: hEF1aprom-F
• 3′ sequencing primer: WPRE-R

Gene/Insert 2

• Gene/Insert name: Porumycin resistance
• Alt name: puromycin N-acetyl-transferase
• Alt name: PAC
• Insert Size (bp): 600
• Promoter: EFS-NS

Cloning Information for Gene/Insert 2

• Cloning method: Restriction Enzyme
• 5′ cloning site: BanHI (not destroyed)
• 3′ cloning site: MluI (not destroyed)
• 5′ sequencing primer: Puro-F2
• 3′ sequencing primer: WPRE-R

Resource Information

• A portion of this plasmid was derived from a plasmid made by: addgene
• Articles Citing this Plasmid:
  • 10 References

Terms and Licenses

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

Depositor Comments

Plasmid is a derivative of Addgene Plasmid #52961, where the Cas9 gene has been mutated at codons 10 and 840 to create a catalytically inactive version of Cas9 (dCas9).