3xFLAG-dCas9/pCMV-7.1
(Plasmid #47948)

• Purpose: Expresses 3xFLAG-dCas9 in mammalian cells for enChIP analysis to purify specific genomic regions of interest.
• Depositing Lab: Hodaka Fujii
• Publication: Fujita et al Biochem Biophys Res Commun. 2013 Sep 13;439(1):132-6. doi: 10.1016/j.bbrc.2013.08.013. Epub 2013 Aug 11.

Backbone

• Vector backbone: pCMV-7.1
• Backbone manufacturer: Sigma-Aldrich
• Backbone size w/o insert (bp): 4636
• Total vector size (bp): 8848
• Vector type: Mammalian Expression, CRISPR

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 30°C
• Growth Strain(s): NEB Stable
• Copy number: High Copy

Gene/Insert

• Gene/Insert name: 3xFLAG-dCas9
• Insert Size (bp): 4212
• Mutation: human codon-optimized, D10A + H840A (catalytically inactive)
• Promoter: CMV
• Tags / Fusion Proteins:
  • 3xFLAG (N terminal on insert)
  • NLS (C terminal on insert)

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ cloning site: Hind III (destroyed during cloning)
• 3′ cloning site: EcoR V (destroyed during cloning)
• 5′ sequencing primer: AAT GTC GTA ATA ACC CCG CCC CGT TGA CGC
• 3′ sequencing primer: TAT TAG GAC AAG GCT GGT GGG CAC

Resource Information

• Articles Citing this Plasmid:
  • 12 References

Terms and Licenses

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

Depositor Comments

This plasmid can be used to isolate specific genomic regions of interest using a catalytically inactive Cas9 fused with a tag(s).

Purify: locus-specific chromatin immunoprecipitation (enChIP)
This system is compatible with gRNA_cloning_vector (www.addgene.org/41824) from the Church lab.

Additional information and protocols can be found at:
http://www.med.hirosaki-u.ac.jp/~bgb/iChIP_protocols/index_e.html

For more information on Fujii Lab CRISPR Plasmids please refer to: http://www.addgene.org/crispr/fujii/