pU6-(BbsI)_CBh-Cas9-T2A-BFP
(Plasmid #64323)

• Purpose: Expression vector for sgRNAs cloned into the BbsI sites and for expression of Cas9 linked to BFP via a T2A peptide
• Depositing Lab: Ralf Kuehn
• Publication: Chu et al Nat Biotechnol. 2015 Mar 24. doi: 10.1038/nbt.3198.

Backbone

• Vector backbone: pX330
• Backbone manufacturer: Zhang lab (Addgene plasmid # 42230)
• Modifications to backbone: The T2A-BFP fragment was cloned into FseI & EcoRI sites of pX330 (Addgene plasmid # 42230). The BFP template was derived from MSCV-IRES-BFP, a kind gift of Frank Rosenbauer and Martin Janz (Charite, Berlin)
• Vector type: Mammalian Expression, CRISPR

Growth in Bacteria

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

Gene/Insert 1

• Gene/Insert name: Cas9
• Alt name: 3xFLAG-NLS-Cas9-NLS-T2A-EBFP2
• Species: Streptococcus pyogenes
• Insert Size (bp): 4500
• GenBank ID: NC_002737.1
• Promoter: CBh
• Tags / Fusion Proteins:
  • 3xFLAG (N terminal on insert)
  • NLS (N terminal on insert)
  • NLS (C terminal on insert)
  • T2A-EBFP2 (C terminal on insert)

Cloning Information for Gene/Insert 1

• Cloning method: Unknown
• 5′ sequencing primer: pCBhPro-F (5'-agggatggttggttggtggg-3') for Cas9

Gene/Insert 2

• Gene/Insert name: sgRNA cassette
• Promoter: U6

Cloning Information for Gene/Insert 2

• Cloning method: Restriction Enzyme
• 5′ cloning site: BbsI (unknown if destroyed)
• 3′ cloning site: BbsI (unknown if destroyed)
• 5′ sequencing primer: hU6-F (5'-GAGGGCCTATTTCCCATGATT-3')

Resource Information

• Supplemental Documents:
  • Plasmid gb file
  • Plasmid sequence in genbank format
  • Cloning protocol
• Articles Citing this Plasmid:
  • 21 References

Terms and Licenses

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

Depositor Comments

Please see the supplemental documents "Plasmid gb file" and "Cloning protocol" for more information on using this plasmid.

To confirm a sgRNA sequence cloned into this plasmid using the BbsI sites, use the hU6-F primer (5'-GAGGGCCTATTTCCCATGATT-3').