pU6-(BbsI)_CBh-Cas9-T2A-mCherry-H1-(BamHI)
(Plasmid #64217)

• Purpose: Expression vector for sgRNAs cloned into the BbsI sites, shRNAs into BamHI & AflII and for Expression of Cas9 linked to mCherry via T2A
• Depositing Lab: Ralf Kuehn
• Publication: Chu et al Nat Biotechnol. 2015 Mar 24. doi: 10.1038/nbt.3198.

Backbone

• Vector backbone: pU6-(BbsI)_CBh-Cas9-T2A-BFP
• Backbone manufacturer: Kuhn lab (Addgene plasmid # 64323)
• Modifications to backbone: The human H1 promoter was cloned into the NotI site of the CRISPR/Cas9-T2A-BFP plasmid
• 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-mCherry
• 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-mCherry (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: hU6 promoter; BbsI sites for sgRNA
• 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')

Gene/Insert 3

• Gene/Insert name: H1 promoter; BamHI site for shRNA
• Promoter: H1

Cloning Information for Gene/Insert 3

• Cloning method: Restriction Enzyme
• 5′ cloning site: BamHI (unknown if destroyed)
• 3′ cloning site: AflII (unknown if destroyed)
• 5′ sequencing primer: H1 (5'-tcgctatgtgttctgggaaa-3')

Resource Information

• Supplemental Documents:
  • Plasmid gb file
  • Plasmid sequence in genbank format
  • Cloning protocol
• Article Citing this Plasmid:
  • 1 Reference

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
  • Takara Bio Limited Use Label License (formerly Clontech)
• Industry Terms:
  • Not Available to Industry