pU6-(BbsI)_CBh-Cas9-T2A-BFP-P2A-Ad4E4orf6
(Plasmid #64220)

• Purpose: Expression vector for sgRNA and Cas9 linked via T2A to BFP linked to the Ad4 E4orf6 gene via P2A
• 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: Mammalian codon–optimized sequence encoding the adenoviral serotype 4 E4orf6 protein and BFP was cloned into the NheI & EcoRI sites of the CRISPR/Cas9-T2A-BFP plasmid by Gibson assembly.
• 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-BFP-P2A-E4orf6
• Species: Streptococcus pyogenes
• 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-BFP-P2A-E4orf6

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: see comments and supplemental documents (unknown if destroyed)
• 3′ cloning site: see comments and supplemental documents (unknown if destroyed)
• 5′ sequencing primer: hU6-F (5'-GAGGGCCTATTTCCCATGATT-3')

Resource Information

• Supplemental Documents:
  • Plasmid gb file
  • Plasmid sequence in genbank format
  • sgRNA cloning information

Terms and Licenses

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

Depositor Comments

Please note that due to the presence of BbsI sites in the Ad4 coding regions it is NOT possible to directly clone new sgRNA sequences into the BbsI sites downstream of the U6 promoter.

Therefore, precloned U6-sgRNA cassettes, isolated as 1.7 kb PvuI-XbaI fragments from pU6-(BbsI)_CBh-Cas9-T2A-BFP (plasmid# 64323) or pU6-(BbsI)_CBh-Cas9-T2A-mCherry (plasmid# 64324) of this deposit, or any other plasmid derived from pX330 (plasmid# 42230), must be ligated into the PvuI-XbaI digested backbone of this Ad4 containing plasmid.