pU6-sgRosa26-1_CBh-Cas9-T2A-BFP
(Plasmid #64216)

• Purpose: Expression vector for a sgRNA against the mouse Rosa26 locus and 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: pU6-(BbsI)_CBh-Cas9-T2A-BFP
• Backbone manufacturer: Kuhn lab (Addgene plasmid# 64323)
• Modifications to backbone: sgRNA targeting mouse Rosa26 locus was cloned into the BbsI sites
• 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
• 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-BFP (C terminal on insert)

Cloning Information for Gene/Insert 1

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

Gene/Insert 2

• Gene/Insert name: sgRNA targeting ROSA26-1
• Alt name: 5'-GACTCCAGTCTTTCTAGAAGA-3'
• Promoter: U6

Cloning Information for Gene/Insert 2

• Cloning method: Restriction Enzyme
• 5′ cloning site: BbsI (destroyed during cloning)
• 3′ cloning site: BbsI (destroyed during cloning)
• 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:
  • 8 References

Terms and Licenses

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