pU6-SacB-Cas9-T2A-mCherry_(BbsI)
(Plasmid #117070)

• Purpose: Encodes Cas9, T2A-mCherry, and AmpR which creates a CRISPR system application with reliable positive selection
• Depositing Lab: Kim Failor
• Publication: CRISPR-Cas9 system with SacB toxin Positive Selection (unpublished)

Backbone

• Vector backbone: pX330
• Backbone manufacturer: Ralf Kuehn (Addgene #64324), originally from Zhang Lab (Addgene #42230)
• Backbone size w/o insert (bp): 9283
• Modifications to backbone: From Kuehn Lab's plasmid, the gene encoding for SacB was placed as a fallout to ease the positive selection process. This is described in US Provisional Patent No. 62/694,458
• Vector type: Mammalian Expression, CRISPR
• Selectable markers: T2A-mCherry

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
• Tags / Fusion Proteins:
  • 3x FLAG (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: 3'-AGGGATGGTTGGTTGGTGGG-5'

Gene/Insert 2

• Gene/Insert name: SacB
• Species: Bacillus subtilis
• 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: 3'-GAGGGCCTATTTCCCATGATT-5'

Terms and Licenses

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

Depositor Comments

This plasmid was created by Arnav Lal, (USPTO no. 62/694,458). Any questions or concerns can be sent to .

I would like to acknowledge Stanford Online High School and Dr. Kim Failor for guidance and mentorship as well as New England Biolabs for their gracious support in making this plasmid.

Please view Addgene plasmid #64324 for instruction on usage.