pSH-Csy4-T2A-SpRFN-P2A-GFP-multi-gRNA
(Plasmid #85756)

• Purpose: All in one plasmid that expresses Csy4, S.pyogenes FokI-dCas9-NLS, and GFP. Also encodes for multiplexed gRNAs. Backbone derived from pSQT1601 (Addgene #53369).
• Depositing Lab: Lawrence Stanton
• Depositing Organization: Genome Institute of Singapore, A*STAR Research Entities
  Located in Singapore
• Publication: Havlicek et al Mol Ther. 2017 Feb 1;25(2):342-355. doi: 10.1016/j.ymthe.2016.11.007.

Backbone

• Vector backbone: pSQT-1601
• Backbone manufacturer: Shengdar Q Tsai from Keith Joung lab
• Backbone size w/o insert (bp): 3565
• Total vector size (bp): 10425
• Modifications to backbone: U6-gRNA expression cassette inserted; CAG promoter exchanged for CMV promoter. P2A-GFP insertion added.
• 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: Csy4-T2A-SpRFN-P2A-GFP
• Alt name: FokI-dCas9
• Alt name: RNA-guided FokI-nuclease
• Species: Streptococcus pyogenes
• Insert Size (bp): 6294
• Mutation: D10A, H840A
• Promoter: CMV
• Tags / Fusion Proteins:
  • FokI fusion via 25 amino acid (GGGGS)5 linker (N terminal on insert)
  • GFP (C terminal on backbone)
  • Csy4 (N terminal on backbone)

Cloning Information for Gene/Insert 1

• Cloning method: Gibson Cloning
• 5′ sequencing primer: gtttgactcacggggatttc
• 3′ sequencing primer: ttttggcagagggaaaaaga

Gene/Insert 2

• Gene/Insert name: gRNA
• Alt name: guide RNA
• Species: Synthetic
• Insert Size (bp): 148
• Promoter: U6
• Tag / Fusion Protein:
  • Csy4 recognition sequence (N terminal on insert)

Cloning Information for Gene/Insert 2

• Cloning method: Gibson Cloning
• 5′ sequencing primer: AGGGTTATTGTCTCATGAGCGG

Resource Information

• A portion of this plasmid was derived from a plasmid made by: Modified from pSQT-1601 (Addgene 53369).

Terms and Licenses

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

DNA (Catalog # 85756-D50)

Purpose

Ready-to-use, high-purity DNA prep. DNA aliquots are suitable for use in mammalian cell transfections and other molecular biology applications.

Delivery

• Amount: 50 μg
• Concentration: 1 μg/μL
• Pricing: $495 USD
• Storage: 4 ℃ (short-term) or -20 ℃ (long-term)

Terms and Licenses

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

Quality Control

Every DNA prep is verified by next-generation sequencing, confirming plasmid identity, sequence integrity, and absence of DNA contaminants. Concentration is confirmed to be within ±2.5% of the stated concentration.

Our production process has been validated to consistently yield endotoxin levels below 1.0 EU/μg. Individual lots are not tested for endotoxin.

DNA (Catalog # 85756-D100)

Purpose

Ready-to-use, high-purity DNA prep. DNA aliquots are suitable for use in mammalian cell transfections and other molecular biology applications.

Delivery

• Amount: 100 μg
• Concentration: 1 μg/μL
• Pricing: $585 USD
• Storage: 4 ℃ (short-term) or -20 ℃ (long-term)

Terms and Licenses

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

Quality Control

Every DNA prep is verified by next-generation sequencing, confirming plasmid identity, sequence integrity, and absence of DNA contaminants. Concentration is confirmed to be within ±2.5% of the stated concentration.

Our production process has been validated to consistently yield endotoxin levels below 1.0 EU/μg. Individual lots are not tested for endotoxin.