Tol2-U6.3-sgRNA-empty-GFP
(Plasmid #221843)

• Purpose: empty vector to clone custom sgRNA into BsaI sites to express sgRNA from chick U6.3 promoter expresses GFP reporter from GAGC promoter
• Depositing Lab: Stephen Terry
• Publication: O’Sullivan et al bioRxiv 2024.03.04.583298

Backbone

• Vector backbone: pTol2{Exp}
• Backbone manufacturer: vectorbuilder
• Backbone size w/o insert (bp): 3000
• Total vector size (bp): 5672
• Modifications to backbone: Chick U6.3 RNA promotor to express sgRNA and EGFP expressed from GACG promoter
• Vector type: Mammalian Expression, CRISPR ; Tol2 transposon optimised for chick expression

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH5alpha
• Copy number: High Copy

Gene/Insert

• Gene/Insert name: EGFP
• Insert Size (bp): 717
• Promoter: CAGC

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ cloning site: EcoRI (not destroyed)
• 3′ cloning site: BamH1 (not destroyed)
• 5′ sequencing primer: gaattcGCCACCATGGTGAGCAAGG
• 3′ sequencing primer: ggatccTTACTTGTACAGCTCGTCCATG

Resource Information

• A portion of this plasmid was derived from a plasmid made by: EGFP was PCR amplified from PCMS28-EGFP-IRES (1) and cloned into Tol2-U6.3-sgRNA-tdTomato a custom made plasmid synthesised by vector builder containing U6.3 chick promoter sequence based on Addgene plasmid 99139 (2) at EcoRI and BamHI sites. (1) Terry SJ, Donà F, Osenberg P, Carlton JG, Eggert US. Capping protein regulates actin dynamics during cytokinetic midbody maturation. Proc Natl Acad Sci U S A. 2018 Feb 27;115(9):2138-2143. doi: 10.1073/pnas.1722281115. Epub 2018 Feb 8. PMID: 29439200; PMCID: PMC5834733 (2) Gandhi S, Piacentino ML, Vieceli FM, Bronner ME. Optimization of CRISPR/Cas9 genome editing for loss-of-function in the early chick embryo. Dev Biol. 2017 Dec 1;432(1):86-97. doi: 10.1016/j.ydbio.2017.08.036. PMID: 29150011; PMCID: PMC5728388.

Terms and Licenses

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

Depositor Comments

Clone sgRNA using dual BsaI sites need to add overhangs of GGAT on forward primer and AAAC on reverse.

Please visit https://doi.org/10.1101/2024.03.04.583298 for bioRxiv preprint.