PurposeExpression vector for sgRNA and Cas9 linked via T2A to BFP linked to the Ad4 E4orf6 gene via P2A
|Item||Catalog #||Description||Quantity||Price (USD)|
|Plasmid||64220||Standard format: Plasmid sent in bacteria as agar stab||1||$65|
This material is available to academics and nonprofits only.
Backbone manufacturerKuhn lab (Addgene plasmid # 64323)
Modifications to backboneMammalian 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 typeMammalian Expression, CRISPR
Growth in Bacteria
Copy numberHigh Copy
- Promoter CBh
/ Fusion Proteins
- 3xFLAG (N terminal on insert)
- NLS (N terminal on insert)
- NLS (C terminal on insert)
Cloning Information for Gene/Insert 1
- Cloning method Unknown
- 5′ sequencing primer pCBhPro-F (5'-agggatggttggttggtggg-3') for Cas9 (Common Sequencing Primers)
Gene/Insert namesgRNA 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') (Common Sequencing Primers)
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.
These plasmids were created by your colleagues. Please acknowledge the Principal Investigator, cite the article in which the plasmids were described, and include Addgene in the Materials and Methods of your future publications.
For your Materials & Methods section:pU6-(BbsI)_CBh-Cas9-T2A-BFP-P2A-Ad4E4orf6 was a gift from Ralf Kuehn (Addgene plasmid # 64220 ; http://n2t.net/addgene:64220 ; RRID:Addgene_64220)
For your References section:Increasing the efficiency of homology-directed repair for CRISPR-Cas9-induced precise gene editing in mammalian cells. Chu VT, Weber T, Wefers B, Wurst W, Sander S, Rajewsky K, Kuhn R. Nat Biotechnol. 2015 Mar 24. doi: 10.1038/nbt.3198. 10.1038/nbt.3198 PubMed 25803306