IRES-GFP Co-Expression HR Targeting Vector [IRES-GFP-pA-loxP-MCS1-EF1α-RFP-T2A-Puro-pA-LoxP-MCS2
- Usually Shipped in 5 Working Days
- 10 ug
- Shipping Temperature:
- Blue Ice/ Dry Ice
IRES-GFP Co-Expression HR Targeting Vector [IRES-GFP-pA-loxP-MCS1-EF1α-RFP-T2A-Puro-pA-LoxP-MCS2]. Cat# HR180PA. Supplier: SBI System Biosciences
**The clever design of these HR Donors enables enrichment for on-target integration events. A PGK-hsvTK cassette is included outside of the homology arms. Because of this configuration, on-target integration that results from homologous recombination will not include the PGK-hsvTK cassette—only randomly-integrated off-target events will lead to integration of PGK-hsvTK and resulting TK activity. Therefore, TK selection will negatively select against off-target integrants. Click on any one of these vectors to see a diagram of how the negative selection works.
At-a-glance—how to use an HR Targeting Vector to tag a gene
Figure 1. Tagging a gene using an HR Targeting Vector. Step 1: Cas9 creates a double-stranded break (DSB) in the genomic DNA at a site that is complimentary to the gRNA. Step 2: The DNA repair machinery is recruited to the DSB. In the presence of an HR Donor with homology to the region adjacent to the DSB (blue areas of the genomic and vector DNA) homologous recombination (HR) is favored over non-homologous end joining (NHEJ). The tag you’d like to co-express with a gene should lie outside of the two LoxP sites. Result: The HR event leads to insertion of the region of the HR Donor Vector between the two homology arms—your tag ends up co-expressed with your gene-of-interest and your selection cassette is integrated. To remove the selection cassette (leaving behind the tag and a single LoxP site), transiently express Cre recombinase.
Genome engineering with CRISPR/Cas9
For general guidance on using CRISPR/Cas9 technology for genome engineering, including the design of HR Targeting Vectors, take a look at our CRISPR/Cas9 tutorials as well as the following application notes:
CRISPR/Cas9 Gene Knock-Out Application Note (PDF) »
CRISPR/Cas9 Gene Editing Application Note (PDF) »
CRISPR/Cas9 Gene Tagging Application Note (PDF) »