System Biosciences
T2A-GFP Co-Expression HR Targeting Vector [T2A-GFP-pA-loxP-EF1α-RFP-T2A-Puro-pA-LoxP-MCS]
- SKU:
- HR130PA
- Availability:
- Usually Shipped in 5 Working Days
- Size:
- 10 ug
- Shipping Temperature:
- Blue Ice/ Dry Ice
Description
T2A-GFP Co-Expression HR Targeting Vector [T2A-GFP-pA-loxP-EF1α-RFP-T2A-Puro-pA-LoxP-MCS]. Cat# HR130PA. Supplier: SBI System Biosciences
Overview
Co-express any gene with GFPLearn more about the expression of any gene using the PrecisionX™ Gene Tagging HR Targeting Vector (T2A-GFP-pA-LoxP-EF1α-RFP-T2A-Puro-pA-LoxP-MCS) which integrates a T2A-GFP cassette into the genome, enabling co-expression of GFP with your gene-of-interest. Clone your homology arms into the EcoRI site upstream of the GFP gene and into the MCS, and use puromycin selection and RFP-positive imaging to find integrants. After you’ve identified clones with your GFP-tagged gene, you can remove the selection cassette using the Cre-LoxP system (learn more about Cre-LoxP excision here).
Why use an HR targeting vector?Even though gene knock-outs can result from DSBs caused by Cas9 alone, SBI recommends the use of HR targeting vectors (also called HR donor vectors) for more efficient and precise mutation. HR donors can supply elements for positive or negative selection ensuring easier identification of successful mutation events. In addition, HR donors can include up to 6-8 kb of open reading frame for gene knock-ins or tagging, and, when small mutations are included in either 5’ or 3’ homology arms, can make specific, targeted gene edits.Choose the right HR Targeting Vector for your project
*All HR Target Vectors except PBHR100A-1 contain LoxP sites. Any sequences that are integrated between the two LoxP sites can be removed through transient expression of Cre Recombinase.
**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.
**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.
How It Works
At-a-glance—how to use an HR Targeting Vector to tag a gene
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) »