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HDR Donor Clones and Cloning Vectors

$995 / HDR donor

For convenient selection and flexible applications

Introduction

Homology-directed repair (HDR) is a natural DNA repair mechanism that is induced by DNA double-strand breaks (DSBs). Site-specific TALENs or CRISPR Cas9-sgRNA complexes generate DSBs in the chromosome, leading to DNA repair. In the presence of donor DNA, HDR occurs and replaces genetic information in the chromosome with new information from the donor.

Donor-mediated HDR makes many applications possible, such as precisely defined gene knockout, in-frame fusion tagging, transgene insertion, and introduction of base pair changes for mutagenesis or disease correction (Figure 1).

Figure 1. CRISPR-Cas9-based gene engineering. A reporter gene and a selection marker are integrated into the targeted genome site via DSB-induced HDR from a donor plasmid.

 

 

HDR Donor Vectors

 

GeneCopoeia offers standard or customized donor cloning vectors for HDR-mediated applications. A wide range of donor vector backbones, promoters, markers, and other useful elements are available to suit your experimental needs, such as:

  • Two multiple cloning sites (MCS) for inserting homologous recombination arms (left and right).
  • Reporter genes such as GFP (optional) for positive clone selection based on cell sorting
  • Selection markers, such as puromycin and neomycin for positive clone selection
  • TK selection marker (optional) for eliminating random integration
  • LoxP sites (optional) for convenient Cre-mediated cassette removal.

 

Buy Vector Promoter Reporter Gene Selection Marker LoxP site
pDonor-D01 EFa1 copGFP Puromycin N/A
pDonor-D02 CMV copGFP Neomycin N/A
pDonor-D03 CMV N/A Neomycin N/A
pDonor-D04 CMV N/A Puromycin N/A
pDonor-D05 EF1a N/A Neomycin N/A
pDonor-D07 EF1a copGFP Puromycin/TK LoxP
pDonor-D08 CMV copGFP Neomycin/TK LoxP
pDonor-D09 EF1a N/A Puromycin/TK LoxP
pDonor-D10 CMV N/A Neomycin/TK LoxP
pDonor-D11 PGK copGFP Puromycin/TK LoxP
pDonor-D12 EF1a copGFP Hygromycin/TK LoxP
pDonor-D13 PGK copGFP Neomycin/TK LoxP
pDonor-D14 PGK N/A Puromycin/TK LoxP

 

Buy Catalog# Product Description Price
FF001 Fast-Fusion™ Cloning Kit
(20 reactions)
For rapid and effective cloning of PCR products inquire
FF002 Fast-Fusion™ Cloning Kit
(60 reactions)
For rapid and effective cloning of PCR products inquire

 

HDR Donor Services

GeneCopoeia also offers custom HDR donor design and construction services, which are provided in two versions (Figure 2):

Gene knockout HDR donor

The first type of custom HDR donor design and construction service is intended for HDR-mediated gene knockout (Figure 2A). These donors are designed in conjunction with our CRISPR and TALEN target sites and can be ordered from our CRISPR/TALEN genome-wide search page, available here. When you search for CRISPR sgRNA or TALEN targets for your gene, you will be presented with options for compatible HDR-mediated knockout donors. While not required for constitutive gene knockout, HDR donors do provide you with the convenience of being able to either apply drug selection or use fluorescence sorting for your knockout cells, which is especially important for cell lines with low transfection efficiency. Gene knockout HDR donors also enable you to define precisely the nature of the knockout mutation.

Knock-in HDR donors for conditional knockout, mutagenesis, and in-frame fusion tagging

The second version of GeneCopoeia's custom HDR donor design and construction services is intended for HDR-mediated knock-in applications (Figure 2B), which require an HDR donor and include 1) conditional gene knockout (flanking an exon with loxP sites for Cre-mediated inactivation); 2) gene mutagenesis (introducing specific base pair changes to either mutate a gene or restore a mutant gene to wild-type); and 3) gene tagging (adding an in-frame fusion protein gene tag to either the 5' or 3' end of a gene). Custom HDR knock-in donors require a custom quote, which is available by filling out the inquiry form.

 

A

 
 
   

B

 
 
 
Figure 2. Two versions of GeneCopoeia's custom HDR donor clone design and construction services. A. Example knockout HDR donor strategy. An HDR donor containing both a drug selection marker (Puro) and a fluorescent reporter (GFP) is constructed. Drug selection or fluorescence sorting enhances the ability to obtain a knockout. Note: Only one allele is shown for simplicity. B. Example knock-in donor strategy for mutagenesis. Top: A donor with a single base pair change is co-transfected with CRISPR sgRNAs or TALENs. A drug selection marker (Puro) and a fluorescent reporter (GFP) are placed into an intron near the mutation site. Drug selection or fluorescence sorting allows isolation of clones with the mutated allele.

 

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