Introduction
The GeneCopoeia EZShuttle™ cloning system uses the site-specific recombination machinery between the E. coli and phage lambda genomes for efficient DNA fragment transfer between
plasmids, the same principle as Gateway® technology. The EZShuttle™ system includes the following major components. Click on each tab above for more details:
1. EZRecombinase™ Mixes for catalyzing attL x attR and attB x attP cloning reactions.
2. pEZ™ expression cloning vectors for creating ready-to-use expression clones.
3. EZShuttle™ Gateway® PLUS ORF clones. More than 35,000 human and mouse ORFs in pShuttle™ plasmid backbones.
The EZShuttle™ cloning system is fully compatible with Gateway® cloning, as shown in Table 1.
Table 1. Compatiblilty between components of GeneCopoeia’s EZShuttle™ recombination cloning system and Gateway® cloning
technology.
Advantages
- Complete system of recombination cloning enzymes, expression cloning vectors, and sequence-verified ORF clones.
- Highly active for EZRecombinase™ LR Mix and EZRecombinase™ BP Mix.
- Choice of pEZ™ expression cloning vectors with a wide variety of selection markers and tags for cloning, processing, protein purification and gene function studies.
- Compatible with existing Gateway® cloning systems.
Applications
EZShuttle™ recombination cloning provides a highly fascile system for many powerful applications, including:
- Create ready-to-express expression clones. Readily shuttle ORFs or other DNA fragments of interest into an expression vector.
- Protein purification. Express your gene of interest in one or more systems for protein purification, leading to crystallization, antibody generation, DNA binding assays, and
more.
- Promoter swapping. Transfer your gene of interest into vectors carrying different promoters.
- Promoter analysis. Test the function of multiple promoter candidates with a reporter such as luciferase.
- Gene tagging. Express your gene either untagged or with a multitude of in-frame fusion tags, such as 6XHis, Flag®, GFP, etc. Useful for co-immunoprecipitation,
subcellular localization, etc.
Mechanism of EZShuttle™ cloning
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GeneCopoeia’s EZShuttle™ recombination-based cloning system for DNA fragment transfer between plasmid vectors is based on the site-specific recombination machinery between the
E. coli and phage lambda genomes, the same principle as Gateway® technology (Figure 1).
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Figure 1. The attachment sites and proteins used for integration and excision reactions between E.coli and phage lambda
genomes |
Note: Gateway® is a registered trademark of Life Technologies.
LR Mix
EZRecombinase™ LR Kit
GeneCopoeia’s EZRecombinase™ LR Kit enable fast, efficient transfer of DNA fragments between compatible plasmids. The EZRecombinase™ LR Kit is used to transfer fragments from
EZShuttle™ or Gateway® Entry clones to pEZ™ or other Gateway®-compatible pDEST vectors.
Advantages
- Highly active. EZRecombinase™ LR Mix exhibits greater activity than Gateway® LR Clonase™.
- Compatible with existing Gateway® technology systems.
EZShuttle™ recombination cloning
In LR recombination cloning, the transfer of a DNA fragment occurs between attL sites in an EZShuttle™ or Gateway® Entry clone and attR sites in a pEZ™ epression or
Gatewayt® pDEST vector using EZRecombinase™ LR Mix, which contains Int (λ Integrase), IHF (E.coli Integration Host Factor), and Xis (Excisionase).
A pShuttle™ or Gateway® entry clone, a pEZ™ cloning destination vector, and EZRecombinase™ LR Mix are incubated at room temperature (25 ºC) for one hour
and used to transform
E. coli competent cells (Figure 2).
Figure 2. Principle for transfering a DNA fragment from an EZShuttle™ or Gateway® Entry clone to a pEZ™ expression or
Gateway® pDEST vector using EZShuttle™ LR recombination cloning.
pEZ™ expression vectors
pEZ™ expression cloning vectors
GeneCopoeia’s pEZ™ cloning vectors allow simple and fast transfer of DNA fragments from EZShuttle™ Gateway® PLUS ORF clones, ORFeome clones, or other Gateway® Entry clones
using EZRecombinase LR Mix, and are functionally equivalent to Gateway® destination vectors. pEZ™ vectors are available with a wide selection of promoters, selection markers, and
tags for ready expression. The series also includes Safe harbor knockin donor cloning vectors, for CRISPR- or TALEN-mediated knockin to the human AAVS1 or mouse ROSA26 loci.
Shuttle clones
EZShuttle™ Gateway® PLUS ORF clones
GeneCopoeia offers more than 35,000 human and mouse EZShuttle™ Gateway® PLUS ORF clones. EZShuttle™ Gateway® PLUS ORF clones carry fully sequence-verified ORFs, and are
available either with stop codons, to permit expression of ORFs that are either untagged or with in-frame N-terminal fusion tags, or without stop codons, allowing expression of ORFs with
C-terminal fusion tags. ORFs are readily transferred to pEZ™ expression or Gateway® destination vectors using EZRecombinase™ LR Mix, as well as multiple cloning sites for
conventional restriction enzyme-based cloning (Figure 3).
Click
here for more information or to search the EZShuttle™ Gateway® PLUS ORF clone
collections.
Advantages
- All ORFs are fully sequence verified.
- ORFs available with or without stop codons.
- Flanked by both attL sites and multiple restriction enzyme cloning sites for cloning flexibility.
- Can be used with pEZ™ expression or Gateway® destination vectors.
Figure 3. EZShuttle™ Gateway® PLUS ORF clones are flanked by sites for recombination-based cloning (attL1
and attL2), as well as multiple cloning sites (MC1 and MC2), and so can be used for both recombination as well as traditional restriction digestion ligation cloning.
Performance comparison of BlazeTaq™ with competitor’s kit. Amplification of the B2M gene using serial diluted cDNA from HeLa cell line. Performance comparison of BlazeTaq™ (red) with BioRad SsoAdvanced™ Universal SYBR® Green Supermix (blue) shows stronger signals with similar amplification performance.
