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.
