EZShuttle™ LR Recombination Cloning System

 

-Efficient cloning system based on phage lambda recombination, the same principle as Gateway® technology.

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

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).

 

 

 

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.

Buy Catalog# Product Description Price User manual
RCBM-1001-020 EZRecombinase™ LR Kit (20 rxn) Catalyzes attL x attR cloning $195
RCBM-1001-100 EZRecombinase™ LR Kit (100 rxn) $875

 

 

 

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.

Buy Catalog# Name Description Type Promotor Selection marker Reporter gene Tag Price
EZ001 pEZ-Lv199 pEZ-Lv199 expression vector for LxR recombination cloning (lentiviral) Lentiviral CMV Puromycin $495
EZ003 pEZ-Lv233 pEZ-Lv233 expression vector for LxR recombination cloning (lentiviral) Lentiviral EF1a Puromycin eGFP $495
EZ004 pEZ-Lv195 pEZ-Lv195 expression vector for LxR recombination cloning (lentiviral) Lentiviral EF1a Puromycin $495
EZ006 pEZ-Lv201 pEZ-Lv201 expression vector for L x R recombination cloning (lentiviral) Lentiviral CMV Puromycin eGFP $495
EZ016 pEZ-Lv235 pEZ-Lv235 expression vector for LxR recombination cloning (lentiviral) Lentiviral CAG Puromycin eGFP $495
EZ017 pEZ-Lv236 pEZ-Lv236 expression vector for LxR recombination cloning (lentiviral) Lentiviral CAG Puromycin $495
EZ018 pEZ-Lv237 pEZ-Lv237 expression vector for LxR recombination cloning (lentiviral) Lentiviral CAG Puromycin C-Flag $495
EZ019 pEZ-Lv238 pEZ-Lv238 expression vector for LxR recombination cloning (lentiviral) Lentiviral CMV Puromycin C-Flag $495
EZ007 pEZ-M02 pEZ-M02 expression vector for LxR recombination cloning Mammalian CMV Neomycin $375
EZ008 pEZ-M13 pEZ-M13 expression vector for LxR recombination cloning Mammalian CMV Neomycin C-Flag $375
EZ009 pEZ-M67 pEZ-M67 expression vector for LxR recombination cloning Mammalian CMV Hygromycin $375
EZ010 pEZ-M68 pEZ-M68 expression vector for LxR recombination cloning Mammalian CMV Puromycin $375
EZ011 pEZ-M98 pEZ-M98 expression vector for LxR recombination cloning Mammalian CMV Neomycin C-monomeric eGFP $375
EZ033 pEZ-M06 pEZ-M06 expression vector for LxR recombination cloning Mammalian CMV Neomycin N-3XHA $375
EZ034 pEZ-M09 pEZ-M09 expression vector for LxR recombination cloning Mammalian CMV Neomycin C-Myc $375
EZ035 pEZ-M12 pEZ-M12 expression vector for LxR recombination cloning Mammalian CMV Neomycin N-3XFlag $375
EZ012 pEZ-SH01 pEZ-SH01 expression vector for LxR recombination cloning (AAVS1 knockin) Human Safe Harbor CMV Puromycin copGFP $425
EZ013 pEZ-SH02 pEZ-SH02 expression vector for LxR recombination cloning (ROSA26 knockin) Mouse Safe Harbor CMV Puromycin copGFP $425
EZ014 pEZ-B01 pEZ-B01 expression vector for L x Rrecombination cloning (E. coli) E. coli T7 N/A N-His $495
EZ015 pEZ-B02 pEZ-B02 expression vector for L x Rrecombination cloning (E. coli) E. coli T7 N/A $495
EZ020 pEZ-P001 pEZ-P001
expression vector for L x R recombination cloning (Plant)
Plant 35S+omega Kanamycin $495
EZ021 pEZ-P002 pEZ-P002 expression vector for LxR recombination cloning (Plant) Plant Kanamycin eGFP C-eGFP $495
EZ022 pEZ-P003 pEZ-P003
expression vector for L x R recombination cloning (Plant)
Plant 35S Kanamycin eGFP C-eGFP $495
EZ023 pEZ-P004 pEZ-P004 expression vector for LxR recombination cloning (Plant) Plant Kanamycin eGFP C-eGFP $495
EZ024 pEZ-P005 pEZ-P005
expression vector for L x R recombination cloning (Plant)
Plant 35S Kanamycin C-Gus $495
EZ025 pEZ-P006 pEZ-P005
expression vector for L x R recombination cloning (Plant)
Plant Hygromycin $495
EZ026 pEZ-P007 pEZ-P007 expression vector for L x R recombination cloning(Plant) Plant 35S+omega Hygromycin $495
EZ027 pEZ-P008 pEZ-P008 expression vector for L x R recombination cloning(Plant) Plant 35S Hygromycin eYFP C-eYFP $495
EZ028 pEZ-P009 pEZ-P009 expression vector for L x R recombination cloning (Plant) Plant 35S Hygromycin eCFP C-eYFP $495
EZ029 pEZ-P010 pEZ-P010 expression vector for L x R recombination cloning(Plant) Plant 35S Hygromycin $495
EZ030 pEZ-P011 pEZ-P011 expression vector for L x R recombination cloning(Plant) Plant 35S Hygromycin eGFP N-eGFP $495
EZ031 pEZ-P012 pEZ-P012 expression vector for L x R recombination cloning(Plant) Plant Hygromycin $495
EZ032 pEZ-P013 pEZ-P013 expression vector for L x R recombination cloning(Plant) Plant Hygromycin C-Gus $495

 

Competent E. coli

Buy Catalog# Product Description Price
CC001 GCI-5α Chemically Competent E.coli Cells, (10 tubes) Competent cells for transformation inquire
CC004 GCI-L3 Chemically Competent E.coli Cells (20 tubes) Competent cells for transformation inquire
CC003 GCI-L3 Chemically Competent E.coli Cells (10 tubes) Competent cells for transformation inquire
CC002 GCI-5α Chemically Competent E.coli Cells, (20 tubes) Competent cells for transformation inquire

 

 

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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.

Publications

2017