MultiSite Gateway Technology

What if you could easily and accurately assemble 2, 3 or even 4 DNA fragments in the order and orientation of your desire into one construct? Imagine reconstituting key element(s) of a pathway or functional complex within one plasmid. Now you can do all of this and more.

Compared to subcloning using restriction enzymes, Gateway® recombination cloning technology is efficient, simple and effective, saving you time and effort (Table 1). Now, with added flexibility to assemble multiple DNA fragments precisely, efficiently, and directionally in a defined order and orientation and without subcloning, Multisite Gateway® technology is the ideal choice for protein expression.

MultiSite Gateway® Pro Kits

Table 1 - Gateway® Recombination Cloning Simplifies Cloning Workflow

Steps Gateway® Recombination Cloning Sub-cloning with Restriction enzymes
Existing primers?YesNo
Vector ready for cloning?YesNo
Ligation reagents included?YesNo
Competent cells separately?IncludedPurchase separately: 0 hours
Prepare:  up to 6 hours
Vector clean up?NoYes
PCR fragment cleanup?NoYes
Recombination efficiencyUp to 99%~50%
Cloning time into expression vector*65 minutesup to 24 hours

*Excludes miniprep time common to both methods

Table 2 - Applications for MultiSite Gateway® Technology

Discover how MultiSite Gateway® Technology gives you the opportunity to perform pathway reconstitution, multiple gene expression and regulation, protein interaction studies, and more. Easily swap and assemble promoters, tags, and genes for your applications, or build libraries of different elements for screening purposes.

See Data Generated with MultiSite Gateway® Pro Kits

Add promoter/tag elements to you Gateway® Entry clones
Combinatorial tagging
Construct biological or biochemical pathways
Expression of enzymatic pathways
Expression of multi-subunit protein complexes
Gene knock-down and rescue
Optimized multi-gene delivery without co-transfection
Screen expression from different promoters
Shuffle protein coding domains
Variable gene expression levels using different expression elements


NEW! Published Gateway® Review Article Available FREE!

Federico Katzen, responsible for the ongoing development of Gateway® Technology at Invitrogen, published a review article in Expert Opinion Drug Discovery about Gateway® enzymes, vectors, and systems. To download a free copy of "Gateway® Recombinational Cloning: A Biological Operating System," simply click here, register with the journal, and enter “Katzen” on the search tab.

MultiSite Gateway® Pro Kits

Simultaneously assemble 2, 3 or 4 DNA fragments into one vector

MultiSite Gateway® Pro Kits:

  • Simplified cloning of multiple DNA fragments—Gateway® recombination eliminates the use of restriction enzymes and ligases
  • Flexible design—clone up to four DNA elements into any Gateway® Destination vector
  • Reliable gene delivery—avoid uncertain cotransfection by creating one plasmid containing all your DNA elements


Multiple Fragment Assembly Simplified 

Figure 1. Overview the MultiSite Gateway® Pro Technology


Figure 2.  An example of using MultiSite Gateway® Pro Technology to study expression of multiple genes in human cells. Entry clones encoding genes for YFP and CFP and the CMV and EF-1α promoters were recombined into pcDNA™ 6.2/V5-PL-DEST (A, C, and E) or into pcDNA™ 6.2/V5-DEST (B and D). The resulting expression clones were transfected into HeLa cells. Expression was verified under a fluorescence microscope. The plasmid pcDNA™ 6.2/V5-PL-DEST is a promoterless derivative of pcDNA™ 6.2/V5-DEST, which carries the CMV promoter.

Easy Experimental design with Vector NTI Advance™ Software

Invitrogen's Vector NTI Advance™ sequence analysis software simplifies designing your primers for Multi-Site Gateway® recombination reactions.  With an user- friendly interface, you select or import the DNA fragment sequences that you wish to clone and choose a destination vector. The Vector NTI Advance™ software generates sequences for PCR primers that amplify your DNA elements, and shows you the vector map and sequences of your Entry Clone(s) as well as the final Expression Clone construct.  It's that simple.