Gateway® cloning is a recombination based cloning method. The primary benefit of Gateway is that the reaction that moves a piece of DNA from one plasmid into another is done via a single recombination reaction, drastically simplifying the process and reducing the time compared to restriction enzyme based cloning. To utilize this approach, the fragment of DNA that you would like to clone must already be surrounded by specific recombination sites (in this regard, not so dissimilar from restriction enzyme cloning). This requires that you first clone your DNA fragment into a Donor plasmid by restriction enzyme cloning. Once your DNA fragment has been cloned into a Donor plasmid, it can then be rapidly shuttled into any compatible Gateway® Destination vector. Thus, you can clone your gene of interest one time by restriction enzyme cloning into a Donor plasmid and then easily move it into a series of plasmids using bacterial recombination. The downside of Gateway® Recombination Cloning is that you can hardly assemble more than 3 fragments simultaneously and your gene of interest is limited to Gateway compatible plasmids significantly reducing your downstream application and lab-to-lab sharing. In addition, the Gateway® Recombination Cloning does not allow to easily excise and replace DNA pieces once the full-length construct is assembled making combinatorial library construction and fragment replacement cumbersome.