Figure 1. Summary of the Golden GATEway cloning kit.

A) Outline of the entire cloning procedure. Eight different entry vectors (pGG-EVx) contain different inserts (colored bars). These inserts are assembled in a predefined order using a Golden Gate reaction into Gateway^TM entry vectors at any position (Threeway Gateway^TM cloning). These can then be assembled to establish a final expression vector in an LR reaction. Compatible overhangs are indicated on each Golden Gate entry vector. B) The principle of Golden Gate cloning is illustrated in the top scheme; the principle of Gateway cloning is illustrated in the bottom scheme. Golden Gate cloning utilizes type II restriction endonucleases to generate compatible overhangs that can be ligated with T4 ligase. The ligation of the two compatible inserts from the entry vectors one and two is illustrated. Gateway cloning relies on recombination of specific att sites using a commercially available enzyme mix (LR Clonase II, Life Technologies).