Type IIS systems, such as Golden Gate, Green Gate , and MoClo , take advantage of the unique properties of type IIS restriction endonucleases, which cut dsDNA at a specified distance from the recognition sequence. This feature allows for the creation of custom overhangs, which is not possible with traditional restriction enzyme cloning. The advantages of this system are two-fold. First, the entire cloning step (digest and ligation) can be carried out in one-pot with a single restriction enzyme, since the resulting overhangs will be distinct and preserve the directionality of the cloning reaction. Second, the restriction site is encoded on both the insert and plasmid in such a way that all recognition sequences are removed from the final product, with no undesired sequence (“scar”) retained. The downside of Golden Gate Assembly is that it does not allow to easily excise and replace DNA pieces once the full-length construct is assembled making post-synthesis manipulation, such as combinatorial library construction and fragment replacement, cumbersome.