Golden Gate Cloning or Golden Gate assembly  is a molecular cloning method that allows a researcher to simultaneously and directionally assemble multiple DNA fragments into a single piece using Type IIS restriction enzymes and T4 DNA ligase. This assembly is performed in vitro. Most commonly used Type IIS enzymes include BsaI, BsmBI, and BbsI.
Unlike standard Type II restriction enzymes like EcoRI and BamHI, these enzymes cut DNA outside of their recognition sites and, therefore, can create non-palindromic overhangs. Since 256 potential overhang sequences are possible, multiple fragments of DNA can be assembled by using combinations of overhang sequences. In practice, this means that Golden Gate Cloning is typically scarless. Additionally, because the final product does not have a Type IIS restriction enzyme recognition site, the correctly-ligated product cannot be cut again by the restriction enzyme, meaning the reaction is essentially irreversible. This has multiple benefits, the first is that it is possible to do digestion and ligation of the DNA fragments in a single reaction, in contrast to conventional cloning methods where these reactions are separate. The second is higher efficiency because the end product cannot be cut again by the restriction enzyme.
A typical thermal cycler protocol oscillates between 37 °C (optimal for restriction enzymes) and 16 °C (optimal for ligases) many times. While this technique can be used for a single insert, researchers have used Golden Gate Cloning to assemble many pieces of DNA simultaneously.