The Golden Gate cloning is an in vitro molecular cloning method that is based on the enzymatic capability of Type IIs restriction enzymes. This methodology enables simultaneous and directional assembly of multiple DNA fragments in a single-tube reaction.
Different from conventional Type II restriction enzymes, Type IIs restriction enzymes can cut DNA outside of their recognition sites and usually generate 4 bp overhangs. Theoretically, 4*4*4*4=256 kinds of overhang sequences can be generated, which enables designing unique fragments and primers to directionally assemble many fragments simultaneously. Once fragments with the complementary overhangs are generated, they can then be annealed and linked together through the enzymatic activity of T4 DNA ligase. The final, correctly-ligated product no longer contains the Type II restriction enzyme recognition sites; meaning that the reaction is essentially irreversible (Figure 1).