Golden Gate assembly is an in vitro molecular cloning technique that is carried out using Type IIs restriction enzymes and T4 DNA ligase. This technique makes it possible to simultaneously and directionally assemble multiple DNA fragments in a single-tube reaction.

BsaI, BsmBI, and BbsI are the most commonly used Type IIs restriction enzymes for Golden Gate cloning. These are different from conventional Type II restriction enzymes. Type IIs restriction enzymes cleave DNA outside of their recognition sites, which offers several advantages including:

• Orderly assembly of multiple fragments simultaneously in one reaction
• Absence of scar sequence since the DNA is cut outside of their recognition sites by the Type IIs restriction enzymes
• Digestion and ligation steps can be carried out simultaneously in a single-tube reaction, saving time and effort