How does Gibson assembly work and when is it preferred over traditional cloning?

Gibson assembly joins multiple DNA fragments in a single isothermal reaction using three enzymes: T5 exonuclease chews back 5' ends creating single-stranded overhangs, Phusion polymerase fills gaps, and Taq ligase seals nicks. Fragments must have ~20-40bp overlapping homology regions. Advantages over traditional cloning include: seamless joining (no restriction sites in product), multiple fragment assembly (up to 6+ fragments), sequence independence (no restriction site limitations), and single-tube reaction. It is preferred for synthetic biology, pathway construction, and when suitable restriction sites are unavailable. Fragments are generated by PCR with primers containing overlap sequences.