Table 2.

Summary of current in vitro seamless assembly methods

Mechanism	Homology				Type IIS
	Overlap		Recombination		Traditional		Modified
Method	USERa	OE-PCRb	SLICc	Gibsond	Golden gate (38)	Golden Braide	PSA (26)	MASTERf
Sequence-independence	+g	+	+	+	−	−	+	+
Hierarchical manner	−h	−	−	+	+	+	+	+
Complicated sequence	−	−	−	−	+	+	+	+
Multiple fragments	+	+	+	+	+	−	−	+
Material sources	PCR	PCR	PCR	Both	Both	Plasmid	Plasmid	Both
Sizes (39)	Parts-Genes- Pathways	Parts-Genes- Pathways	Genes- Pathways	Genes-Pathways- Genome	Parts-Genes- Pathways	Parts-Genes- Pathways	Genes-Pathways- Genome	Parts-Genes- Pathways

^aUSER is a representative of USER (6), PLICing (40), Ribocloning (41) and so forth.

^bOE-PCR is a representative of OE-PCR (42), CPEC (43), SHA (44) and so forth.

^cSLIC is a representative of SLIC (5), In-Fusion (45), Cold Fusion (System Biosciences), Fast Seamless Cloning (DoGene), CloneEZ (GenScript) and so forth.

^dGibson is a representative of Gibson Assembly (8) and SLiCE (46).

^eGolden Braid is a representative of Golden Braid (47), MoClo (48), NOMAD (49) and so forth.

^fAs the efficiency drops remarkably when large DNA fragments are used for ligation, the application of MASTER Ligation in genome assembly remains to be established.

^gPlus represents techniques applicable in relevant context.

^hMinus represents techniques unsuitable in relevant context.