Table 1.

Comparison of the utility of the different approaches for pathway construction

Approach	Principle of method	Efficiency	Size Assembled (Numbers of Fragments)
MoClo system	based on Golden Gate cloning	90–100% for 10 fra gments	50 kb (68 fragments)
MASTER Ligation	using MspJI, which specifically recognizes methylated 4-bp sites and generates a 4-bp arbitrary overhang	NA	29 kb
DNA Assembler (any-gene-any-plasmid (AGAP))	based on yeast in vivo homologous recombination mechanism	100% for 2 fragments	50 kb
Reiterative Recombination	endonuclease-induced homologous recombination in conjunction with recyclable markers	99% for less than 10 kb	less than 10 kb, could be larger
site-specific recombination-based tandem assembly (SSRTA)	based on φBT1 integration system
RecET-mediated linear-plus-linear homologous recombination (LLHR)	linear-linear homologous recombination		10–52 kb
phage wBT1 integrase-mediated site-specific recombination	requiring both homologous and site-specific recombinations. The homologous recombinations were used for targeted integration of the mutated attB and attP into Streptomyces chromosome, while the wBT1 integrase-mediated site-specific recombination was employed to excise targeted region of interest from the chromosome	90% for 23 kb, 80% for 157 kb	157 kb
transformation-associated recombination (TAR)	in vivo homologous recombination of Saccharomyces cerevisiae		21.3 kb and 67 kb