Table 1.
Type IIS restriction enzyme-based hierarchical assembly systems
| Assembly system | Enzyme | Forbidden site frequency | Mode of assembly |
|---|---|---|---|
| MoClo (9,12) | BsaI, BpiI | 1 in 1 kb | Multiplicative or iterative linear addition assemblya |
| GoldenBraid (10) | BsaI, BsmBI | 1 in 1 kb | Multiplicative assembly |
| EcoFlex (13) | BsaI, BsmBI | 1 in 1 kb | Multiplicative assembly |
| CIDAR (14) | BsaI, BpiI | 1 in 1 kb | Multiplicative assembly |
| Yeast Toolkit (15) | BsaI, BsmBI | 1 in 1 kb | Multiplicative assembly |
| TNT (16) | LguI, EarI | 1 in 2 kb | Multiplicative assemblyb |
| GreenGate (17) | BsaI | 1 in 2 kb | Iterative linear addition assembly |
| PODAC (18) | BsaI | 1 in 2 kb | Iterative linear addition assembly |
| MetClo-BsaI | BsaI | 1 in 2 kb | Multiplicative or iterative linear addition assembly |
| MetClo-BpiI | BpiI | 1 in 2 kb | Multiplicative or iterative linear addition assembly |
| MetClo-LguI | LguI | 1 in 8 kb | Multiplicative or iterative linear addition assembly |
aThree enzymes required for linear addition assembly.
bMaximum three parts per reaction with constraints in design of adaptor sequence.