Fig. 2.

Sequence diversification using restriction enzyme-based methods. Techniques to create libraries for both A) single genes and B) pathways follow the same general procedure. 1. Restriction digestion of the target sequence using a type IIS restriction endonuclease (e.g. BsaI). 2. Ligation is performed using a DNA ligase with directionality controlled by the single-stranded overhang sequences to yield (3.) gene and pathway combinatorial libraries.