Timothy A. Whitehead

Protocols for the construction of large, deeply mutagenized protein encoding libraries via Golden Gate assembly of synthetic DNA cassettes employ disparate, system‐specific methodology. Here we present a standardized Golden Gate method for building user‐defined libraries. We demonstrate that a 25 μL reaction, using 40 fmol of input DNA, can generate a library on the order of 1 × 10 6 members and that reaction volume or input DNA concentration can be scaled up with no losses in transformation efficiency. Such libraries can be constructed from dsDNA cassettes generated either by degenerate oligonucleotides or oligo pools. We demonstrate its real‐world effectiveness by building custom, user‐defined libraries on the order of 10 4 –10 7 unique protein encoding variants for two orthogonal protein engineering systems. We include a detailed protocol and provide several general‐use destination vectors.