Schematic presentation of substrate-linked directed molecular evolution for zinc-finger domains (ZF-SLiDE). Evolution cycles start with cloning the ZFD library between residues 278 and 279 of the recombinase sequence encoded in the pEVO vector. Additionally, the vector carries two lox-zif target sites of interest (lox sites are indicated as triangles; zif motifs are indicated as ellipses flanking the lox sites). After expression of the ZFD–recombinase fusion, plasmid DNA is isolated and analyzed. Upon successful recombination, a unique restriction site (indicated as scissors) between two target sites is excised. By applying restriction digestion, the non-recombined plasmids are linearized, whereas the recombined plasmids remain circular. The digestion is followed by a PCR using indicated primers (arrows), which generates product only from recombined plasmids. Successful ZF variants are then subjected to the next round of directed evolution. Counter-selection is applied with vectors containing the lox sites of interest alone, without the zif motifs.
