Overview of the method. The left flowchart is the standard strategy and the right flowchart is the strategy described in this study. Brackets on the left indicate the two stages of the strategy: the epPCR library construction (stage1) and the sub-cloning of mutant inserts from reporter to non-reporter expression plasmid (stage 2). ER1 and ER2 denote restriction sites used to clone the sequence to evolve and create the PCR template of the standard strategy. Inner arrows with continuous lines are the core of the method. Outer arrows with dashed lines indicate the pathways used to transfer the wt sequence to the non-reporter expression plasmid, and (in the right flowchart only) to create the internally deleted wt NTAIL in pDEST17O/I. Mutated and wt sequences are represented by thick dark and light grey lines, respectively. The internally deleted wt sequence is denoted by an asterisk. Antibiotic resistance markers are indicated: A, ampicillin resistance; K, kanamycin resistance.