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Site‐directed combinatorial construction of chimaeric genes: General method for optimizing assembly of gene fragments

Site‐directed construction of chimaeric genes by in vitro recombination “mixes‐and‐matches” precise building blocks from multiple parent proteins, generating libraries of hybrids to be tested for structure–function relationships and/or screened for favorable properties and novel enzymatic activities. A direct annealing and ligation method can construct chimaeric genes without requiring sequence identity between parents, except for the short (≈3 nt) sequences of the fragment overhangs used for specific ligation. Careful planning of the assembly process is necessary, though, in order to ensure effective construction of desired fragment assemblies and to avoid undesired assemblies (e.g., repetition of fragments, fragments out of order).