Figure 2.

Mutations introduced into an evolved DNA fragment after 50 rounds of directed evolution. The sequence of the original “preevolved” DNA, AR0, is aligned with that of AR50–3. PCR primers used for DNA shuffling anneal to sequences just outside the region shown here. The mutated nucleotide positions are color shaded: missense mutations in the truncated coding region are in blue, and other mutations are in red. Asterisks indicate mutations found in all five AR50 mutants. Start and stop codons are underlined. The nucleotide position, at which the coding and 3′ regions of AR50–3 are separated and the coding region of AR50–3 was linked to the 3′ region of AR0 in Fig. 3, is shown by an arrowhead.