Markerless replacement of a target. (A) A linear DNA cassette containing a positive selection marker (cat), an I-Sce I recognition site (I), a gene to be replaced (thrA*BC or rhtA23), and three homology arms (represented as a, b, and c) were amplified by recombinant PCR (refer to Materials and Methods). PCR primers are labeled with lower case, italicized letters (P-f1, P-r1, P-f2, and P-r2) and arrows. (B) The target of the E. coli genome was replaced by the constructed DNA cassette, and recombinants were selected on LB plates containing chloramphenicol. (C) The cat gene introduced was excised from the selected recombinants by double-strand break repair mediated by I-Sce I cleavage.