The plasmid Co1E2-P9 (Co1E2) origin (32 bp) is specifically recognized by the plasm id-specified Rep protein that initiates DNA replication. The Co1E2 origin is divided into at least three functional subregions (I, II, and III), and three sites (a, b, and c) found in subregions I and II play important roles in Rep protein binding. We performed SELEX experiments of plasmid Co1E2 to determine the optimal sequences for specific binding of the Rep protein. From these experiments, we obtained a common 16-bp sequence (5-TGA GACCANATAAGCC-3'), which corresponds to about one half of the minimal Co1E2 origin and contains sites a and b. Gel mobility shift assays using single-point mutant origins and the Rep protein further indicated that high affinity sequence-specific recognition by the Rep protein requires sites a, b, and c, but that mutations in site c were less disruptive to this recognition than those in sites a and b. (c) 2006 Elsevier Inc. All rights reserved.