We constructed a new cosmid vector suitable for the previously developed nested deletion method which used the in vitro DNA packaging system of bacteriophage T3. The first step of this method is linearization of a cosmid clone to be packaged, and we previously introduced cleavage at the cos site using lambda-Terminase, but optimization of the reaction conditions was required for complete digestion because of its instability. In the newly constructed vector, pAT5, the sites of 4 different restriction enzymes, Sse8387I, Asc I, Fse I and Pme I, each of which recognizes an 8-bp sequence (8-base cutter) were introduced in the vicinity of the cos site. In addition, the species of restriction sites for cloning were increased to broaden its application. The cosmid clone constructed by this new vector could be linearized at one of the 8-base cutter sites which are assumed to rarely occur in the genome, and followed by in vitro packaging, nested deletion clones were successfully prepared.