Abstract: The present invention provides a method for producing nested deletions in vitro in desired DNA, comprising: 1) preparing a vector containing a DNA fragment in which nested deletions are to be generated and the terminal repeat of a transposon; 2) incubating the vector in vitro with transposase and a DNA replication system; 3) obtaining the vector incorporating the DNA fragment with nested deletions as a reaction product; and optionally, 4) transforming a host cell with the reaction product and growing it; and 5) recovering a vector incorporating said DNA fragment with nested deletions from the grown host cell.