Schematic overview of the subcloning procedure. The upper box contains the two vectors the reaction starts with, i.e. the entry vector with its two key elements flanking an insert and the destination vector with its NheI recognition site. By the enzymatic action (arrows) of Esp3I and NheI these vectors are linearized to form linear intermediate products as shown in the central box. These intermediates are subject to T4 ligase activity and can be ligated to yield a range of products: the initial vectors (upper box), circular intermediate products (lower box) and the desired product vector. Note that all circular intermediate products are again substrate for Esp3I and thus again linearized. There is a sole stable product in this reaction system, which is the desired product vector (circular products only shown if carrying at least one resistance marker). Shaded boxes termed KEY: key element as shown in Fig. 1.