FIG. 5.

Substrate for monitoring precise religation. Shown at the top of the figure is the 47-bp oligonucleotide inserted into the TK gene on pTKSce2. The oligonucleotide causes a frameshift mutation in the TK gene and contains two I-SceI recognition sites (5′-TAGGGATAACAGGGTAAT-3′) flanking an XbaI site (5′-TCTAGA-3′). The actual sites of cleavage by I-SceI and XbaI on the top strand of the construct are indicated. As illustrated, simultaneous cleavage of the substrate at both I-SceI sites followed by loss of the intervening 23-bp fragment and precise religation of the two outermost I-SceI sticky ends produces a functional, and thus recoverable, TK gene containing a 24-bp insert harboring a single I-SceI site.