LE/RE mutant lox system. (a) Wild-type loxP sequence. The loxP site is composed of an asymmetric 8-bp spacer flanked by 13-bp inverted repeats that the Cre recombinase recognizes and binds to. (b) Integrative recombination with LE/RE mutant lox sites. The asterisks represent the mutations. RE mutants have mutations in the right inverted repeat region, and LE mutants have mutations in the left inverted repeat region. Through recombination between LE and RE mutant lox sites, a wild-type loxP site and a double mutant lox site carrying mutations in both ends are produced. The double mutant lox site has a lower affinity for the Cre enzyme; therefore, recombination of the product rarely occurs, and the recombination reaction tends toward integration. (c) Sequences of mutant lox sites used in this study. Lox71 carries 5 bp of mutation in the left element. The sequence of the right element of lox66 is complementary to the left element sequence of lox71. Mutated sequences are indicated by bold and are underlined.