lox66 is an engineered recombination target site derived from the wild-type loxP site (the target site for the recombinase encoded by the cre locus of the bacteriophage P1 genome). It is composed of two 13bp inverted repeats flanking an 8bp asymmetric spacer sequence (5'-TACCGTTCGTATAatgtatgcTATACGAAGTTAT-3') and contains mutations in the left-hand inverted repeat element (LE) compared to loxP (PMID:7742860, FBrf0188158). Recombination occurs between a pair of target sites oriented in the same direction; the 13bp repeats each act as binding sites for the recombinase, while the asymmetric 8bp spacer is the site of DNA strand exchange and determines the orientation of the target site (PMID:3856690). Since the spacer sequence is not altered compared to loxP, lox66 is expected to be able to recombine with the wild-type loxP site and any of its engineered derivatives which retain the wild-type spacer sequence. However, due to the mutation in the LE, binding affinity of the P1cre recombinase towards this site is expected to be reduced compared to loxP. This property can be exploited to drive the directionality of the recombination reaction; recombination between the lox66 LE variant and an engineered loxP variant containing mutations in the right-hand inverted repeat element (RE), such as lox71, will result in a wild-type loxP site and a doubly mutant 'LE+RE' site containing both the LE and RE variant sequences. The double mutant LE+RE site is expected to have much reduced binding affinity for the cre recombinase, effectively making it inert, such that the 'forward' recombination reaction between the LE and RE single mutant variants is favoured over the reverse reaction (PMID:7742860). This strategy can be used to engineer stable insertions of DNA into the genome or to produce stable inversions (PMID:7742860, reviewed in FBrf0231034).