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Double Ala substitution at each Gln90 and Gln94 carried out equally to the Gln90→Ala single mutant (Fig. 2e), suggesting that the DNA-contacting residue Gln90 plays the dominant function in defining DNA specificity amongst the 2 residues. As well as, Rec-seq identified a contribution from a secondary residue beforehand unknown to take part in specifying positions 5-7. Ala substitution at Gln94 resulted in lower specificity at positions 6 and 7 but compensatory will increase elsewhere (Fig. 2e), despite the fact that Gln94 does not directly contact the DNA, but as an alternative engages in hydrogen bonds with Gln9035. Rec-seq revealed the sequence desire of Cre to be asymmetric, as is clear when the left and proper half-site enrichment profiles are superimposed (Supplementary Fig. 6a). To ensure that an asymmetric sequence preference is a property of the enzyme and not due to the totally different DNA sequences flanking the library oligonucleotides (Fig. 1b), we carried out Rec-seq using a substrate library similar to the original except that the non-palindromic loxP core was replaced with its reverse complement (Supplementary Fig. 6b). The Rec-seq enrichment profile of this "inverted core" loxP library mirrored, relatively than duplicated, the profile on the original substrate library (Supplementary Fig. 6b), indicating that the oligonucleotide sequence context was not accountable for the asymmetry of the Cre specificity profile

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