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The specificity profile for Cre exhibits its relative desire for the canonical base at every place in the loxP site. We sought to develop a system for profiling recombinase specificity by means of identification of bona fide recombinase substrates from an enormous in vitro library of potential targets. Recombinase specificity profiling of wild-type Cre. In addition, Rec-seq recognized 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), even though Gln94 does in a roundabout way contact the DNA, however as a substitute engages in hydrogen bonds with Gln9035. Together, Rec-seq profiling clarifies the numerous interactions that collectively define Cre recognition at positions 5-7, and highlights the essential roles of secondary and oblique interactions. These long-vary results cannot be inferred from the Cre:loxP structure, highlighting the utility of unbiased, high-decision specificity profiling. Determinants of Cre:loxP specificity identified by Rec-seq on wild-type Cre and Ala-substituted Cre variants

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