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We co-transfected HEK293T cells with the reporter plasmid and a plasmid expressing either Tre or Brec1, then used the fraction of cells exhibiting EGFP fluorescence to evaluate the activity on each goal. To generate candidate off-goal substrates for Tre and Brec1, we first identified non-target half-site sequences that appeared with excessive abundance within the post-recombinase-handled dataset. We additionally assessed whether Rec-seq knowledge alone may predict the activity of Tre and Brec1 on endogenous human genomic sequences. We assessed the activity of Tre and Brec1 on these artificial substrates in human cells utilizing a reporter plasmid containing pairwise mixtures of L1-L4 and R1-R4 half-sites flanking a poly-A terminator that blocks EGFP transcription (Fig. 5a). In this reporter system, recombinase-mediated deletion of the terminator restores EGFP expression. Cells had been transfected with recombinase expression plasmid and an EGFP reporter plasmid containing candidate recombinase substrates flanking a poly-A terminator that blocks EGFP transcription. Both sites comprise a G•C base pair at position 4 and 4′, and, in agreement with the literature41, we noticed nearly absolute specificity for these positions in both substrates (Fig. 4d). Rec-seq profiles additionally showed enrichment of the ACNAC motif present at positions 6-10 in both the attP and attB half-sites (Fig. 4d), in line with the presence of specifying protein:DNA interactions operating on each targets

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