Fig. 1: Effect of LoxPsym spacer sequence on recombination efficiency.

a Cre-LoxP recombination. Cre monomers form a tetrameric complex comprising two active (light) and two inactive (dark) units interacting with two LoxP targets (scissile base pairs in pink). b The orientation of LoxP (green triangle) determines the recombination outcome (left), whereas LoxPsym (pink diamond) recombination stochastically results in deletion or inversion (right). c LoxPsym-NNN variants were obtained by editing spacer nucleotides 1, 2 & 3 (dark pink). Complementary nucleotides of the second strand, and complementary spacer positions 8, 7 & 6 (light pink) were modified accordingly. d The (genomically integrated) fluorescent reporter construct (TDH3 promoter – yECitrine – CYC1 terminator) used to determine recombination efficiency. The plasmid encoding GAL1 promoter –Cre – CYC1 terminator allowed galactose-inducible recombination. e Recombination efficiency of LoxPsym variants after 6 h induction. LoxPsym variants are represented by their spacer nucleotides 1, 2 & 3. Three control samples (black, left) indicate reporters flanked by LoxPsym-ATG sites with C and G at spacer positions 4 & 5 (‘ATGc’), original LoxP sites in inverted (‘LoxP_INV’) and same (‘LoxP_DEL’) orientation. Bars and error bars represent average and standard deviation of three biological replicates, respectively. Reference LoxPsym-ATG in blue. The map (bottom) graphically represents spacer nucleotides 1, 2 & 3. f Effects of single nucleotides on recombination efficiency for each position of the spacer, calculated by generalized linear mixed-effects model fit 3 (Supplementary Table 1), based on data of three biological repeats shown in (e). Odds ratios >1 (dotted line) indicate events are more likely to occur as the predictor increases, odds ratios <1 indicate the opposite. Dots and error bars represent average and standard error, respectively. Statistics by multiple pairwise-comparison by two-sided Tukey honest significant differences test on the log odds ratio scale (‘***p < 0.001, ‘**’p < 0.01, ‘*’p < 0.05, ‘.’p < 0.1, see Supplementary Data 1, GLME for exact p values). g Effects of interactions between nucleotides at two positions of the LoxPsym spacer. Data and statistics similar to (f). Source data are provided as a Source Data file.