Fig. 3: Limited cross-reactivity between 16 LoxPsym variants simultaneously.

a Each construct (16 test and 2 control) included all 16 LoxPsym variants and ADE2 and URA3 expression cassettes. The URA3 cassette was flanked by LoxPsym-TCA (selection for recombination). Controls tested 2 locations of the ADE2 cassette and should not result in ADE2 deletion. Test constructs differed in the LoxPsym-NNN variant upstream of ADE2 (pink) and verified cross-reactivity between all sites and recombination efficiency between identical LoxPsym-NNN. LoxPsym variants in the array were separated by 100 bp⁷⁰. Constructs were inserted at the CAN1 locus of BY4741 ΔADE2 carrying plasmid pSH47-His-Cre or pSH47-His-Vec (negative control). b After 6 h induction, cells were plated on SC+FOA plates (URA3 deletion with LoxPsym-TCA). Red clones (deletion of ADE2) were selected for PCR and sequencing. c Percentage of the population with ADE2 deletion (red phenotype). Dots represent plate counts of six biological replicates, bars and error bars indicate average and standard deviation, respectively. Color indicates pairwise recombination efficiency (Fig. 1e). Control strains showed a negligible frequency of ADE2 deletions (0.61 ± 0.70% (control 1) and 0.37 ± 0.22% (control 2)). Statistics by analysis of variance and two-sided Dunnettx’s multiple comparisons of means (‘***’p < 0.001, ‘.’p < 0.1, ‘ns’ p > 0.1, Supplementary Data 1, counts) to control 1 (black) and control 2 (gray). No colonies were observed for strains carrying pSH47-His-Vec (Supplementary Data 1, counts). d Pearson correlation test between average recombination efficiency (error bars indicate standard deviation) and degree of LoxPsym insulation (distance to array border). Colors similar to (a). e Measured (dots/triangles) and expected (crosses) length of recombined constructs of 36 randomly selected red clones from each strain. Three random samples of correct size and all fragments of unexpected size (illegitimate recombination) were analyzed (Sanger sequencing, triangles). Color indicates sequencing result (green if correct, red-scaled if incorrect, in which case interacting LoxPsym-pairs are indicated). Bars indicate the number of illegitimate recombination events per LoxPsym variant. f Recombination efficiencies of red clones normalized by the number of possible observations. Source data are provided as a Source Data file.