Extended Data Fig. 3: Domain insertion screening of PAC and CAT confirms ProDomino predictions.

a, c, ProDomino inferred insertion scores are mapped onto the primary sequence of PAC (a) and CAT (c). Insertion sites selected for experimental testing are marked by vertical lines and color coded as indicated. b, Assessment of insertion tolerance in PAC. HEK293T cells were transfected with vectors encoding the respective PAC variants carrying PDZ insertions after the indicated residue or a negative control expressing enhanced green fluorescent protein (eGFP). Cells were treated with 5 µg/mL puromycin and incubated for 48 hours before cell viability was assessed by MTT assay. Cells treated with different concentrations of toxic Triton X-100 served as controls for the assay itself. Bars indicate means, error bars the standard deviation, and black dots individual data points from n = 3 independent experiments. d, Assessment of the CAT insertion permissibility. Bacteria were transformed with plasmids expressing the indicated CAT variant or an empty control plasmid. Liquid cultures were grown in the presence of 25 µg/mL chloramphenicol for 7 hours and cell density was assessed by measuring OD at 600 nm. Light gray bars represent PDZ insertions behind the indicated residue and dark gray bars correspond to LOV2 insertions at the same position. Bars indicate means, error bars the standard deviation, and black dots individual data points from n = 3 independent experiments.