UASt regulatory sequences drive expression of two coding regions that are separated by a 2A linker (this results in separate translation of each protein). The first coding sequence consists of a consensus substrate peptide (HVRAHSSPASLQ) that is specifically phosphorylated by yki, the EGFP fluorescent protein and 'HOTag3' (a homo-oligomeric coiled-coil sequence that forms hexamers). The second coding sequence consists of a phosphoserine/threonine-binding domain (from 14-3-3ΞΆ) fused to 'HOTag6' (a homo-oligomeric coiled-coil sequence that forms tetramers). Together, the two proteins form 'YAP-SPARK', a phase separation-based kinase reporter that reports the activity of yki. Upon activation of yki, the substrate peptide in ORF1 is phosphorylated and interacts with the phosphoserine/threonine-binding domain in ORF2. The presence of the 'HOTag3' and 'HOTag6' homo-oligomeric coiled coils on the respective proteins results in multivalent protein-protein interaction, leading to phase-separation into intensively fluorescent droplets.
