UAS 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 peptide (PDVPRTPVDKAKLSFQFP) that is specifically phosphorylated by extracellular signal-regulated kinase (ERK), the EGFP fluorescent protein and 'HOTag3' (a homo-oligomeric coiled-coil sequence that forms hexamers). The second coding sequence consists of a phosphothreonine-binding WW domain, the EGFP fluorescent protein and 'HOTag6' (a homo-oligomeric coiled-coil sequence that forms tetramers). Together, the two proteins form 'ERK-SPARK', a phase separation-based kinase reporter that reports the activity of ERK. Upon activation of ERK, the PDVPRTPVDKAKLSFQFP substrate peptide in ORF1 is phosphorylated and interacts with the WW 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.