UASt regulatory sequences drive expression of a transcript that encodes two open reading frames separated by a T2A linker (this results in separate translation of each protein). The first ORF encodes the 'ERK-KTR' kinase translocation reporter of MAPK activity (PMID:24949979). 'ERK-KTR' is a fusion protein composed of the ERK docking site from the Mmus\Elk1 gene, a nuclear localization signal (NLS), a nuclear export signal (NES) and the Clover fluorescent protein (sequences codon-optimized for Drosophila). Both the NLS and NES have been mutated to contain consensus phosphorylation sites for MAPK, such that phosphorylation decreases the activity of the NLS and increases the activity of the NES. This results in the protein shuttling from the nucleus to the cytoplasm in response to MAPK phosphorylation. The second ORF encodes His2Av tagged with mCherry, a red fluorescent nuclear marker that can be used to segment and track cells and as a normalization reference so that nuclear ERK-KTR levels can be used as a readout for MAPK kinase activity.