20xUAS regulatory sequences drive expression of 'FLIM-AKAR' (also known as AKAR3.4), a genetically encoded A-kinase activity reporter that is compatible with 2-photon fluorescence lifetime imaging microscopy. "FLIM-AKAR" is derived from the AKAR3 reporter and consists of two fluorescent proteins (mEGFPΔ and circularly permuted sReaCh) that are separated by sequence encoding a phospho-binding domain (FHA1) and a protein kinase A (PKA) substrate domain. When phosphorylated by PKA, the PKA substrate domain interacts with the phospho-binding domain, resulting in fluorescence resonance energy transfer (FRET). The phosphorylated reporter leads to an increase of acceptor:donor emission ratio, as well as a decrease in donor fluorescence lifetime because of an additional energy transfer pathway. mEGFPΔ, a truncated monomeric EGFP (amino acids 1-227), acts as the donor fluor, circularly permuted sReaCh, a circularly permuted dark YFP, acts as the acceptor fluor (details from PMID:24765076).