cAMPFIRE-M is a genetically encoded fluorescent cAMP sensor. It is a fusion protein that consists of a FRET (fluorescence resonance energy transfer) donor (mTurquoise2 with a truncation of the C-terminal 11 amino acids) and a FRET acceptor (two tandem copies of a circularly permuted version of Venus) which are separated by sequence from the Homo sapiens RAPGEF3 (Epac1) cAMP-binding protein. The RAPGEF3 sequence lacks the N-terminal DEP domain, is catalytically dead (contains amino acid replacements T781A and F782A), and contains a number of mutations that in combination improve the properties of the sensor (amino acid replacements Q270E, M312L, L777A, K778A). The RAPGEF3 portion undergoes a conformational change upon binding to cAMP, which causes a robust decrease in FRET (FBrf0254910).