cAMPFIRE-M-dark is a genetically encoded fluorescent cAMP sensor based on cAMPFIRE-M, except that dark tdVenus is used as the FRET (fluorescence resonance energy transfer) acceptor. It is a fusion protein that consists of a FRET donor (a form of mTurquoise2) and a FRET acceptor (two tandem copies of a dark version of Venus) which are separated by sequence from the Homo sapiens RAPGEF3 (Epac1) cAMP-binding protein. The RAPGEF3 sequence is catalytically dead and contains a number of mutations that in combination improve the properties of the sensor. The RAPGEF3 portion undergoes a conformational change upon binding to cAMP, which causes a robust decrease in FRET. The use of dark tdVenus in cAMPFIRE-M-dark allows the collection of a broader bandwidth of mTurquoise2 spectrum compared to cAMPFIRE-M and reduces the bleedthrough signal at the orange and red spectrum, facilitating the multiplexed use of a sensor or cytosolic marker at those wavelengths (FBrf0256297).