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Note: Research in this publication was not performed at Janelia.
Far‐red emitting fluorescent labels are highly desirable for spectral multiplexing and deep tissue imaging. Here, we describe the generation of frFAST (far‐red Fluorescence Activating and absorption Shifting Tag), a 14‐kDa monomeric protein that forms a bright far‐red fluorescent assembly with (4‐hydroxy‐3‐methoxy‐phenyl)allylidene rhodanine (HPAR‐3OM). As HPAR‐3OM is essentially non‐ fluorescent in solution and in cells, frFAST can be imaged with high contrast in presence of free HPAR‐3OM, which allowed the rapid and efficient imaging of frFAST fusions in live cells, zebrafish embryo/larvae and chicken embryo. Beyond enabling genetic encoding of far‐red fluorescence, frFAST allowed the design of a far‐ red chemogenetic reporter of protein‐protein interactions, demonstrating its great potential for the design of innovative far‐red emitting biosensors.