Abstract: The present disclosure provides fluorescent polypeptides containing a fusion of a circularly permuted, first fluorescent protein and a second fluorescent protein, the first fluorescent protein containing a first fluorescent moiety and the second fluorescent protein containg a second fluorescent moiety, wherein the second fluorescent protein is contained in the circularly permuted first fluorescent protein to form a cassette that can be inserted into sites within a sensing protein of interest to form novel biosensors; alternatively, the reference domain can be inserted into an existing single-fluorescent protein-based, intensiometric biosensor in order to make a ratiometric biosensor in one cloning step; nucleic acid sequences encoding the fluorescent polypeptides, fluorescent sensors including the fluorescent polypeptides, and methods of making and using same are described herein.