pECFP-N1 encodes an enhanced cyan fluorescent variant of the Aequorea victoria green fluores-cent protein gene (GFP). The ECFP gene contains six amino acid substitutions. The Tyr-66 to Trpsubstitution gives ECFP fluorescence excitation (major peak at 433 nm and a minor peak at 453 nm)and emission (major peak at 475 nm and a minor peak at 501 nm) similar to other cyan emissionvariants. The other five substitutions (Phe-64 to Leu; Ser-65 to Thr; Asn-146 to Ile; Met-153to Thr; and Val-163 to Ala) enhance the brightness and solubility of the protein, primarily due toimproved protein folding properties and efficiency of chromophore formation. In addition to the chromophore mutations, ECFP contains >190 silent mutations that create an open reading framecomprised almost entirely of preferred human codons. Furthermore, upstream sequences flanking ECFP have been converted to a Kozak consensus translation initiation site. These changes increasethe translational efficiency of the ECFP mRNA and consequently the expression of ECFP in mammalian and plant cells.