pEYFP-N1 encodes an enhanced yellow-green variant of the Aequorea victoria green fluorescent protein (GFP). The EYFP gene contains four amino acid substitutions previously published as GFP-10C (1). The fluorescence excitation maximum of EYFP is 513 nm, and the emission spectrum has a peak at 527 nm (in the yellow-green region). When excited at 513 nm, the Em of EYFP is 36,500 cm–1M–1 and the fluorescence quantum yield is 0.63 (1), resulting in a bright fluorescent signal. The fluorescence level observed from EYFP is roughly equivalent to that from EGFP.

In addition to the chromophore mutations, EYFP contains >190 silent mutations that create an open reading frame comprised almost entirely of preferred human codons (2). Furthermore, upstream sequences flanking EYFP have been converted to a Kozak consensus translation initiation site (3). These changes increase the translational efficiency of the EYFP mRNA and consequently the expression of EYFP in mammalian and plant cells.

The MCS in pEYFP-N1 is between the immediate early promoter of CMV (PCMV IE) and the EYFP coding sequences. Genes cloned into the MCS will be expressed as fusions to the N-terminus of EYFP if they are in the same reading frame as EYFP and there are no intervening stop codons. The inserted gene should include an initiating ATG codon. EYFP with N-terminal fusion moieties retains the fluorescent properties of the native protein and thus can be used to localize fusion proteins in vivo. The vector contains an SV40 origin of replication and a neomycin resistance (Neor) gene for selection (using G418) in mammalian cells. A bacterial promoter upstream of this cassette (P) expresses kanamycin resistance in E. coli. The vector backbone also provides a pUC19 origin of replication for propagation in E. coli and an f1 origin for single-stranded DNA production.

The recombinant EYFP vector can be transfected into mammalian cells using any standard transfection method. If required, stable transformants can be selected using G418 (4). pEYFP-N1 can also be used simply to express EYFP in a cell line of interest (e.g., as a transfection marker). EGFP, EYFP, and EBFP variants can be used independently or in combination for flow cytometry analysis