pEYFP-C1 encodes an enhanced yellow-green variant of the?Aequorea victoria?green fluorescent protein (GFP). The EYFP gene contains the four amino acid substitutions previously published as GFP-10C (1): Ser-65 to Gly; Val-68 to Leu; Ser-72 to Ala; and and Thr-203 to Tyr. The fluorescence excitation maximum of EYFP is 513 nm; 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 fluorescent quantum yield is 0.63 (1), resulting in a bright fluorescent signal. The fluorescence observed is roughly equivalent to that from EGFP.
A mixture of EYFP- and EGFP-expressing cells can be sorted by flow cytometry using a single excitation wavelength (i.e., 488 nm). EYFP emission is detected using a 525-nm dichroic shortpass mirror and a 530/30-nm bandpass filter; EGFP emission is detected using a 510/20-nm bandpass filter.
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-C1 is between the EYFP coding sequence and the stop codon. Genes cloned into the MCS will be expressed as fusions to the C-terminus of EYFP if they are in the same reading frame as EYFP and there are no intervening in-frame stop codons. EYFP with a C-terminal fusion moiety 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 for replication and a neomycin resistance (Neor) gene for selection (using G418) in eukaryotic cells. A bacterial promoter (P) upstream of Neor?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-C1 can also be used simply to express EYFP in a cell line of interest (e.g., as a transfection marker).