Codon adaptation of <i>eGFP</i> for medaka is not improving fluorescence.

(A) Relative comparison of in vivo fluorescence in medaka with in vivo fluorescence in E. coli. Indicated are red, green and yellow fluorescent proteins and a diagonal line for orientation. The red, green and yellow fluorescent proteins have been normalized to the same relative values published of mRFP1*, eGFP and Venus, respectively [18]. While many of the fluorescent proteins have similar properties, some are significantly different, reinforcing the need of a systematic review of fluorescent proteins in medaka. (B) Codon adaptation indices (CAIs) of all prior used sequences. Codon adaptation index of each sequence is plotted for medaka and human along with a diagonal line for orientation. Labelled are the two normalizing fluorescent proteins eGFP and mCherry, and the outlier mRuby2. For a plot with full labelling, refer to S2 Fig. (C) Pure codon usage table driven codon averaging does not improve fluorescence intensity of fluorescent proteins. OleGFP (medaka codon-averaged eGFP) fluorescence intensity is overall lower than SceGFP (yeast codon-averaged) and 25 to 30-fold lower than eGFP. (D) Codon adaptation indices of sequences used in order to score for the effect of codon averaging. Additional to the prior used sequences are an eGFP codon optimized for medaka (OleGFP) and an eGFP codon optimized for yeast (SceGFP).