PME-mCherryCAAX H80D**

Construction Details

pME-mCherryCAAX was made by PCR amplification of mCherry-CAAX, using primers to add att sites, followed by a BP reaction. mCherry- CAAX is mCherry with a C-terminal fusion of 21 amino acids of human Harvey Ras, encoding a prenylation signal. Sequencing shows that there is a single missense mutation in the mCherry (H80D).

25 June 2007: Apparently our original mCherry-CAAX miniprep ( which we sequenced) was a mixture of two clones, one correct and one with a H80D point mutation. Functional tests of expression clones made from the miniprep gave red fluorescence, but these clones lack the H80D mutation. Sequencing confirms that the maxiprep that we grew for distribution retained the point mutation. Gage Crump has made expression clones from the maxiprep (presumably bearing H80D) which give weak red fluorescence. However, we believe that the H80D mutation in fact diminishes fluorescence of this construct (data from Seok-yong Choi).

This clone was replaced by clone 450, and then by 550, which lacks this mutation.