Difference between revisions of "PME-mCherryCAAX H80D"

Revision as of 22:53, 2 November 2007

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, there is a good chance that the H80D mutation in fact diminishes fluorescence of this construct.

We are currently working towards resolving this issue (i.e., obtaining a clone without this mutation).

Crudely annotated sequence

FASTA file with the full-length sequence as well as sequences of individual components:
pME-mCherryCAAX sequence

Crude map

Screenshot from Sequencher showing locations of components: