(→Construction details) |
(→Construction details) |
||
| Line 2: | Line 2: | ||
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 confirms that all bases of the insert are correct. | 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 confirms that all bases of the insert are correct. | ||
| − | + | 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 === | === Crudely annotated sequence === | ||
Revision as of 22:52, 25 June 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 confirms that all bases of the insert are correct.
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:
