(New page: === 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-term...) |
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=== Construction details === | === 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. | 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. | ||
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| + | 18 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. We believe that the maxiprep that we grew for distribution retained the point mutation, but the functional tests that we did ourselves (which gave fluorescence) used the miniprep. So, while expression clones made with the middle clone miniprep gave red fluorescence, we can't say for sure whether these clones had the point mutation or not. We are going to go back and sequence our expression clone to look for H80D. | ||
=== Crudely annotated sequence === | === Crudely annotated sequence === | ||
Revision as of 20:51, 18 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.
18 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. We believe that the maxiprep that we grew for distribution retained the point mutation, but the functional tests that we did ourselves (which gave fluorescence) used the miniprep. So, while expression clones made with the middle clone miniprep gave red fluorescence, we can't say for sure whether these clones had the point mutation or not. We are going to go back and sequence our expression clone to look for H80D.
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:
