PME-mCherryCAAX H80D**: Difference between revisions
Hggenusrwki (talk | contribs) Created page with "== Construction Details == This template is used to display DNA sequence information in a standardized format. == Crude Map == photo showing locations of components == Sequence == * Annotated Sequence: (Genbank format) link * Full-Length Sequence with individual component sequences included: (FASTA file) link == Reference == This template is used to display DNA sequence information in a standardized format." |
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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 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This clone has now been replaced by clone 450, which lacks this mutation. | |||
== Crudely Annotated Sequence == | |||
FASTA file with the full-length sequence as well as sequences of individual components: pME-mCherryCAAX H80D sequence | |||
== Crude Map == | |||
== | Screenshot from Sequencher showing locations of components: | ||
PME-mCherryCAAX H80D.png | |||
Revision as of 14:44, 14 March 2025
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 has now been replaced by clone 450, which lacks this mutation.
Crudely Annotated Sequence
FASTA file with the full-length sequence as well as sequences of individual components: pME-mCherryCAAX H80D sequence
Crude Map
Screenshot from Sequencher showing locations of components: PME-mCherryCAAX H80D.png