Part:BBa_K1075026
AraC-pBad-RBS34-mCherry-(Ec)ssrA(DAS+4)-TT
a
construction
The part contains the promoter plac, a ribosomal binding site, the red fluorescent protein mCherry fused to the ssrA (DAS+4) tag and a double terminator.
biology
AraC-pbad is an arabinose inducible regulatory promoter/repressor unit.
The ribosomal binding side is a sequence of mRNA where the ribosome binds to start translation.
The ssrA tag is a short peptide sequence, which is fused to the C-terminus of proteins, which should be degraded. It relates to the protease ClpXP complex in E.coli and it also allows the adaptor proteins SspB binding and delivering substrates to the proteases in order to make the process more efficient. The mutated ssrA (DAS+4) weakens a direct binding between proteases and ssrA and increases the dependance of sspB. [http://www.ncbi.nlm.nih.gov/pubmed/16762842]
mCherry is a red fluorescent protein with the excitation maximum at 587 nm and the Emission maximum at 610 nm. [http://www.ncbi.nlm.nih.gov/pubmed/15558047]
The double terminator stops the transcription at this point.
application
As we want to control protein degradation by controlling the function of SspB, we tag the red fluorescent protein mCherry with ssrA (DAS+4) to measure the degradation rate.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 2726
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 1144
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 979
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 961
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