Part:BBa_K1923006
NLS-FLAG-EGFP-dCas9-NLS encoding gene
This composite part is a fusion protein composed of a triple FLAG tag, two SV40 nuclear nuclear localization sequences, a GFP and a dCas9. Sv40 NLS enables the fusion protein to localize in the nuclear. GFP provides fluorescence signal convient for observation under microscope. Protein purification and expression detection can be easily achieved with the help of FLAG tag. dCas9 can be used for CRISPR interference and DNA imaging. We can also utilize dCas9's RNA-binding ability with the help of sgRNA and PAMmer, a NGG-containing DNA oligo.
We have experimentally validated that this new BioBrick Part of our own design and construction works, documented the characterization of this part here, and submitted this part to the iGEM Parts Registry. Since this construc contains a nuclear localization sequence, the most direct supporting evidence is the imaging result. Here, as is illustrated in figure below, we transformed this plasmid into yeasts and indeed observed a nuclear localization of GFP signal, suggesting that after being translated, this fusion protein is relocated into the nucleus guided by the signal on its N-terminal.
Figure 1. Visualization of nuclear localization of suvCas9 proteins guided by NLS.
For more information, please refer to the Wiki page of Team Tsinghua.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1972
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 4251
Illegal XhoI site found at 145 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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