Composite Parts
Part-NLS-FALG-EGFP-dCas9-NLS-Silver-1
This composite part is a fusion protein composed of a triple FLAG tag, two SV40 nuclear localization
sequences, a GFP and a dCas9. Sv40 NLS enables the fusion protein to localize in the nuclear. GFP provides
fluorescence signal convenient for observation under microscope. Protein purification and expression
detection is pretty 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.
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Part-GAL4BD-NLS-FLAG-EGFP-dCas9-NLS-GAL4AD-Gold-1
This composite part is a fusion protein composed of GAL4, triple FLAG tag, two sv40 nuclear localization
sequence, dCas9 and GFP. Triple FLAG tag and GFP is designed for observation and expression detection. The
protein produced by this parts can be sequestered in the cytoplasm with the help of sgRNA and PAMmer,
utilizing dCas9's mRNA binding ability. However, when there is a mutation in the target sequence of the
mRNA, SV40 NLS would drag this protein into the nuclear. Then GAL4 would active the gene downstream UAS
promoter. Thus this parts can be used for gene mutation surveillance.
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Part-msr-msd-Reverse transcriptase-Bronze-2
The msr-msd sequence in the part is flanked by two inverted repeats. Once transcribed, the msr-msd RNA
folds into a secondary structure guided by the base pairing of the inverted repeats and the msr-msd
sequence. The RT enzyme recognizes this secondary structure and uses a conserved guanosine residue in the
msr as a priming site to reverse transcripe the msd sequence and produce a hybrid RNA-ssDNA molecule
called msDNA. The two BsaI sites enable researchers to design ssDNA sequence produced by this cassette,
thus providing a programmable in-vivo ssDNA expression system.
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Part-Promoter-HDV ribozyme-sgRNA-Terminator-Gold-2
This part expresses small guide RNA targeting yeast Actin mRNA. With the assistance of PAMmer, a
nucleotide oligo containing NGG sequence, it is able to help dCas9 to bind to the Actin mRNA transcripts,
thus sequestering dCas9 in the cytoplasm. The transcription of this sgRNA is driven by tRNATyr promoter.
HDV ribosome is a self-splicing element, which would process sgRNA to form a mature transcript.
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