Device
Part:BBa_K1150022
Designed by: M. Stammnitz Group: iGEM13_Freiburg (2013-09-17)
uniCAS Repressor (CMV promoter)
| CMV:HA-NLS-dCas9-Linker-KRAB-NLS:BGH | |
|---|---|
| Function | Transcriptional Repression |
| Use in | Mammalian cells |
| RFC standard | RFC 25 |
| Organism | Human cytomegalovirus Streptococcus pyogenes Homo sapiens |
| Source | iGEM Team Freiburg 2012 Feng Zhang, MIT AG Weber, University of Freiburg |
| Submitted by | [http://2013.igem.org/Team:Freiburg Freiburg 2013] |
Krüppel-associated Box repressor domains - commonly termed as KRAB - are highly conserved polypeptide motifs and were first functionally characterized in 1991 (Rosati et al., 1991). As they constitute about one third of all human zinc finger transcription factors, key regulatory features in higher eukaryotic transcriptomics are suggested (Witzgall et al., 1994). Even in terms of tetrapod evolution, the role of their great abundance has been extensively discussed (Birtle, 2006). Even though KRAB minimal domains are usually no longer than 50-75 amino acids, their mechanism of function remains complex.
Common biochemical models suggest a key role in epigenetic silencing, by recruiting a scaffold of diverse proteins - amongst others histone deacetylases and histone methyltransferases (Urrutia, 2003). Til date in 2013, KRAB repressor domains were attached to several DNA binding proteins such as tetR, TAL effectors and [http://2013.igem.org/Team:Freiburg dCas9] - thereby efficiently silencing gene expression downstream of desired target promoters.
In this attempt, a CMV promoter was cloned in front of a RFC25-conformal dCas9. Flanked by two NLS, complemented with an HA-Tag for Western blot detections and fused to KRAB with a net 7 amino acid linker, this resulted in the here-shown composite part.
In this attempt, a CMV promoter was cloned in front of a RFC25-conformal dCas9. Flanked by two NLS, complemented with an HA-Tag for Western blot detections and fused to KRAB with a net 7 amino acid linker, this resulted in the here-shown composite part.
Theoretically, this functional repressor can be directed towards almost any gene or promoter of interest, by harnessing a specifically designed [http://2013.igem.org/Team:Freiburg/Project/crrna#rnaimer RNAimer plasmid]. Two corresponding 30-basepair oligos, corresponding to the desired target, must therefore be annealed and inserted into the RNAimer plasmid. Team Freiburg 2013 provides an easy [http://2013.igem.org/Team:Freiburg/Project/crrna#design_tool designing tool] for such an attempt. Furthermore, [http://2013.igem.org/Team:Freiburg/Project/crrna#multiple_targeting multiple transcriptional regulation] can be achieved by an idempotent cloning strategy, utilizing various crRNA plasmids and co-transfecting this final vector with dCas9-effectors.
Sequence and Features
Assembly Compatibility:
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 576
Illegal BglII site found at 900 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 5020
Illegal SapI.rc site found at 4984
References
Rosati, M. et al. (1991). Members of the zinc finger protein gene family sharing a conserved N-terminal module. Nucleic acids research 19, 5661-5667.
Witzgall, R. et al. (1994). The Krüppel-associated box-A domain of zinc finger proteins mediates transcriptional repression. Proc Nati Acad Sci 91, 4514-4518.
Birtle, Z. and Ponting, C. (2006). Meisetz and the birth of the KRAB motif. Bioinformatics 22, 2841-2845.
Urrutia, R. (2003). KRAB-containing zinc-finger repressor proteins. Genome Biology 4, 4:231.
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Categories
Parameters
| None |