Part:BBa_K1150021

uniCAS Repressor (SV40 promoter)

SV40:HA-NLS-dCas9-Linker-KRAB-NLS:BGH
Function	Transcriptional Repression
Use in	Mammalian cells
RFC standard	RFC 25
Backbone	pSB1C3
Organism	Streptococcus pyogenes, Homo sapiens
Source	Feng Zhang, Addgene Konrad Müller, 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, an SV40 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 device.

Fig. 1 Schematic overview of dCas9-KRAB composite part with all features.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 664
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 4784
  Illegal SapI.rc site found at 4748