Part:BBa_K1150017

dCas9 with CMV promoter

pCMV:HA-NLS-dCas9-G9a-NLS:tBGH
Function	DNA binding protein
Use in	Mammalian cells
RFC standard	RFC 25
Backbone	pSB1C3
Organism	Streptococcus pyogenes
Source	Feng Zhang, Addgene
Submitted by	[http://2013.igem.org/Team:Freiburg Freiburg 2013]

Cas9 is a protein that binds DNA via an protein-RNA-DNA interaction. This version of Cas9 (dCas9) is working as a carrier for effectors that want to be brought to DNA in a sequence specific manner. The native nikase activity of the protein has been mutated so that it allows DNA binding without cutting. The sequence is human codon optimized. The protein is part of team Freiburg's (2013) the [http://2013.igem.org/Team:Freiburg uniCAS toolkit].

The usage of the strong CMV promoter enables this device to be expressed in a strong manner. If weaker expression levels are needed, we recommend using our device with SV40 promoter.

Usage and Biology

The dCas9 protein is able to target several loci at once as it interacts with small RNAs to build up a complex that will interact with complementary DNA strands. Its origin is the adaptive immune system of Streptococcus pyogenes called CRISPR. Hijacking this system leads to a whole new approach for multiple gene targeting.

The iGEM team Freiburg 2013 combined effectors with this protein to create a toolkit, that is able to activate or repress specific and multiple target loci.

This approach offers new possibilities for fundamental epigenetic research, tissue engineering and cancer research.

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
  Illegal BglII site found at 5139
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]