Part:BBa_K1150063

HA-NLS-Cas9-Linker 3 AA

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HA-NLS-dCas9-Linker
Function	A new effector can be fused to this dCas9 intermediate part
Use in	Mammalians
RFC standard	RFC 25
Backbone	pSB1C3
Submitted by	[http://2013.igem.org/Team:Freiburg Freiburg 2013]

This intermediate part can be used to engineer your own dCas9 fusion proteins. [http://2013.igem.org/Team:Freiburg Freiburg 2013] already fused VP16, KRAB, G9a, UVR8, PIF6 and CRY2 to dCas9. With the HA tag you can verify via wester blot that your fusion protein is expressed or you can purify the protein with affinity chromatography. The NLS makes sure your fusion protein is transported into the nucleus of mammalian cells. The short 3 amino acids linker will make a connection beween dCas9 and your effector domain.

Usage

At the front of this intermediate part you can ligate any favored promoter (Freiburg 2013 used SV40 and CMV promoters). At the back of this part you can fuse an effector domain without frameshift by using the RFC 25 standard.

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 312
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]