Part:BBa_K812013

GFP-AID OsTirI polysistronic system for auxin detection in tadpole

This part is the combination of the BBa_K812010(GFP-AID) and BBa_K812012 (OsTirI) for co-expression in eukaryotic cells such as the one of frogs for example. It is difficult to use internal Ribosome Entry Site (iRES) to create polysistronic mRNA in tadpole. In order to overcome this limitation, we fused the proteins with a self-cleavable peptide, Pep2A. The protein is transcribed as a single protein and the the peptide cleave itself or with the help of an endogeneous protease giving the two protein, colocalized in the same cell.

This system is a modified version of a patented device by Kanemaki Masato, Kakimoto Tatsuo, Nishimura Kohei, Takisawa Haruhiko and Fukagawa Tatsuo for yeast and mammalian cells use (http://www.freepatentsonline.com/y2012/0115232.html). However the patent does not cover the use for oviparian such as frogs and chicken.

This part can be micro-injected using pSC2+ ( BBa_K812000) or related plasmid as a backbone.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 1990
  Illegal NotI site found at 1844
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 3205
  Illegal XhoI site found at 1913
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 1612
  Illegal NgoMIV site found at 1721
  Illegal AgeI site found at 1021
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 1406
  Illegal BsaI site found at 1636