Part:BBa_I756028

cmv - IgM signal- VH-VL-EporD2-EporTm mut- gp130i- dnaEC-VP16-SV40PA

SCFV-erythropoietinR-GP130 chimeric receptor

When the SCFV is activated by contact with exposed MHC on cardiomyocytes that have been damaged in an infarction, the receptors will dimerise via interactions of the EporD2 domains. EporTransmembrane mutant version was used, L241G and L242P, to introduce a kink into the leucine zipper and to decrease the interchain interaction of EpoR for a tight on/off regulation of the receptor. Leucine zipper formation induces alpha helical structures that induce self-assembly by close packing of TM structures. Oligermerisation of wild-type EporTM is considerably high, producing a high basal rate of signaling which was not desired.

Upon dimerisation of the receptors, the intracellular portion of the chimeric receptor, GP130i (intracellular)will activate downstream signaling. JAK, a tryosine kinase, phosphorylates the gp130i which then Shp2 (a Src homology 2-containing tyrosine phosphatase) binds to. In our novel signaling system we have designed a version of Shp2 fused to dnaEN-mLexA. DnaEN and dnaEC are part of the split intein system which are designed to reconsitute mLexA-VP16 in vivo. MLexA-VP16 translocates to nucleus where is functions as the switch to upregulate expression of the rescue circuit, death circuit and cell adhesion circuit via the binding to the lexA operator minimal promoter.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 1
  Illegal XbaI site found at 7
  Illegal SpeI site found at 3033
  Illegal PstI site found at 2299
  Illegal PstI site found at 2460
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 1
  Illegal NheI site found at 2704
  Illegal SpeI site found at 3033
  Illegal PstI site found at 2299
  Illegal PstI site found at 2460
  Illegal NotI site found at 15
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 1
  Illegal BglII site found at 546
  Illegal BglII site found at 1963
  Illegal BglII site found at 2227
  Illegal BamHI site found at 1212
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 1
  Illegal XbaI site found at 7
  Illegal SpeI site found at 3033
  Illegal PstI site found at 2299
  Illegal PstI site found at 2460
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 1
  Illegal XbaI site found at 7
  Illegal SpeI site found at 3033
  Illegal PstI site found at 2299
  Illegal PstI site found at 2460
  Illegal NgoMIV site found at 1514
  Illegal NgoMIV site found at 2609
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1776
  Illegal SapI site found at 1041