Part:BBa_K2549061

CMV-CD8α-V5-LaG16-mN1ce-tTA-P2A-EGFP

This part contains our SynNotch receptor. The extracellular domain of SynNotch is LaG16 which is a nanobody with high affinity and high specificity to EGFP. Mouse Notch1 extended core region is served as the Notch core. tTA is a transcription activator. A CD8α signal peptide is placed on the N terminal to the SynNotch receptor for membrane localization. V5 tag is fused to the SynNotch receptor for easy determination of surface expression. A CMV promotor which can conduct a strong constitutive expression is set before SynNotch. A P2A-EGFP is placed into the same open reading framework with SynNotch to ensure its expression. This part is copied from a viral vector which can be expressed in mammalian cells.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 3156
  Illegal XbaI site found at 2154
  Illegal PstI site found at 924
  Illegal PstI site found at 968
  Illegal PstI site found at 1287
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 3156
  Illegal NheI site found at 531
  Illegal PstI site found at 924
  Illegal PstI site found at 968
  Illegal PstI site found at 1287
  Illegal NotI site found at 2085
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 3156
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 3156
  Illegal XbaI site found at 2154
  Illegal PstI site found at 924
  Illegal PstI site found at 968
  Illegal PstI site found at 1287
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 3156
  Illegal XbaI site found at 2154
  Illegal PstI site found at 924
  Illegal PstI site found at 968
  Illegal PstI site found at 1287
  Illegal NgoMIV site found at 723
  Illegal NgoMIV site found at 1428
  Illegal NgoMIV site found at 1605
  Illegal NgoMIV site found at 2143
  Illegal NgoMIV site found at 3198
  Illegal AgeI site found at 540
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 1764
  

Our characterization

Please refer to our previous wiki http://2017.igem.org/Team:Fudan/Demonstrate for more experimental details. Last year, we have tested this SynNotch using surface-expressed EGFP.

SynNotch receptor shows an antigen-dependent behaviour. We used fluorescence confocal microscopy to capture images for this figure. maxZ is the max intensity projection of all slices (about 80 in total) at a given position, while slice⁵¹ was a middle bottom optical slice. OE means overexposed images to reveal details. EGFP signals in green is the antigen, and mCherry signal in red is the output. We present this data in 2017 iGEM. And, this plasmid has been requested by others since then.