Part:BBa_K4686069

pMCherry+AmylaseDegrader_Link_EGF_mEGFP

This plasmid contains the pMLB_mCherry plasmid backbone along with the Amylase Degrader signal peptide. This signal peptide is linked to an EGF ligand bound to mEGFP via a GGSG linker. As a signal peptide, this protein contains a signal sequence that will interact with signal recognition particles to then direct ribosomes to the endoplasmic reticulum for co-translational insertion to take place.

Used primers BBa_K4686027 and BBa_K4686028 on the pMLB_mCherry Backbone and primers BBa_K4686035 and BBa_K46036 on the T7_PB_SFFV_CD4_EGFf_mEGFP_TEVs_CD28_ plasmid. The BBa_K4686035 primer contained the Amylase Degrader sequence sourced from our mentor Jeffery Lee, so the resulting insert contains the EGF ligand sequence from T7 as well as the signal peptide sequence. The pMLB_mCherry_GGBsMTS plasmid backbone was sourced from Piet de Boer. The T7 plasmid was sourced from our mentor Beatrice Ramm.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 1306
  Illegal EcoRI site found at 2456
  Illegal XbaI site found at 1382
  Illegal PstI site found at 7959
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 1306
  Illegal EcoRI site found at 2456
  Illegal PstI site found at 7959
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 1306
  Illegal EcoRI site found at 2456
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 1306
  Illegal EcoRI site found at 2456
  Illegal XbaI site found at 1382
  Illegal PstI site found at 7959
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 1306
  Illegal EcoRI site found at 2456
  Illegal XbaI site found at 1382
  Illegal PstI site found at 7959
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1637
  Illegal BsaI.rc site found at 7785
  Illegal SapI site found at 6702