Part:BBa_K5036032

dCas9(C)_NES-Syn-VEGFR-2 (VEGF-R2, N-TEV, NES, TCS (Q, L), HA, dCas9(C),mCherry

Part Description

In our second receptor's chain, we've engineered a system that responds to tissue injury. An external domain, VEGF-R2, is attached to an internal domain composed of N terminal domain of TEV protease, a nuclear export signal (NES), a TEV cleavage site(TCS(Q,L)), and dCas9(C).

Usage

this is our receptor's second chain. our receptor is activated after binding of VEGF to the external domain which is designed to attach specifically to it. after activation the two domains of TEV dimerizes forming catalytically active TEV protease which will cleave the two chains at TCS. upon cleavage of the two chains the two domains of dCas9 dimerize and is released attached to transcription activator to be guided to its direction

this figure illustrates variant of our receptor's second chain whare dCas9 (C) is attached to it. .

Software Characterization

we had the chance to match the external domains with different internal domain components to select single suitable receptor chain. The whole chain affinity is affected by the internal domain, thus we had to try VEGFR2Cdcas with VEGFA:

VEGFR2Cdcas-VEGFA

The interaction between the chain composed of VEGFR2 as an external domain and Cdcas9 as an internal domain with VEGFA yields ΔG of -13.6 kcal mol-1 .

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 982
  Illegal BglII site found at 2341
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 3976
  Illegal NgoMIV site found at 4049
  Illegal NgoMIV site found at 4534
  Illegal NgoMIV site found at 5443
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 1854
  Illegal BsaI.rc site found at 664
  Illegal BsaI.rc site found at 1442
  Illegal BsaI.rc site found at 2731
  Illegal SapI.rc site found at 3289