Part:BBa_K5036027
dCas9(C)_NLS-Syn-VEGFR-1(VEGF-R1, C-TEV, NLS, TCS(Q,G),HA,dCas9(C), VP64, GAL4, GFP)
Part Description
In our first receptor chain, we've engineered a system that responds to tissue injury. An external domain, VEGF-R1, is attached to an internal domain composed of C terminal domain of TEV protease, a nuclear localization signal (NLS), a TEV cleavage site(TCS(Q,G)), and dCas9(C) which is linked to transcription activators: VP64 and GAL4
Usage
this is our receptor's first 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 our receptor's first chain structure which contain VP64 and GAL4 as our transcription factor activators.
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 VEGFR1Cdcas with VEGFA:
VEGFR1Cdcas-VEGFA
The interaction between the chain composed of VEGFR1 as an external domain and Cdcas9 as an internal domain with VEGFA yields ΔG of -11.2 kcal mol-1 .
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 2173
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 6308
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 3973
Illegal NgoMIV site found at 4046
Illegal NgoMIV site found at 4531
Illegal NgoMIV site found at 5440 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 6227
Illegal BsaI.rc site found at 7175
Illegal SapI.rc site found at 3268
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