Difference between revisions of "Part:BBa K3147010:Design"
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<partinfo>BBa_K3147003 short</partinfo>
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<partinfo>BBa_K3147010 short</partinfo>
  
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<partinfo>BBa_K3147003 SequenceAndFeatures</partinfo>
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<partinfo>BBa_K3147010 SequenceAndFeatures</partinfo>
  
 
===Design note ==  
 
===Design note ==  

Revision as of 11:28, 14 October 2019

Mutant TEV (PE10) protease fused to anti-sfGFP VHH with TEV-cleavable maltose binding protein


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 77
    Illegal BsaI.rc site found at 2377
    Illegal SapI.rc site found at 1868

=Design note

The TEV cutting site used is a modified site from the original consensus sequence: ENLYFE/G [3]. The TEV protease used is a mutant TEV with the mutation T17A, T146A, D148P, S153C, S168T, S170A, T173A, S219P [3]. The Terminator used is a double terminator (BBa_0015). The VHH is a VHH directed against the sfGFP [4], it recognizes a conformational pattern.

Reference

[3] Yi, L. et al. 2013. « Engineering of TEV Protease Variants by Yeast ER Sequestration Screening (YESS) of Combinatorial Libraries ». Proceedings of the National Academy of Sciences 110(18): 7229‑34

[4] Kubala, M. H., Kovtun, O., Alexandrov, K., & Collins, B. M. (2010). Structural and thermodynamic analysis of the GFP:GFP-nanobody complex. Protein science : a publication of the Protein Society, 19(12), 2389–2401. doi:10.1002/pro.519