Difference between revisions of "Part:BBa K3505026"
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<partinfo>BBa_K3505026 short</partinfo> | <partinfo>BBa_K3505026 short</partinfo> | ||
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===Usage and Biology=== | ===Usage and Biology=== | ||
− | + | The TANGO GPCR-assay, takes advantage of the b-arrestin capabilities to normally bind to the GPCR receptor and facilitate its endocytosis, desensitization or GPCR-independent signaling effects. The second composite of our TANGO system is a fusion of the b-arrestin-2 with the TEV protease. Upon SCFAs-induced activation of the FFAR2 receptor, β-arrestin 2 is recruited and binds to the intracellular part of the receptor. This low proximity allows the TEV protease that is tagged to the βarrestin-2 carrier to cleave its substrate (TCS) and subsequently release lac repressor. Then, the lac repressor is free to inhibit expression of downstream elements of our signaling system, by binding to a lac operator sequence. | |
===Design Notes=== | ===Design Notes=== |
Revision as of 17:56, 27 October 2020
ParaBAD:RBS- β-arrestin2:TEVp:terminator
Usage and Biology
The TANGO GPCR-assay, takes advantage of the b-arrestin capabilities to normally bind to the GPCR receptor and facilitate its endocytosis, desensitization or GPCR-independent signaling effects. The second composite of our TANGO system is a fusion of the b-arrestin-2 with the TEV protease. Upon SCFAs-induced activation of the FFAR2 receptor, β-arrestin 2 is recruited and binds to the intracellular part of the receptor. This low proximity allows the TEV protease that is tagged to the βarrestin-2 carrier to cleave its substrate (TCS) and subsequently release lac repressor. Then, the lac repressor is free to inhibit expression of downstream elements of our signaling system, by binding to a lac operator sequence.
Design Notes
The coding sequence was domesticated . We removed BsmBI ,BsaI , BtgZI, BpiI sites in order to be compatible with GoldenBraid and MoClo. The sequence is cloned in alpha2 vector BBa_K3505009 and has overhangs compatible for Golden Braid cloning.
Verification of Cloning
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal SpeI site found at 2854
Illegal PstI site found at 1546
Illegal PstI site found at 1633 - 12INCOMPATIBLE WITH RFC[12]Illegal SpeI site found at 2854
Illegal PstI site found at 1546
Illegal PstI site found at 1633 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1446
Illegal BamHI site found at 1148
Illegal XhoI site found at 1843
Illegal XhoI site found at 2237 - 23INCOMPATIBLE WITH RFC[23]Illegal SpeI site found at 2854
Illegal PstI site found at 1546
Illegal PstI site found at 1633 - 25INCOMPATIBLE WITH RFC[25]Illegal SpeI site found at 2854
Illegal PstI site found at 1546
Illegal PstI site found at 1633
Illegal AgeI site found at 983
Illegal AgeI site found at 1829 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 965
Illegal SapI.rc site found at 2788
Illegal SapI.rc site found at 3136