Part:BBa_K2980011:Design
CRY2(R489E)
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 723
Illegal PstI site found at 533 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 723
Illegal PstI site found at 533 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 723
Illegal BglII site found at 393
Illegal BglII site found at 852
Illegal BamHI site found at 1331 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 723
Illegal PstI site found at 533 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 723
Illegal PstI site found at 533
Illegal AgeI site found at 277
Illegal AgeI site found at 1006 - 1000COMPATIBLE WITH RFC[1000]
Design Notes
We introduce mutations on C terminal of CRY2, charges on which is probably reasonable for its oligomerization. According to theoretical postulation, positive charge on C terminal facilitates oligomerization while negative charge inhibits. Upon knowing that, the arginine on 489 is mutated to glutamate (R489E) and aspartate (R489D), or amino acids from 489 to 498 are completely deleted(Δ489-498), making the mutant C terminal more negative than wild type. Mutants and its principle is shown in Table1. CRY2wt and its mutants are all tagged with mCherry, in order to show the distribution of CRY2 in cells. Meanwhile, charge of N terminal is critical for CRY2-CIB1 interaction. We also change the N terminal charge of CRY2 to weak its interaction with CIB1 and increase light threshold of the system.
Source
CRY2 is from Arabidopsis thaliana