Part:BBa_K365002:Design
Phytochrome B from A. thaliana (aa 1-908)
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 490
Illegal BamHI site found at 572
Illegal XhoI site found at 523
Illegal XhoI site found at 542
Illegal XhoI site found at 2677 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 2062
Illegal BsaI site found at 2468
Illegal SapI site found at 739
Design Notes
To create the BioBrick part the sequence was amplified with primers containing the standard prefix with ATG and the fusion suffix of the Fusion Protein Assembly Standard.
Forward primer (5’->3’): (41bp)
GGATCCgaattcgcggccgcttctagATGGTTTCCGGAGTC
Reverse primer (5’->3’): (52 bp)
CAGCTGctgcagcggccgctactagtattaaccggtGCTCGGGATTTGCAAG
In order to get a sequence without an internal restriction sites of one of the BioBrick standards the SpeI-restriction site was altered without changing the encoded amino acid (ACT=Threonine (AC(T,A,G,C)).
(We had to delete one SpeI restriction site by directed mutagenesis on position 2313.)
Primers for Pfu-mutagenese:
Forward primer (5’->3’): (28 bp)
GGACAAGACGTTACGAGTCAGAAAATCG
Reverse primer (5’->3’): (28 bp)
CGATTTTCTGACTCGTAACGTCTTGTCC
Source
The plasmid containing the PhyB-sequence was provided by the laboratory of Wilfried Weber from the University of Freiburg.
References
Quail, P. H., R. Khanna, et al. (2004). "A novel molecular recognition motif necessary for targeting photoactivated phytochrome signaling to specific basic helix-loop-helix transcription factors." Plant Cell 16(11): 3033-44.
Voigt, C. A., A. Levskaya, et al. (2009). "Spatiotemporal control of cell signalling using a light-switchable protein interaction." Nature 461(7266): 997-1001.