Part:BBa_K2722004:Design
J23106 Promoter - mTurquoise - 380 Terminator
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 301
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
This is a short and simply to clone sequence. When we added His-Tags on this protein, this was added on the C-terminus. Also the mRNA was tagged on the 3' part on a fluorescent aptamer, which did not affect protein expression.
During the cloning of this part we accidently inserted a Mutation on position 27. A cytosin was mutated to an alanin. This changes the amino acid sequence on position 9 from a Phenylalanin (F) to a Leucin (L). This Mutation does not affect protein functionality, solvability nor brightness, nor did we realise any other change of characteristics.
Source
The protein sequence was obtained from Goedhart, J. et al., (2012), codon optimized for E.Coli and ordered as a g-Block by IDT.
The complete sequence including the correct scars and spacers is:
gatctttacggctagctcagtcctaggtatagtgctagcGAAGCTTCAGTCATAAGTCTGGGCTAAGCCCACTGATGAGTCGCTGAAATGCGACGAAACTTATGACCTCTACAAATAATTTTGTTTAAAGTCAGTaaagaggagaaaTAGACATGGTTAGCAAGGGTGAAGAACTGTTAACCGGCGTCGTGCCGATTCTGGTTGAGCTGGATGGTGATGTCAACGGTCACAAGTTTAGCGTTAGCGGTGAGGGCGAGGGCGACGCCACCTACGGTAAATTGACCCTGAAGTTTATCTGCACGACCGGTAAGCTGCCGGTTCCGTGGCCGACCCTGGTGACGACTCTGTCGTGGGGCGTGCAATGTTTCGCGCGCTATCCGGATCACATGAAACAGCATGACTTCTTTAAGAGCGCGATGCCGGAAGGCTACGTTCAGGAACGTACGATCTTTTTCAAAGACGACGGTAACTATAAGACCCGCGCAGAAGTCAAGTTCGAGGGTGACACGCTGGTGAATCGTATTGAGCTGAAAGGTATTGACTTTAAAGAGGACGGTAACATCCTGGGTCACAAACTGGAGTATAATTACTTCAGCGACAATGTGTACATCACCGCTGATAAACAGAAAAACGGCATTAAAGCAAACTTCAAGATCCGTCACAATATTGAAGATGGCGGCGTGCAATTGGCCGATCACTATCAACAGAACACCCCGATTGGCGATGGTCCGGTCCTGCTGCCAGATAATCACTACTTGAGCACGCAATCCAAACTGTCCAAAGATCCGAACGAAAAACGTGACCACATGGTCCTGCTGGAATTTGTTACCGCGGCGGGTATCACGCTgggtatggacgaactgtacaagTAAttcagccaaaaaacttaagaccGCCGGTCTTGTCCACTACCTTGCAGTAATGCGGTGGACAGGATCGgcggttttcttttctcttctcTGG
References
Markwardt, M. L., Kremers, G. J., Kraft, C. A., Ray, K., Cranfill, P. J., Wilson, K. A., ... & Rizzo, M. A. (2011). An improved cerulean fluorescent protein with enhanced brightness and reduced reversible photoswitching. PloS one, 6(3), e17896.
Goedhart, J., Van Weeren, L., Hink, M. A., Vischer, N. O., Jalink, K., & Gadella Jr, T. W. (2010). Bright cyan fluorescent protein variants identified by fluorescence lifetime screening. Nature methods, 7(2), 137.
mTurquoise, FPbase, under https://www.fpbase.org/protein/mturquoise/. (retrieved on 10.10.2019)
Goedhart, J., Von Stetten, D., Noirclerc-Savoye, M., Lelimousin, M., Joosen, L., Hink, M. A., ... & Royant, A. (2012). Structure-guided evolution of cyan fluorescent proteins towards a quantum yield of 93%. Nature communications, 3, 751.
Müller, S. M., Galliardt, H., Schneider, J., Barisas, B. G., & Seidel, T. (2013). Quantification of Förster resonance energy transfer by monitoring sensitized emission in living plant cells. Frontiers in plant science, 4, 413.