Part:BBa_K3781016:Design
mCerulean, MocloMania B5
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 679
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
This part was directly derived from mCerulean-B3_B4 by introducing a different set of MoClo overhangs as well as an additional stop codon via PCR. The original sequence was adapted from a Chlamy-optimized plasmid, in which it contained a RBCS2 intron. We thus had to design special primers that would render the genetic regions upstream and downstream of the intron as individual parts via PCR. These then could be re-assembled into an intact mCerulean gene with the help of invertedly oriented BbsI restriction sites introduced with the primers. Due to the high resemblence in codon usage between Chlamydomonas reinhardtii and Leishmania tarentolae, the codons were not further optimized.
Source
The sequence was directly derived from the plasmid pCM0-081 of the MoClo Toolkit distributed by Crozet et al.[1] The protein is a mutated version of the cyan fluorescent protein ECFP which in itself is derived via mutagenesis from the green fluorescent protein avGFP isolated from the jellyfish Aequorea victoria.[2] mCerulean's genetic sequence can be found in the Addgene Plasmid #27796.[3]
References
- ↑ Crozet et al. (2018) Birth of a Photosynthetic Chassis: A MoClo Toolkit Enabling Synthetic Biology in the Microalga Chlamydomonas reinhardtii. ACS Synthetic Biology 7:2074–2086)
- ↑ M. Ormo, A. B. Cubitt, K. Kallio, L. A. Gross, R. Y. Tsien, S. J. Remington, Crystal structure of the Aequorea victoria green fluorescent protein. Science 273, 1392–1395 (1996)
- ↑ Koushik SV, Chen H, Thaler C, Puhl HL, Vogel SS. Cerulean, Venus, and VenusY67C FRET reference standards. Biophys J. 2006 Dec 15. 91(12):L99-L101. 10.1529/biophysj.106.096206 PubMed 17040988