Coding

Part:BBa_K3781015:Design

Designed by: Nicolas Bayer   Group: iGEM21_TU_Kaiserslautern   (2021-10-06)

mVenus, MocloMania B5


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 202
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 202
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 202
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 202
    Illegal NgoMIV site found at 679
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

This part was directly derived from mVenus-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 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 mVenus 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 underlying sequence was derived from the plasmid pCM0-086 of the MoClo Toolkit distributed by Crozet et al.[1] The protein is a mutated version of the yellow fluorescent protein EYFP which in itself is derived via mutagenesis from the green fluorescent protein avGFP isolated from the jellyfish Aequorea victoria.[2] mVenus's amino acid sequence can be found in the NCBI GenBank under AAZ65844.1.


References

  1. 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
  2. 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)