Part:BBa_K3781018:Design
3xHA, MocloMania B5
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
This part was directly derived from 3xHA-B2 by introducing a different set of MoClo overhangs via PCR. The part functioning as a template was de novo synthesized and thus easily codon optimized towards Leishmania tarentolae. We generated that part by designing and ordering two opposing oligomeres that carried the sequence of interest and were furthermore flanked by <two invertedly oriented BbsI recognition sites. The single-stranded oligomeres were fused together according to a standard heat-cycle ligation protocol. The double-stranded part could then be introduced into its respective plasmid backbone with a simple MoClo ligation.
Source
The genetic sequence to this part was originally derived from a publically available Addgene plasmid #105075 named pNXgate33-3HA. [1] As a genetic source, we furthermore relied on the Chlamydomonas-based MoClo plasmid from our supervising working group, pCM0-058, that was built in the style of the Crozet et al. MoClo toolkit.[2] The part's amino acid sequence relates to an epitope found on the Influenza A viral protein hemagglutinin, NCBI: 956529
References
- ↑ Obrdlik P, El-Bakkoury M, Hamacher T, Cappellaro C, Vilarino C, Fleischer C, Ellerbrok H, Kamuzinzi R, Ledent V, Blaudez D, Sanders D, Revuelta JL, Boles E, Andre B, Frommer WB. K+ channel interactions detected by a genetic system optimized for systematic studies of membrane protein interactions. Proc Natl Acad Sci U S A. 2004 Aug 17;101(33):12242-7. Epub 2004 Aug 6. 10.1073/pnas.0404467101 PubMed 15299147
- ↑ 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