Part:BBa_K4275036:Design
pLAC4-KmarxMFα-NpaBGS-t-tTDH1
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 331
- 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 331
Illegal NheI site found at 89
Illegal NheI site found at 1783 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 331
Illegal XhoI site found at 1252
Illegal XhoI site found at 2858
Illegal XhoI site found at 3937 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 331
- 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 331
Illegal NgoMIV site found at 2355
Illegal AgeI site found at 1369
Illegal AgeI site found at 1387 - 1000COMPATIBLE WITH RFC[1000]
Design Notes
1. The whole expression construct is assembled via goldengate assembly. The plasmid backbone, pLAC4 promoter, MFalpha, mTurquiose and tTDH1 terminator are firstly assembled as the vector for later assembly operations.
2. The CDS fragment is inserted into the previously assembled K.marxianus secretory expression vector via the enzymatic cleavage of the fragment termini by BsaI, and the subsequent creation of TTCT DNA overhang and the ATCC DNA overhang.
3. the pLAC4 promoter is an galactose-inducible promoter derived from K.marxianus whilst the tTDH1 terminator is derived from S.cerevisiae.
Source
Kluyveromyces marxianus DMKU3-1042
Neocallimastix patriciarum W5
Clostridium thermocellum (Dockerin-I domain)
Saccharomyces cerevisiae
References
1. Rajkumar, Arun S. et al. "Biological Parts For Kluyveromyces Marxianus Synthetic Biology". Frontiers In Bioengineering And Biotechnology, vol 7, 2019. Frontiers Media SA, https://doi.org/10.3389/fbioe.2019.00097.
2.“Mating Factor Alpha-1 [Kluyveromyces Marxianus] - Protein - NCBI.” National Center for Biotechnology Information, U.S. National Library of Medicine, https://www.ncbi.nlm.nih.gov/protein/QGN17207.1
3. Chen, Hsin-Liang et al. "A Highly Efficient Β-Glucosidase From The Buffalo Rumen Fungus Neocallimastix Patriciarum W5". Biotechnology For Biofuels, vol 5, no. 1, 2012. Springer Science And Business Media LLC, https://doi.org/10.1186/1754-6834-5-24.
4. "Part:Bba K2753052 - Parts.Igem.Org". Parts.Igem.Org, 2022, https://parts.igem.org/Part:BBa_K2753052.