Part:BBa_K3841094

Expression cassette of yeast optimized Venus from methanol-dependent promoter

BBa_K3841094 is an expression cassette designed for Komagataella phaffii GS115 (previously referred to as Pichia pastoris. The Venus protein is the Part:BBa_K165005 from the distribution kit 2021. The TEF terminator is codon-optimized for K. phaffii under the control of the methanol-induced AOX1 promoter.

Usage
For chromosomal integration, flank the expression cassette by 65bp long homology arms. The homology arms should match upstream and downstream to an induced DSB. Visit 2021 DTU-Denmark’s Toolbox for K. phaffii to find homology arms. Insert the fragment into a vector backbone of choice for instance the pGGAselect destionation plasmid (BBa_K3841023). To make the Venus compatible with RFC[1000] and remove BsaI cut site, the following reverse primer can be used for amplification of the part: Rv: 5' - tgcggtaccagaacctttgtatagttcatccatgccatgtgtaatcccagcagctgttacaaattccaagagtaccatgtgatctctttt

The part is designed for measuring the AOX1 promoter strength in K. phaffii. Upon cultivation in methanol contaning medium, the system is activated and the transciptional activity can be monitored using a flourescent filter for Venus. Venus has an excitation wavelength of 515 nm and emission wavelength of 528 nm [1].

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1598
  

References
[1] Shaner, Nathan C., et al. “A Guide to Choosing Fluorescent Proteins.” Nature Methods, vol. 2, no. 12, NATURE PUBLISHING GROUP, 2005, pp. 905–09, doi:10.1038/nmeth819.