Part:BBa_K3841050

Homology arm for neutral insertion site 2 in Komagataella phaffii

This part is a homology arm complementary to a neutral region in the genome of Komagataella phaffii ideal for chromosomal integration in this yeast.

Origin
The homology arm is complementary to chromosome 2 (position 666161-666225) within the assembled genome of K. phaffii GS115 strain [1] (Accession number FN392320).

Usage
Co-transformation with the CRISPR-Cas9 plasmid and a repair oligo will mediate homology directed repair (HDR) [2]. This part anneals 65 base pairs upstream of the cut-site of the gRNA BBa_K3841081 and is used to mend DSBs induced by the CRISPR-Cas9-BBa_K3841081 complex. For insertion, flank the expression cassette of interest by this homology arm in combination with homology arm BBa_K3841049 annealing downstream of the cut-site.

Conceptual illustration of appropriate usage of BBa_K3841050 for chromosomal integration

Functionality
This part has not been experimentally tested.

References
[1] De Schutter, Kristof, et al. “Genome Sequence of the Recombinant Protein Production Host Pichia Pastoris.” Nature Biotechnology, vol. 27, no. 6, NATURE PUBLISHING GROUP, 2009, pp. 561–66, doi:10.1038/nbt.1544.
[2] Jakociunas, Tadas, et al. “CRISPR/Cas9 Advances Engineering of Microbial Cell Factories.” Metabolic Engineering, vol. 34, Academic Press Inc., 2016, pp. 44–59, doi:10.1016/j.ymben.2015.12.003.