## 2 Materials and Methods
### Cloning and Vector Construction  
The construct used for transforming the yeast was prepared in three steps: insert preparation, vector construction and ligation. To insert the amplified gene, as previously described, the insert was purified after gel electrophoresis using a QIAquick Gel Extraction kit (QIAGEN, Cat. 28706X4, Hilden, Germany). The purified insert (2 \(\mu\)g) was treated with _Bam_H1 (New England BioLabs, Cat. R3136S, Ipswich, MA, USA) and _XhO_1 (New England BioLabs, Cat. R0146S, Ipswich, MA, USA) restriction enzymes (2 \(\mu\)L each) with Cutsmart buffer (4 \(\mu\)L) (New England BioLabs, Cat. B7204S, Ipswich, MA, USA) and incubated at 37 \({}^{\circ}\)C for 4 h. To digest methylated DNA, 2 \(\mu\)L of _Dpn_1 enzyme was added to the restriction digest, which was then incubated at 37 \({}^{\circ}\)C for 2 h. Additionally, the pRSk42 vector (3 \(\mu\)g) was also treated with _Bam_H1 and _XhO_1 enzymes (2 \(\mu\)L each) by incubating at 37 \({}^{\circ}\)C for 4 h. After cutting with the restriction enzymes, the vector was then treated with CIP (New England BioLabs, CatM0525S, Ipswich, MA, USA) to dephosphorylate the ends of the vector. Finally, the insert was ligated to the vector at an insert:vector ratio of 5:1 in the presence of Quick Ligase and 2\(\times\) Quick Ligase Reaction buffer (New England BioLabs, B6058S, Ipswich, MA, USA). This construct was then transformed and propagated in _E. coli_ JM109 cells. The plasmid used in this experiment was pRS42k, which derives from the pRS series of yeast episomal plasmids and acts as a shuttle vector between yeast and _E. coli_. Usually, pRS42k is not used for gene expression as it has no promoter and terminator sequences. To construct this plasmid as the expression vector pRS42k-PGK1p, we performed the following steps: the PGK1 promoter and CYC1 terminator site, along with the _OsCM_ gene (Accession No. XM_015793648.2), were inserted into the pRS42K plasmid, as presented in 5 kb with the appropriate restriction sites. The restriction enzymes _Kpn1_ and _Sac1_ were used for the insertion of the whole fragment (promoter + gene + terminator). This plasmid backbone is mostly used as an expression vector in yeast because it can be easily manipulated to introduce foreign DNA into yeast. It is an independent and high copy number replicating plasmid containing the 2\(\upmu\) circle cloned at one of the 2\(\upmu\) circle _Eco_RI sites [26; 27]. The 2\(\upmu\) circle fragment allows for the efficient replication of the plasmid in yeast [26]. It has been reported that the 2\(\upmu\) circle is present in almost all strains of _S. cerevisiae_ at 50-100 copies per haploid cell [28; 29]. The replication of pRS42k in bacteria, as well as in yeast, eases the isolation of specific genes of interest. Here, we propagated our gene of interest in _E. coli_ and then used yeast for functional expression experiments. Due to the presence of the 2\(\upmu\) circle, the pRS42k plasmid can be efficiently transformed into yeast by the lithium acetate/single-stranded carrier DNA/polyethylene glycol (PEG) method [29], with slight changes. The colonies were grown for 6 days until the growth rate decreased, thereby showing a dependency on the substrate [30]. Often, yeast transformants may be affected by the structural instability of the vector due to a large foreign gene, which could be a reason for its decreased replication rate. Furthermore, episomal vectors are known to be structurally unstable when they contain a large foreign gene [31].