4.6.4. Construction of the pH6 Expression Vector

The pH6 expression vector was constructed by cloning a PCR product designated as H6 and containing a sequence of the IEF2 promoter from D. macquariensis D50 into the pH3 expression vector. The PCR was performed using a genomic DNA of D. macquariensis D50 as the template and primers designated as pIEF2pH6_forward and pIEF2pH6_reverse (Table 10). The obtained PCR product of 684 bp was purified using an Extractme DNA Clean-Up Kit (Blirt S.A., Gdansk, Poland). Next, the purified PCR product and the pH3 vector were digested with BamHI and VspI (AseI) restriction enzymes (Thermo Fisher Scientific Baltics UAB, Vilnius, Lithuania), purified through DNA precipitation, and ligated. The correct construction of the pH6 expression vector was confirmed by DNA sequencing (Genomed S.A., Warsaw, Poland).