The authors designed primers at two ends of the c-FLIP(L) open reading frame, and inserted Bg/II Restriction Enzyme site in the upstream prime and four protective bases before ATG, meanwhile they inserted a Kozak sequence, which could increase inserted gene expression level, in the eukaryotic cell.
Therefore the Kozak sequence was introduced after upstream primer's Bg/II site, to make sure that c-FLIP(L) gene was highly expressed in recombinant plasmid.
The presence of the -5C allele increases the mean level of GPIb[alpha] on the platelet plasma membrane (roughly a 50% increase in homozygous individuals and a 33% increase in heterozygous individuals), presumably because it enhances the interaction of the cellular protein translation machinery to the adjacent Kozak sequence.
Kozak sequence polymorphism of the glycoprotein (GP) Ib[alpha] gene is a major determinant of the plasma membrane levels of the platelet GP Ib-IX-V complex.
Variability in these regions, especially in the so-called KOZAK sequence
around the start codon (33), may influence efficiency of translation initiation and, thus, protein expression and virus production.