Is a Kozak sequence necessary in Sindbis replicon vector?
I amplified a viral gene by PCR and cloned it into a Sindbis virus replicon plasmid vector.The vector has a human cytomegalovirus (hCMV) immediate-early promoter/enhancer element. The total plasmid side is 16kb. There is no kozak sequence before the viral gene start codon. I have been electroporating in HEK and BHK cells but the level of protein expresson is very low, around 5%. Could it be that the low level of protein expression is because of the absence of the kozak sequence? Do i need to incoporate it inorder to improve protein expression levels or what else can i do improve protein expression?