this is a schematic of the mentioned vaccine construct.

Hello Mohamad, how are you doing?

Well, usually in the manual you will see the plasmid sequence with the location of T7 and SP6 promotes. Between then, there are several restriction sites where you will insert your construct (amplified by PCR). Usually the plasmid comes linearized and just to perform a single ligase reaction with DNA ligase of the kit. However, according with PGEM®-11Zf(+) manual, the plasmid did not come linearized, so you need to use one of the restriction enzymes to open the plasmid to insert your construct.

Regarding to which promoter you will use for mRNA synthesis, your vaccine construct can be inserted in both ways, for instance 5’T7 promoter+5’vaccine construct3’ in the main strain of DNA or in reverse way 5’SP6 promoter+5’vaccine construct in the other strain. So, you need to chose one of the promoters to use for mRNA synthesis. There are different mRNA synthesis kits that use both or each one promoter.

Regarding to your last question, I am sorry, but I cannot answer for sure about it.

I’ll hope helped. Good luck

Hi, dear Yoshinaga,

Thank you so much for your time and effort.

You described a part of my question very well and it was completely helpful for me. I am very thankful to you.

Best regards

T7 promoter should be upstream the non-structural proteins of the self-amplifying mRNA vaccine, the non-structural proteins need to be transcribed in vitro and translated in cells to replicate the RNA.