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Plasmid, gene orientation, insert orientation. - (Jan/30/2012 )


I have been doing some readings on vector production. But I have some questions hopefully you guys can help me out :-)

So far I know that the orientation of my insert is important since this determines that the initial start codon would be on the 5' region during transcription translation for my protein production. However I don't understand why that some genes such as resistance genes can be placed on the opposite direction, without causing problems s.a. in pET-28a. Since the mRNA is produced along the 5' to 3' site and translation as well, how can than a sequence be reversed inserted into the vector for translation?

I am very confused about this right now.. hopefully some of you can help me out :-)




resistance genes have their own promoter so the gene is oriented according to it promoter.