How could I express two genes in opposite orientation - (Jan/27/2010 )

Hi Folks,

Recently, I would like to clone 4 genes into one expression vectors under control of one promoter in the vector, but the problem is one of gene (gene1) have opposite transcriptional orientation with others. I could clone the 3 genes together into vector first, but I'm not sure if I should put the upstream spacer of gene 1, which may include original promoter and SD sequence, into the vector with gene1. I mean should I add the any upstream sequence of gene1 or I just simply clone the ORF of gene1 behind the three genes into the vector.

Does Any body have experience for that?

Thanks

Hi there,

I don't exactly understand what you want to do: Do you want to do some genetics, i.e. put some chromosomal region that includes 4 genes and a promoter for all of them into cells (I've got no experience for that), or do you want to over express 4 different genes at the same time from one promoter? If the latter, I would recommend you using 2A peptides, for example here: PMID 19252477. For that you'd need only the ORF.

Cheers,
Minna

Leonid on Jan 27 2010, 10:01 PM said:

Thanks Minna. I meant co-expression of 4 genes under a promoter in vector. but the 4th gene has opposite transcriptional orientation. Do I need add additional E.coli SD sequence and any flanking spacer around the SD sequence upstream in my Start codon?

Thanks

Minna on Jan 27 2010, 11:44 PM said: