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How could I express two genes in opposite orientation


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#1 Leonid

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Posted 27 January 2010 - 09:01 PM

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

#2 Rsm

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Posted 27 January 2010 - 10:44 PM

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

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


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#3 Leonid

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Posted 28 January 2010 - 11:01 AM

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

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

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






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