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cloning into F1 ori of expression vector???


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#1 dan_

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Posted 20 January 2011 - 07:37 AM

hi everyone,

I want to know if it is possible to cloned insert into F1 ori sequence of expression vector, such as pEGFP-N1 vector?

Thanks!

#2 pDNA

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Posted 20 January 2011 - 02:46 PM

it always depends on your intention ...what you are going to achieve with your cloning step
...but in general it is not a good idea
since the purpose of pEGFP-N1 is a N-terminal fusion of EGFP to your gene of interest. So if you are going to use pEGFP because of that, because you want your gene N-terminaly fused to EGFP ...and your goal is expression in eukaryotics cells ...then you should use the multiple cloning site and clone your gene in frame. If you use a restriction site within the f1ori you will end up with your gene inserted into the vector with no promoter in front and no polyA signal at the end ...and consequently with no expression in eukaryotic cells!
Therefore not advisable!

Regards,
p




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