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When designing a Mammalian Expression Vector, Should you include the complete CD


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#1 SimoneM

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Posted 26 February 2015 - 03:05 PM

I'm currently designing a mammalian expression vector.

 

When I look up the sequence for my gene of interest on Genbank I notice that the sequence has 18 base pairs upstream from its Start (ATG) codon, and 180 base pairs after its Stop Codon (TGA).

 

DNA20 provides a really neat service to design a vector. I copy and pasted my Gene from its start codon to stop codon, and added the EcoR1 restriction sites to either side of this sequence.

 

Is this a common technique in the designing of a vector, or should I be including the full sequence of my gene from Genbank (base pairs before the start codon and after the stop codon)? I'm fairly certain that these regions wont be translated, but is it common practice to insert the whole sequence anyway?

 

Also, the vector I am using has a multiple cloning site with ten or so sites to choose from. None of those restriction sites exist in my gene of interest. I chose Eco R1 because we have plenty of this enzyme in our lab. But are there any restriction sites to avoid using in mammalian expression vector design.

 

Thanks,

 

any help on this is much appreciated

 

 

 

 



#2 Smog187

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Posted 26 February 2015 - 08:48 PM

If you are cloning this into something for cell culture, then the plasmids already have a 5' and 3' UTR  that will ensure you're gene gets transcribed. However you may want to encode a Kozak sequence or an intron to ensure you get efficient splicing and translation. 

 

If you plan on re-purifing your plasmid from your organism, then I would check to make sure certain types of meythlation does not negatively affect your restriction enzymes. 



#3 bob1

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Posted 01 March 2015 - 11:48 AM

As Smog indicated, you don't usually include UTRs unless you have a specific reason to.

 

I would suggest using two enzymes to cut your plasmid, this makes it directional cloning, which means that you (should) only get your insert going in one direction. If you use the same site at both ends, then you can get the insert in either orientation. To do this choose one enzyme that is nearer the promoter, and use this on the 5' end of your sequence, put the other site on the 3' end.



#4 miST32

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Posted 02 March 2015 - 11:11 AM

You could approach the restriction problem by amplifying your gene of interest with PCR using primers containing two enzyme recognition sequences and extra bases on the 5' end.  i.e. 
5'-nnnnnn[cut site](forward primer annealing sequence)-3'-->
<--3'-(reverse primer annealing sequence)[cut site]nnnnnn-5'

 

This will let you choose a good enzyme pair for directional cloning as suggested by bob1.  Consider enzymes/buffers you have in the lab, if they work for double digests, and if they can be heat-denatured.  The latter makes your cloning a little bit easier as you can proceed directly from the digestion of your purified PCR product to ligation without re-purifying to remove active enzyme.

Regarding the incorporation of UTRs, if you might ever be interested in studying regulatory elements or RNA structure, then it might be worth keeping them.  It might make no difference, but in my experience (so far) I haven't found it to be problematic.  I have endogenous 5' and 3' UTRs on my current project's gene of interest and have found no problems with expression.






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