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Cloning into pVIVO2 vector

Cloning mammalian expression Expression vector Molecular Cloning

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#1 SG10

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Posted 23 April 2014 - 12:52 PM

Well, I want to clone a gene in one of the two MCS of pVIVO2 vector, between BspHI and Avr II sites. This vector from Invivogen has an ATG in MCS itself (BspHI). So suppose if I want to clone a gene that has an ATG as starting codon, will it has to be in frame with the ATG on the vector ? What about the same in another MCS i.e. between sites NcoI and NheI ?

File is attached for your convenience.

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#2 bob1

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Posted 23 April 2014 - 02:29 PM

Ideally yes, especially as the vector has a kozak sequence built into the NcoI site  This is not the case for the BspH1 site though, but it would be a good plan to or you may end up with the insert being mis-translated by being out of frame.

 

You can leave the ATG off the insert.



#3 SG10

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Posted 23 April 2014 - 10:17 PM

Thanks Bob1, 

 

If I leave my ATG on insert off, and as the first base of the second codon is a "T" and not a "G/A", my second amino acid will be a different one. An Isoleucine, wouldn't it cause any problem with the protein folding or its activity ?

 

 

 

Secondly, the other site which doesn't seem to have a kozak sequence around ATG, is it possible to add "GCC RCC ATG" sequence on my insert (Primer) so the first ATG of vector is omitted (Being not having a Kozak) and translation starts from the second strong ATG site on my insert ?

 

 

And what if I do not remove my ATG on the insert and i just let it to be there, though in frame with the ATG on vector??

 

i.e. ATG (vector)...XXX ...ATG (Insert)..  ?


Edited by SG10, 23 April 2014 - 10:21 PM.


#4 bob1

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Posted 24 April 2014 - 01:44 AM

To get your first amino acid to be the correct one you could look for an isoschizomer (if there is one) that has the right base.  Another option is to add a short tag such as FLAG to get around this problem.

 

It is possible to add the kozak sequence to the primer.  Another option is to clone into a different restriction site, and add the kozak etc to this site.

 

Leaving the initial methionine there for your insert is also fine, it just means that you will have more than one met at the start of your sequence.



#5 SG10

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Posted 24 April 2014 - 02:09 AM

Well, what i am planing to do is to clone insert at the Nco I site. Now the problem is my insert also has NcoI site, but then Nco I is compatible to Bsp HI and my insert does not have it :). Now the sequence will become

 

from ATG "G" to ATG "A"  (Nco I site C/CATGG, Bsp HI -T/CATGA). So now the second amino acid should start with AXX. Now I am again in the same mess!!

 

Anyways If add a ATG again after the start ATG on vector, would it cause a problem being two ATG back to back ?



#6 bob1

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Posted 24 April 2014 - 07:04 PM

personally if I was trying to do what you are doing, I would be cloning into the EcoRI and BamH1 sites of MCS2.  This should solve most of your problems...



#7 SG10

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Posted 25 April 2014 - 06:25 AM

Then you will have 3-4 amino acids extra at N-terminal  ? (Would you use insert ATG in that case or you will start with ATG on vector surrounded by Kozak ?)



#8 bob1

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Posted 25 April 2014 - 05:18 PM

Use a kozak.   



#9 SG10

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Posted 26 April 2014 - 11:47 PM

Thank you very much Bob1 for your kind suggestions :)







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