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Kozak sequence and mammalian expression vector

Kozak Subcloning Mammalian expression Lentivirus vector

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3 replies to this topic

#1 Epigeneticist

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Posted 13 January 2014 - 03:48 PM

I am going to subclone an ORF from a vector that has a Kozak sequence. The destination vector is an inducible lentivirus vector. Should I subclone the ORF with the Kozak sequence or just include the start codon? Kozak sequence will enhance translation but will it cause problems in a inducible lentivirus vector? Or will the lack of the kozak sequence have a minimal impact on translation efficiency? Goal is to produce lentivirus from this vector and transduce mammalian cells. 

 

Thanks!



#2 bob1

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Posted 14 January 2014 - 11:59 AM

I'm not sure about the lenti thing, but I suspect that you should include the kozak as the gene will ultimately end up being transcribed by the cellular components.



#3 Osibisa

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Posted 14 January 2014 - 07:55 PM

The importance of the Kozak consensus sequence will vary, but I can't see why it would cause any problems for you. Most genes I have looked at are far from ideal, but normally you want ACC-ATG... ideally GCCGCCACCATG

 

Also spread the word that ATGG is not important, merely an observation on the effiency of cleavage of the signalling peptide with proteins starting with an amino acid coded by GNN. I can't find the paper right now, but take my word for it :)



#4 vladCH

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Posted 02 February 2014 - 03:12 AM

In my experience, the presence or absence of Kozak consensus sequence has only minimal effect on the resulting protein level. It is, however, very important that no other ATG is present upstream of your starting ATG. I recommend you to check this carefully by cloning digitally (i.e. on a computer) first. You can do it easily without any installed software with the tools available on the www.cloninghelp.com website.







Also tagged with one or more of these keywords: Kozak, Subcloning, Mammalian expression, Lentivirus vector

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