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Is Kozak's full sequence necessary? or just ACCATG is enough?


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4 replies to this topic

#1 Curtis

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Excellent

Posted 14 February 2012 - 07:16 PM

I usually only introduce ACC before the start codon in my forward primers. I have managed to get quite good expressions but most of the time I have low expression. Just wondering if this is because of the lack of having the full Kozak's sequence (GCCGCCACC) before the stop codon? that GCCGCC is really disturbing :D

#2 scolix

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Neutral

Posted 15 February 2012 - 12:09 PM

usually i add (CCACC ) and expression is fine. The promoter can make a huge difference in expression.

#3 bob1

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Posted 15 February 2012 - 01:45 PM

Furtering Scolix's comment, it has been found that the critical sequence in the kozak is not necessarily the whole sequence, but that some bases are more important than others. IIRC the important ones are the GCCACCatgG, with the final G after the start codon being quite important. Obviously you can't always use a G in that postition though.

#4 Curtis

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Excellent

Posted 27 February 2012 - 04:39 AM

guys, Promega just emailed me that the Kozak's sequence is not really neededPosted Image . Here's the email:

From: sgtechserve<sgtechserve@promega.com>

Dear Curtis,
I do apologise this reply is late. I realize that I had not replied your email.

A KOZAK sequence is not absolutely essential for expression- it enhances expression in some cases. The pCI-neo has CMV promoter and an intron upstream of MCS, so I would expect a cDNA with a ATG start site to work well. Regarding the distance between the ATG and promoter, that is already taken care of by the intron found on the plasmid.

I hope this helps!

Cheers,
Jessi

#5 bob1

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Posted 27 February 2012 - 12:35 PM

Yep, that's correct, it isn't absolutely necessary, however, in many cases a kozak will enhance expression.




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