Is a Kazark sequence needed in a upper primer? - (Dec/05/2006 )

I need to insert a gene into a vector through a double digetion liagtion after a PCR.
Should I add a "CCACC"before "ATG" when I design the upper primer ?
The vector is pcDNA3.1. The gene is inserted into the MCS domain.
Thank you in advance!

Check the pcDNA 3.1 Manual, in “Cloning Considerations”. It says:
“pcDNA3.1(+) and pcDNA3.1(-) are nonfusion vectors. Your insert must contain a Kozak translation initiation sequence and an ATG start codon for proper initiation of translation (Kozak, 1987; Kozak, 1991; Kozak, 1990). An example of a Kozak consensus sequence is provided below. Please note that other sequences are possible (see references above), but the G or A at position -3 and the G at position +4 are the most critical for function (shown in bold). The ATG initiation codon is shown underlined. (G/A)NNATGG Your insert must also contain a stop codon for proper termination of your gene. Please note that the Xba I site contains an internal stop codon (TCTAGA).”

Check your gene sequence, may be it has a Kozak sequence already.

Hi,

You can just add CCACC before [ATG] or
GCCACC then [ATG]. Most of the genes I worked with contain a G (+4) after ATG, but some don't. So adding extra Kozak sequence would help, I guess. E.g. CGCCACC [ATG] (G?)

Recommendation by pcDNA3.1 manual such as (G/A)NNATGG should work too, but since most papers I've browsed through added GCCACC most of the time, I guess it's no harm following them.

Ok.
I see now. Thank you!

Does Kozak Seq reallly make sense?
I mean, if the insert CDS doesn't contain Kozak Seq or +4G,
what will happen after this plasmid is transfected into mammalian cells?
Low expression or no expression at all, or cann't function properly?

Thanks a lot.

i just checked one plasmid we bought for mammalian expression, it already has my protein inserted into it. it has NO kozak sequence not even necessary G and A. i never checked the level of protein expression of that plasmid though. maybe we should contact the company.

Hi Kat,

Most (I'm guessing??) mammalian expression vector will not include the Kozak sequence. Normally, I'd just have to include the Kozak sequence into my CDS. Though you can also opt for translation initiation sequence that is intrinsic to the CDS under study.

Well... exception being vectors with N-terminal epitope/tag.

MSG, what is CDS? i mean we bought this "ready to use" plasmid with our DNA in it already. I never checked it though. you mean even in this case if the translation is not so good i should cut it and include kozak sequence?