Is kozak sequence necessary for cloning? - (Apr/26/2005 )
Hi everybody!
i want to clone several cDNA in the pcDNA3 expression vector. my genes don't present any kozak consensus sequence or even kozak like sequence upstream of the ATG.
do i need to insert it in my construction or not?
thanks a lot for your answers.
bye
Xenia
i want to clone several cDNA in the pcDNA3 expression vector. my genes don't present any kozak consensus sequence or even kozak like sequence upstream of the ATG.
do i need to insert it in my construction or not?
thanks a lot for your answers.
bye
Xenia
Hi
I think it is not necessary to insert the kozak sequence since i have have successfully cloned some cDNA only with ATG start codon and they were expressed in Hela cells, but it would be better to have it at the begining of your cDNA since this assures your DNA expression. hope that helps
The original paper by Kozak describing Kozak sequence http://www.ncbi.nlm.nih.gov/sites/entrez?D...Pubmed_RVDocSum
Kozak M.
Department of Biological Sciences, University of Pittsburgh, PA 15260.
Sequences flanking the AUG initiator codon influence its recognition by eukaryotic ribosomes. From a comparison of several hundred mRNA sequences, CCA/GCCAUGG emerged as the consensus sequence for initiation in higher eukaryotes. Systematic mutagenesis of a cloned preproinsulin gene confirmed the facilitating effect of A or G in position -3 (i.e. 3 nucleotides upstream from the AUG codon), C in positions -1 and -2, and G immediately following the AUG codon. The analysis of a new set of mutants now reveals that sequences slightly farther upstream are also influential, the optimal context for initiation being (GCC)GCCA/GCCAUGG. Possible mechanistic implications of the repeating GCC motif are discussed.
Dear Xenia,
the Kozak sequence is not essential for expression in mammalian cells (not all genes have them) but for some genes, it can improve expression levels.
If expression level is important, do a comparison between presence or absence of the sequence.
If you just need some expression, and it works without the kozak sequence, then your fine :-)
Good Luck.
i want to clone several cDNA in the pcDNA3 expression vector. my genes don't present any kozak consensus sequence or even kozak like sequence upstream of the ATG.
do i need to insert it in my construction or not?
thanks a lot for your answers.
bye
Xenia
Hi
I think it is not necessary to insert the kozak sequence since i have have successfully cloned some cDNA only with ATG start codon and they were expressed in Hela cells, but it would be better to have it at the begining of your cDNA since this assures your DNA expression. hope that helps
Hi! i am going to subclone my cDNA clone into pcDNA3.1(+). it seems that there is a kozak seq few base pair in front of my ATG. The problem is this kozak sequence will be located around 110bp away from cmv promoter in the pcDNA3.1(+) eukaryotic expression vectors once it is subclone. I wonder wether the expression of the protein will happen efficiently. please help!!!