I made a fused gene using gene 1 promoter to drive gene 2 expression. Because 3' of the gene 1 promoter is GC only, it was difficult to design primer for promoter amplification. To avoid this problem, I omitted the last 30 bp sequence upstream the gene 1's start codon and fused the fragment I got with gene 2's CDS. Now I am worrying about if the fused gene can be translate properly due to the destruction of kozak sequence of the fused gene. Does anyone can tell if the construct will work? Many thanks!
It might depend on the organism, but for plants I don't think it is a problem. I had similar problem creating 24 different promoter reporter constructs and for primer design missed 0-40bp (depending on the specific promoter) of the region just upstream of the start codon. All 24 promoters conferred clear reporter expression.
As Eric says, probably fine for plants, but would undoubtedly be a major problem for a mammalian construct, since transcription initiation is likely to be disrupted. Because of atypical sequence composition around transcriptional regulatory sequences and start codons, I have tended to get around this by synthesising the atypical region as long complementary olignucleotides with judiciously inserted restriction sites, allowing them first to be cloned upstream of the gene that I wish to express and subsequently for the desired promoter to be inserted upstream of the oligonucleotide sequence. Oligo design without disrupting crucial regulatory sequences can be a bit of a challenge, but one can usually find convenient 4/6 or 5/6 base matches to a restriction site that can be mutated without causing too much disruption.
Hope thathelps
Best iwhes
Steve Morley
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