Model-guided random assembly PCR for the synthesis of eukaryotic promoters

摘要

The current invention is concerned with a method for the manufacture of a polynucleotide comprising a synthetic promoter specifically controlled by at least one selected transcription factor, comprising a) determining the distance between the transcriptional start point and the binding site for said at least one transcription factor in a given number of promoters comprising at least one binding site for said at least one transcription factor, b) selecting a distance range comprising the distances determined in a) to occur most frequently, c) determining the accompanying transcription factors in a given number of promoters, d) selecting a suitable number of accompanying transcription factors determined in c) to occur most frequently, e) synthesizing the synthetic promoter by randomly assembling i) at least one binding site for each of the at least one transcription factor, ii) at least one binding site for each of the selected accompanying transcription factor(s), and iii) a suitable number of stuffer fragments and by arranging said randomly assembled binding sites in the distance range from the transcriptional start point selected in b). The invention is further concerned with a polynucleotide comprising the synthetic promoter manufactured by the method of the current invention, a vector comprising the polynucleotide of the current invention, and a cell comprising the polynucleotide or the vector of the current invention. Furthermore, the invention relates to a device for the manufacture of oligonucleotides suitable for manufacturing a synthetic promoter of the current invention and a device for the manufacture of the synthetic promoter of the current invention.