pGL3 Promoter replacing TSS bases - (Nov/09/2012 )

I'm interested in cloning various 5'untranslated regions (UTR) into the pGL3-Promoter vector so that the resulting mRNAs are all controlled by the same SV40 promoter. According to PNAS (1983); 80(3): 721–725, the transcriptional start site (TSS) of the SV40 promoter is primarily a sequence starting with GGCCTCTGAG... starting at the corresponding position 184 pGL3-Promoter, 21 bp downstream of the TATA box TATTTAT. If I simply replace everything in the pGL3-Promoter from 184 to 279 (AUG starts at 280) with my 5'UTR from another gene, will transcription occur at the proper place?

In other words can the SV40 promoter promote the transcription of any mRNA starting 21 bp downstream of the TATA box? The same paper (PNAS 1983; 80(3): 721–725) identified many important GC-rich regions upstream of the TATA box, so I wonder if the transcribed bases are important as well.

I think it is fine to replace the sequence between promoter and ATG with your own 5'UTR sequence as long as it does not contain ATG and you keep the Kozak sequence which lies immediately upstream of ATG. The sequence between promoter and ATG usually is just linker sequence.

Be ware that there may be regulator elements in your UTR sequence which may affect promoter activity. There are additional explanations on this page