heat shock overexpression construct

hi everyone, i have a very silly basic molecular cloning question. i hope someone could help me. i am trying to make a pPD49.83-cDNA with stop codon construct to be used for heat-shock promoter driven overexpression. i have already got the successful clones but haven’t got to microinject. while i was analyzing my sequencing data, i noticed that heat shock promoter is not inframe with the ATG of my coding region. is this necessary? ???
thank you for anyone who could shed light to my confusion. :slight_smile:

What matters is the first AUG in the predicted transcript. The transcription start sites were mapped to sequences that are some 50bp upstream of the BamHI site and there are no ATGs in between. After the BalI site there is a synthetic intron of sequence GTATGTTTCGAATGATACTAACATAACATAGAACATTTTCAG that contains an ATG, but that will be spliced out. There is also an ATG in the NcoI site in the polylinker, just before EcoRV, so if you cloned downstream of that ATG in the wrong frame, you will probably have problems. What enzyme(s) did you use to clone your cDNA into pPD49.83?


Thank you for your time to answer my curious question.
I have cloned my insert in between the BamHI site (both 5’ and 3’) and this restriction site has been regenerated as my primers have the restriction sites. i have already checked for correct orientation and got the one which is in correct orientation with hsp promoter upstream of my gene’s ATG. i have checked the sequence and there’s no ATG in between the hsp promoter until my predicted gene’s ATG… so I guess I can go ahead with my microinjection then?

Yes, I would proceed with making transgenics. Good luck!