I am facing issues in expression of my gene of interest. I have custom synthesized a single fragment (AseI -- lacI-pLacIq-pTac-SD-GOI-stop codon-terminator ---EcoRI). the pLacIq and ptac are in reverse direction with a gap of 6 base pairs. I have cloned this fragment into a vector. after digestion with enzymes, I am getting the right fragments also. I am even able the grow the transformed cells in the presence of 100ug/mL Amp BUT there is no protein expression even after inducing with 1mM IPTG (have tried 50/100/200/500/1000um IPTG). I am unable to guess why is this happening, is this happening because pLacIq and pTac are too close???. Please suggest.