I designed a synthetic construct on paper and got it synthesized from a company. The objective was to make a vector which can be used to study both transcriptional and post-transcriptional regulation by different cis-elements (2-3 different cis-elements can be studied simultaneously).
The construct is like this:
RES1 = KpnI [GGTACC] RES1’= SalI [GTCGAC] MCS1 = EcoRI+BamHI [GAATTCTGGATCC] RES2 = ClaI [ATCGAT] RES2’= NheI [GCTAGC] MCS2 = SpeI+HindIII [ACTAGTAAGCTT] RES3 = XbaI [TCTAGA] RES3’= PvuI [CGATCG] MCS3 = NotI+SacI[GCGGCCGCGAGCTC] Kozak consensus sequence = GCCACCATGG Linker (100bp) = AATTCTGGATCCTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCGAATTTCGATTCCACCGCCGCCTTCTATGAAAGGTTGGGCTTCGGAATCGTTTTCCGGGACGCCGGCTGGATGATCCTCCAGCGCGGGGATCTCATGC
GFP, RFP and YFP are fast turnover variants with MODC pest sequence. I took the sequences for these proteins from Evrogen website. I took the Sv40 polyA signal sequence from one of their vectors in order to avoid any event of incompatibility. Kozak sequence was also taken from those vectors only. The total size it ~4.8kb. For CMV promoter I took the standard sequence used in most vectors.
With this vector I could clone any post-transcriptional regulatory sequence between stop codon and SV40PA, and could change promoters as well.
With the CMV promoter and no 3'UTRs I see a very faint expression of GFP, but could not see any RFP expression. I used pMaxGFP and dsRed as a control for filter sensitivity.
I transfected the plasmid in Hela and Neuro2a and at various concentrations (1-5μg) using lipofectamine. The controls express well (transfected 1μg each) but I see very faint GFP expression and dont see any RFP expression.
I checked the sequencing QC report given by the company and there were no indels or other mismatches. I am clueless about why the construct fails to express.
Is the linker a problem; is 100bp not sufficient ?