As a first step, two plasmid types were generated. Plasmid type number one, assembled via Gibson Assembly, contained from 5’ end to 3’ direction one of the enhancers (HRE/CRE/pCTLA4), a minimal promoter to ensure better recruitment of the transcription machinery, one reporter protein (eCFP/SEAP/Luciferase) and a polyA signal sequence. The polyA signal was included to ensure better release of the transcribed mRNA from the transcription machinery and longer mRNA half-life (Hager et al., 2008). These plasmids were already suitable for pretests but were lacking a transfection control or a selection marker for lentiviral transduction (Fig. 1).

The second plasmid type, also assembled via Gibson Assembly, contained from 5’ end to 3’ a constitutive promoter (CMV/SV40), a selection marker (mCherry/Zeocin/Neomycin) and a polyA signal sequence. These plasmids sole purpose was to be assembled with type one into type three plasmids. In a second step, plasmids type one and type two were assembled via a Gibson assembly into type three plasmids (Fig. 1).

Considering that the enhancers one may only lead to very weak transactivation and therefore low reporter gene expression, plasmids containing an enhancer flanked by compatible restriction sites, suitable for compatible end assembly, were designed. To generate four enhancer elements in succession, compatible end assembly was performed twice. Additionally, two restriction sites, that were compatible with type three plasmids, were included, so that in a third step, type three plasmids and the four enhancer elements in succession could be combined via T4 ligation (Fig. 1).