Schematic illustration of BiDi promoter system for antibody production. (A) The MD vector was designed to exhibit a 200-bp stuffer, flanked by Esp3I restriction sites (Esp3I sites A and B), adjacent to a SV40 polyA signal sequence and the regions encoding for hinge-CH2-CH3, terminated by a SV40 polyA signal sequence. (B) VL-CL and VH-CH1 amplicons can be inserted into MD by Golden Gate cloning utilizing Esp3I restriction sites (Esp3I sites A and B). The BiDi promoter can be chosen individually and is flanked by BbsI sites (BbsI sites A–D), compatible with the VL and VH sequences. (C) Golden Gate assembly results in a fully functional and re-circularized vector, with the light chain under the control of promoter I and the heavy chain under the control of promoter II. (D) Schematic representation of the resulting heterotetrameric IgG1 antibody using the same colour code as for the genetic elements.