Additional file 2: of Restriction site free cloning (RSFC) plasmid family for seamless, sequence independent cloning in Pichia pastoris

Figure S2. Simple strategy for confirming the orientation of the insert. The forward or reverse primer used for amplifying the insert can be used together with the forward or reverse sequencing primer of the vector to confirm the correct orientation. Upon correct primer choice only the forward orientation gives a PCR fragment. The sequencing primers designed for Sanger sequencing allow sequencing of the insert from both sides. Depending on the vector, different primers should be used (e.g. when the MFalpha signal sequence or a fusion protein is present, see the primer list for all sequencing primers available)

Additional file 4: of Restriction site free cloning (RSFC) plasmid family for seamless, sequence independent cloning in Pichia pastoris

Figure S3. Fluorescence measurements of fusions of HRP to eGFP. Samples are labeled in the same way as in Figure 4. eGFP fluorescence of supernatants and cell pellets of methanol induced cells were normalized per cell density (OD600)

Prozesstechnik der DME und OME-Synthese und Life Cycle Assessment

Table S1. Primers used in this study and detailed plasmid construction. Primers used for creating the S. pombe and P. pastoris plasmids are separated. Also primers for construction of the plasmids are separated from primers for sequencing and insertion of GOIs. In addition separate spreadsheets are providing information on the exact construction of the plasmids by listing the PCR products and restriction enzymes used for assembly