USER Cloning and Fusion Protocol

USER cloning and USER fusion was performed as previously described [9] (link), [11] (link) with minor modifications: Vector backbones holding a PacI/Nt.BbvCI USER cloning compatible cassette were digested as previously described [13] (link). 1 µl of USER enzyme mix, 0.5 µl NEB Buffer 4, 0.5 µl BSA x10 (all purchased from New England Biolabs), and 0.1 pmol digested vector backbone were mixed in a 0.2 ml PCR tube. If vector backbone was amplified by PCR, 1 µl purified DNA element was added. Finally, 7 µl of purified DNA elements were mixed to a total reaction volume of 10 µl. If more than one DNA element was to be inserted, all elements were added in equal volumes. The reaction mixture was incubated for 40 min at 37°C, followed by 30 min at 25°C. Subsequently the 10 µl reaction mixture was used directly to transform chemically competent E. coli DH5α cells. Transformants were selected in Luria Broth (LB) medium supplemented with 100 µg/ml ampicillin. Three transformants were picked randomly for each construct and validated by DNA sequencing (Star SEQ, Mainz, Germany). For each construct, a validated expression vector was purified by Plasmid Plus Maxi kit (Qiagen, Hilden, Germany) following the manufacturer's instruction and dilution to a concentration of 1 µg/µl DNA in MilliQ water.