T4 ligase kit

Plasmid DNA were purified by plasmid mini kit (Omega), isolation of genomic DNA and other manipulations of Streptomyces were performed using the method described by Keiser et al. (2000) . Restriction enzymes NdeI, EcoRI and HindIII and DNA polymerase were purchased from New England Biolabs (Beijing) Ltd. Primers (Supplementary Table S1) were synthesized by JIE LI BIOLOGY (Shanghai, China), PCR products were purified from agarose gels (0.8%) using DNA Gel Extraction Kit (Omega). T4 ligase kit for construction of recombinant plasmid was purchased from TaKaRa (China). The PCR-targeting method for in-frame deletion of blsL was done according to Bertolt Gust (Gust et al., 2002 ).

One set of primers (Table 1E, 1F) were used to amplify the kifc1 gene with truncated motor domain (ΔC) from testis cDNA profile with PrimerSTAR^® HS DNA Polymerase (TaKaRa). The PCR procedures were as follows: 34 cycles of 98°C 15s and then 68°C 2min. The PCR product was then tested on the agrose gel and purified with SanPrep column PCR Purification Kit (Sangon Biotech). Digest the PCR product and the pCMV-N-FLAG with final concentration of 0.75U/μl restriction endonucleases (each): XhoI and XbaI (TaKaRa). We used TaKaRa T4 ligase Kit for ligation overnight at 18°C and transformed the ligation reaction into DH5α. We used one set of primers that are priming at sequence on the pCMV-N-FLAG backbone (Table 1G, 1H) to do the colony PCR for screening, and sequencing was performed by Biosune. We then use EZNA Plasmid Miniprep Kit II (Omega) to prepare endotoxin-free plasmids.