Part:BBa_K3683002
Plasmids
The psPAX2 was purchased from addgene, and the pLOVE-Luciferase-EGFP was purchased from GenScript (Nanjing, China), the full length Spike gene (S) from the SARS-CoV-2 (previously 2019-nCoV) strain Wuhan-Hu-1 (GenBank: MN908947) was codon-optimized (sequence shown in Supplementary Table 1), synthesized, and cloned into pCAGGS vector (pCAGGS S(1-1254aa)) using seamless cloning by GenScript. The primers S-D614G-F, 5′-CTGTACCAGGgCGTGAATTGCACCGAGGTGC-3′ and S-D614G-R 5′- TGCAATTCACGcCCTGGTACAGCACGGCCACC-3′ were used to generate S-D614G mutant by PCR-based direct mutagenesis using High-fidelity DNA polymerase Mix (P525, Vazyme) with the following condition: 95℃ 5min, 95℃ 30s, 56℃ 30s, 72℃ 4min for 28 cycles, 72℃ 10min. We next purified the exact size of S-D614G PCR product by gel extraction, then we used Exnase II (C214, Vazyme) to make the linearized product circled. Then circled S-D614G plasmids were transformed into DH5α competent cells, single clones were select to grow recombinant plasmids in culture. The information for these maps are shown in Supplementary map.
DNA and Protein sequence matching of S-D614 and S-D614G are attached in this part (BBa_K3683002).
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