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Fig. 2 | Virology Journal

Fig. 2

From: Efficient generation of influenza virus with a mouse RNA polymerase I-driven all-in-one plasmid

Fig. 2

Construction of the all-in-one plasmid. a Using primers engineered with unique restriction enzyme cleavage sites, the NP, PB1, NS, and NA cassettes from pYA4968 (NP), pYA4965 (PB1), pYA4972 (NS), and pYA4970 (NA) were amplified and cloned into the pYA4518-derived T-vector to obtain four 1-unit plasmids pYA4974 (NP), pYA4973 (PB1), pYA4976(NS), and pYA4975 (NA). b The GFP cassettes were amplified from pYA4964 and inserted into the four 1-unit plasmids between SmaI, and the adjacent enzyme site downstream each influenza gene cassette. The resulting plasmids were pYA4978 (NP, GFP), pYA4977 (PB1, GFP), pYA4980 (NS, GFP), and pYA4979 (NA, GFP). c The PA, PB2, M, and HA cassettes were amplified from pYA4967 (PA), pYA4966 (PB2), pYA4971 (M), and pYA4969 (HA) to replace the GFP cassettes in the 1-unit plasmids from step b. The resulting 2-unit plasmids were pYA4982 (NP, PA), pYA4981 (PB1, PB2), pYA4984 (NS, M), and pYA4983 (NA, HA). d The GFP cassettes were amplified from pYA4964 and inserted into three of the 2-unit plasmids. The resulting plasmids were pYA4986 (NP, PA, GFP), pYA4985 (PB1, PB2, GFP), and pYA4987 (NS, M, GFP). e The AsiSI-XmaI fragments from plasmids pYA4986 (NP, PA, GFP) and pYA4985 (PB1, PB2, GFP) were combined to obtain a 4-unit plasmid pYA4988 (NP, PA, PB1, PB2, GFP). The AsiSI-XmaI fragments from plasmids pYA4987 (NS, M, GFP) and pYA4983 (NA, HA) were combined to obtain another 4-unit plasmid pYA4989 (NS, M, NA, HA). f The two 4-unit plasmids were digested with BssHII and XmaI. Combination of the fragments containing influenza cassettes resulted in the all-in-one plasmid pYA4990. The PA, PB2, M, HA, and GFP cassettes had triple C (CCC) at their 3′-end for connecting to the SmaI-generated blunt end (as shown by primer sequence in Table 3). The SmaI and XmaI endonucleases were isoschizomers. For clarity, each element is labeled at only one location in plasmid pYA4990

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