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gfp containing an n-terminal membrane targeting sequence (nts)  (GenScript corporation)
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GenScript corporation gfp containing an n-terminal membrane targeting sequence (nts)
Primers used to create recombinant BAC
Gfp Containing An N Terminal Membrane Targeting Sequence (Nts), supplied by GenScript corporation, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Primers used to create recombinant BAC

Journal: Journal of Virology

Article Title: The nsp3 Macrodomain Promotes Virulence in Mice with Coronavirus-Induced Encephalitis

doi: 10.1128/JVI.02596-14

Figure Lengend Snippet: Primers used to create recombinant BAC

Article Snippet: GFP containing an N-terminal membrane targeting sequence (NTS) was amplified from pUC57-NTS-GFP (Genscript) while FRT-Kan r -FRT was amplified from plasmid C006 (a generous gift from Dong Yu).

Techniques: Recombinant, Sequencing, Construct

rJ virus mutated at residue 1347 (N1347A) is minimally attenuated in vitro. (A) To compare replication kinetics of WT (GFP−) and WT (GFP+) viruses, 17Cl-1 cells were infected at an MOI of 0.1 PFU/cell. Progeny virus was collected at the indicated times postinfection, and titers were determined by plaque assay. (B) pBAC-JHMV encoding the N1347A mutant was created using a two-step linear recombination with a Kanr-I-SceI dual marker cassette as described in Materials and Methods. Individually shaded boxes indicate regions of homology to viral sequence; the asterisk identifies the location of the active-site asparagine. Revertant rJ was also engineered (revN1347) using the same procedure. (C) Growth kinetics of rJIA. 17Cl-1 cells were infected with the indicated viruses. Progeny virus was collected at the indicated times postinfection, and yields were determined by plaque assay. (D) To assess RNA accumulation, 17Cl-1 cells were infected with the indicated viruses, and total RNA was collected at the indicated times postinfection. RNA was quantified by RT-qPCR with primers specific for genomic RNA and normalized to HPRT (Table 2 lists qPCR primers). The normalized amount of viral transcript at 6 hpi during revN1347 infection was set to 1. (E) Viral genome RNA/PFU ratio was determined by dividing the relative gRNA of revN1347 and N1347A virus from cells at 0 hpi by the PFU count of the viral stocks. The normalized ratio of revN1347 virus was set to 1. Data shown are mean values and standard errors of the means from two independent experiments with two different virus stocks performed in duplicate. (F) To examine viral fitness in vitro, 17Cl-1 cells were coinfected with N1347A (GFP+) and wild-type (GFP−) virus or wild-type (GFP+) and wild-type (GFP−) virus at a ratio of ∼4:1 and at a combined MOI of 0.1 PFU/ml. Virus was passaged three times in 17Cl-1 cells, with titers determined after each passage so that at each subsequent passage cells were infected at an MOI of 0.1 PFU/cell. The percentage of GFP-expressing virus was determined by dividing the number of GFP+ plaques by the number of total plaques. Passage 0 represents input virus. Data shown are mean values ± standard errors of the means (SEM) from a representative experiment performed in duplicate. *, P ≤ 0.05; **, P ≤ 0.005.

Journal: Journal of Virology

Article Title: The nsp3 Macrodomain Promotes Virulence in Mice with Coronavirus-Induced Encephalitis

doi: 10.1128/JVI.02596-14

Figure Lengend Snippet: rJ virus mutated at residue 1347 (N1347A) is minimally attenuated in vitro. (A) To compare replication kinetics of WT (GFP−) and WT (GFP+) viruses, 17Cl-1 cells were infected at an MOI of 0.1 PFU/cell. Progeny virus was collected at the indicated times postinfection, and titers were determined by plaque assay. (B) pBAC-JHMV encoding the N1347A mutant was created using a two-step linear recombination with a Kanr-I-SceI dual marker cassette as described in Materials and Methods. Individually shaded boxes indicate regions of homology to viral sequence; the asterisk identifies the location of the active-site asparagine. Revertant rJ was also engineered (revN1347) using the same procedure. (C) Growth kinetics of rJIA. 17Cl-1 cells were infected with the indicated viruses. Progeny virus was collected at the indicated times postinfection, and yields were determined by plaque assay. (D) To assess RNA accumulation, 17Cl-1 cells were infected with the indicated viruses, and total RNA was collected at the indicated times postinfection. RNA was quantified by RT-qPCR with primers specific for genomic RNA and normalized to HPRT (Table 2 lists qPCR primers). The normalized amount of viral transcript at 6 hpi during revN1347 infection was set to 1. (E) Viral genome RNA/PFU ratio was determined by dividing the relative gRNA of revN1347 and N1347A virus from cells at 0 hpi by the PFU count of the viral stocks. The normalized ratio of revN1347 virus was set to 1. Data shown are mean values and standard errors of the means from two independent experiments with two different virus stocks performed in duplicate. (F) To examine viral fitness in vitro, 17Cl-1 cells were coinfected with N1347A (GFP+) and wild-type (GFP−) virus or wild-type (GFP+) and wild-type (GFP−) virus at a ratio of ∼4:1 and at a combined MOI of 0.1 PFU/ml. Virus was passaged three times in 17Cl-1 cells, with titers determined after each passage so that at each subsequent passage cells were infected at an MOI of 0.1 PFU/cell. The percentage of GFP-expressing virus was determined by dividing the number of GFP+ plaques by the number of total plaques. Passage 0 represents input virus. Data shown are mean values ± standard errors of the means (SEM) from a representative experiment performed in duplicate. *, P ≤ 0.05; **, P ≤ 0.005.

Article Snippet: GFP containing an N-terminal membrane targeting sequence (NTS) was amplified from pUC57-NTS-GFP (Genscript) while FRT-Kan r -FRT was amplified from plasmid C006 (a generous gift from Dong Yu).

Techniques: Virus, Residue, In Vitro, Infection, Plaque Assay, Mutagenesis, Marker, Sequencing, Quantitative RT-PCR, Expressing