psmart bac vector  (New England Biolabs)

Journal: Journal of Virology

Article Title: Porcine Epidemic Diarrhea Virus Deficient in RNA Cap Guanine-N-7 Methylation Is Attenuated and Induces Higher Type I and III Interferon Responses

doi: 10.1128/JVI.00447-20

Figure Lengend Snippet: Strategy to construct the infectious PEDV cDNA clone and mutants. (A) Assembly of the full-length genomic cDNA of PEDV in the pSMART-BAC vector. The pSMART-BAC vector was modified as described in Materials and Methods. The full-length genomic cDNA of PEDV was amplified by RT-PCR using seven overlapping fragments designated A to G, and was assembled by using the GeneArt High-order genetic assembly system. The diagram of PEDV genes encoding ORF1a, ORF1b, spike (S), ORF3, envelope (E), membrane (M), and nucleocapsid (N) is shown. Regulatory elements, including the cytomegalovirus (CMV) promoter, hepatitis delta virus (HDV) ribozyme sequence, and bovine growth hormone (BGH) polyadenylation and terminator, are indicated in the plasmid. (B) Strategy to introduce mutations to PEDV genome. Diagrams of the two-step selection recombineering are shown. The first step is to place the rpsl+-Kana cassette at the locus of nsp14 via positive kanamycin selection. The second step is to replace the rpsl+-Kana cassette in the BAC intermediate construct with an nsp14 mutant cassette containing the same homology arms via negative selection of streptomycin. A and B indicate homology arms; nsp14 and nsp14-mutation indicate wild-type nsp14 and nsp14 mutations, respectively.

Article Snippet: The pSMART-BAC vector was modified to insert a yeast replication origin from the plasmid pYES1L (Thermo Fisher Scientific), a cytomegalovirus (CMV) promoter from pCI vector (Promega), a hepatitis delta virus ribozyme (HDVRz) sequence, and a bovine growth hormone (BGH) polyadenylation and terminator.

Techniques: Construct, Plasmid Preparation, Modification, Amplification, Reverse Transcription Polymerase Chain Reaction, Sequencing, Introduce, Selection, Mutagenesis

Journal: bioRxiv

Article Title: T7 RNA polymerase-independent expression of reporter genes from a T7 promoter-driven SARS-CoV-2 replicon-encoding DNA in human cells

doi: 10.1101/2024.03.01.582915

Figure Lengend Snippet: (A) Western blot showing N expression in HEK293T cells two days following PEI transfection of pEZY3-N, and in HEK293T cells lentivirally transduced with N. “Neg” are non-transfected, non-transduced HEK293T cells. (B) Effect of SARS-Cov-2 N transfection on SARS-CoV-2 replicon RNA expression. HEK293T cells were untransfected (Control, left dot plot), or were electroporated with SARS-CoV-2 replicon RNA (center plot), or were co-electroporated with the replicon and with SARS-CoV-2 N (right plot). Cells were analyzed for GFP expression by flow cytometry two days later. (C) Effect of SARS-CoV-2 N transduction on SARS-CoV-2 replicon RNA expression. Cells stably transduced with SARS-CoV-2 N were left untransfected (left plot), or were electroporated with SARS-CoV-2 replicon RNA in the absence (center plot) or the presence (right plot) of 1 μM remdesivir. GFP expression was analyzed by flow cytometry two days later. (D) Effect of SARS-CoV-2 N transduction on GFP expression from BAC replicon DNA. Control untransduced cells (top three plots) and N-transduced cells (bottom three plots) were left untransfected (left plots), or were electroporated with SwaI-linearized pSMART-BAC-T7-scv2 in the absence (center plots) or the presence (right plots) of 1 μM remdesivir. GFP expression was analyzed by flow cytometry two days later.

Article Snippet: The pSMART BAC v2.0 vector containing the SARS-CoV-2 (Wuhan-Hu-1) non-infectious replicon ( ) was obtained from BEI resources (#NR-54972).

Techniques: Western Blot, Expressing, Transfection, Transduction, RNA Expression, Flow Cytometry, Stable Transfection

Journal: BMC Oral Health

Article Title: Functional screening of a human saliva metagenomic DNA reveal novel resistance genes against sodium hypochlorite and chlorhexidine

doi: 10.1186/s12903-021-02000-5

Figure Lengend Snippet: Minimum inhibitory concentration (MIC) and the range of antimicrobials tested against E. coli BacRep containing empty pSMART BAC vector

Article Snippet: The pSMART BAC HindIII vector (7.6 kb) was fully digested and dephosphorylated by using HindIII restriction enzyme and calf intestinal alkaline phosphatase (CIAP) enzyme (NEB, UK) at 37 °C for 60 min.

Techniques: Concentration Assay

Journal: BMC Oral Health

Article Title: Functional screening of a human saliva metagenomic DNA reveal novel resistance genes against sodium hypochlorite and chlorhexidine

doi: 10.1186/s12903-021-02000-5

Figure Lengend Snippet: HindIII digestion of plasmids extracted from resistant clones identified from the oral metagenomic library. pSMART BAC vector backbone was indicated with the green arrow. Lane M, HyperLadder™ 1 kb. U, undigested plasmid; D, digested plasmid. The digested product was run on a GelRed® precast gel

Article Snippet: The pSMART BAC HindIII vector (7.6 kb) was fully digested and dephosphorylated by using HindIII restriction enzyme and calf intestinal alkaline phosphatase (CIAP) enzyme (NEB, UK) at 37 °C for 60 min.

Techniques: Clone Assay, Plasmid Preparation

Journal: Journal of Insect Science

Article Title: Construction of the Antheraea pernyi (Lepidoptera: Saturniidae) Multicapsid Nucleopolyhedrovirus Bacmid System

doi: 10.1093/jisesa/ieaa088

Figure Lengend Snippet: Schematic representation of the construction strategy of the AnpeNPV bacmid. (A) A fragment carrying the bacterial transposon Tn7 target site flanked by lacZα reporter gene ( lacZα :mini- att Tn7: lacZα ) was inserted into pSMART BAC-N1 to generate the plasmid pSMART BAC-N1/ lacZα :mini- att Tn7: lacZ α. (B) Two DNA fragments, one containing partial orf144 ( lef-2 ), full-length orf145, and the polyhedrin promoter of AnpeNPV genomic DNA (correspond to nt 124957–126242*) with AvrII and HindIII restriction sites and the other containing partial orf1 ( polyhedrin ) and orf2 ( 1629capsid ) (corresponding to nt 254–1393*) with BamHI and AvrII restriction sites were cloned into pSMART BAC-N1/ lacZα :mini- att Tn7: lacZ α to create the transfer vector pSMARTBAC-N1/ lacZα :mini- att Tn7: lacZα / phΔN . *The numbering system begins with the first nucleotide (A) of the initiation codon of orf1 ( polyhedrin ) based on the sequence of AnpeNPV L2 (GenBank accession number: EF207986 ). (C) Treatment of the genomic DNA of AnpeNPV PhEGFP -Avr II with AvrII endonuclease generated the linear AnpeNPV DNA . (D) Treatment of pSMARTBAC-N1/ lacZα :mini- att Tn7: lacZα / phΔN with AvrII generated the linearized transfer vector DNA. (E) The recombinant AnpeNPV was obtained by homologous recombination between the linear AnpeNPV DNA (C) and the linearized transfer vector DNA (D) in Tn-Hi5 cells, and the recombinant virus was amplified in A. pernyi pupae. (F) The genomic DNA of the recombinant AnpeNPV was extracted from hemolymph of A. pernyi pupae and transformed into E. coli BAC-Optimized Replicator v2.0 Cells by electroporation. Positive colonies that contained AnpeNPV genomic DNA with chloramphenicol resistance gene and lacZα reporter gene were selected. The bacmid is named AnpeNPV bacmid.

Article Snippet: The CopyRight pSMART BAC cloning vector harboring chloramphenicol resistance gene (42030–1, Lucigen) utilizes the single-copy origin of the E. coli F plasmid to maintain large inserts of up to 350 kb with the highest stability possible in E. coli .

Techniques: Plasmid Preparation, Clone Assay, Sequencing, Generated, Recombinant, Homologous Recombination, Amplification, Transformation Assay, Electroporation