Pcmv lgbit expression plasmid

Manufactured by Promega

The PCMV-LgBiT expression plasmid is a tool used for genetic engineering and protein expression studies. It contains the CMV promoter to drive the expression of the LgBiT gene, which is a component of the NanoBiT protein complementation assay.

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LgBit (7 citations)

3 protocols using pcmv lgbit expression plasmid

SARS-CoV-2 Spike Protein Expression and Characterization

Cited 2 times

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Full-length SARS-CoV S (GenBank: AY278741.1) and SARS-CoV-2 S, E, M, and N (GenBank: NC_045512.2) genes were synthesized by Genscript, Inc. as human codon-optimized cDNAs, and inserted into pcDNA3.1 expression vectors. All S mutations were introduced via site-directed mutagenesis, except for omicron NTD, which was synthesized (Intergrated DNA Technologies). HiBiT-N was constructed by fusing HiBiT peptide (VSGWRLFKKIS) coding sequences with linker (GSSGGSSG) to the 5′ end of the N gene, as described in (Kumar et al., 2021 (link); Qing et al., 2021 (link)). The pCMV-LgBiT expression plasmid was purchased from Promega. pcDNA3.1-hACE2-C9 was obtained from Dr. Michael Farzan, Scripps Florida. pcDNA3.1-hACE2-LgBiT was constructed by fusing the coding sequence of LgBiT to the 3′ end of hACE2 gene.

Qing E., Li P., Cooper L., Schulz S., Jäck H.M., Rong L., Perlman S, & Gallagher T. (2022). Inter-domain communication in SARS-CoV-2 spike proteins controls protease-triggered cell entry. Cell Reports, 39(5), 110786.

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SARS-CoV-2 Construct Cloning and Expression

Cited 4 times

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Full-length SARS-CoV-2 S, E, M, and N genes (GenBank: NC_045512.2) were synthesized by Genscript, Inc. as human codon-optimized cDNAs and inserted into pcDNA3.1 expression vectors. Omicron BA.1 S was synthesized (Integrated DNA Technologies). All S recombinants were constructed via gene fragment Assembly (NEB, Ipswich, MA USA; #E2621S). HiBiT-N was constructed by fusing HiBiT peptide (VSGWRLFKKIS) and linker (GSSGGSSG) coding sequences to the 5′ end of the N gene, as described in references (47 (link), 133 (link)). Nluc-PS9 was constructed by fusing PS9 SARS-CoV-2 RNA packaging sequence to the 3′ end of an Nluc gene, as described in reference (74 (link)). The pCMV-LgBiT expression plasmid was purchased from Promega. pcDNA3.1-hACE2-C9 was obtained from Dr. Michael Farzan, Scripps Florida. pcDNA3.1-hACE2-LgBiT was constructed by fusing the coding sequence of LgBiT to the 3′ end of hACE2 gene. pHEF-VSVG-Indiana was constructed previously (134 (link)). hACE2-Fc expression plasmid was constructed previously (47 (link), 52 (link)).

Qing E, & Gallagher T. (2023). Adaptive variations in SARS-CoV-2 spike proteins: effects on distinct virus-cell entry stages. mBio, 14(4), e00171-23.

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Constructing SARS-CoV and SARS-CoV-2 Expression Vectors

Cited 3 times

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Full-length SARS-CoV S (GenBank accession no. AY278741.1) and SARS-CoV-2 S, E, M, and N (GenBank accession no. NC_045512.2) genes were synthesized by GenScript, Inc., as human-codon-optimized cDNAs and inserted into pcDNA3.1 expression vectors. C9-tagged versions of the S genes were generated by replacing the 19 3′-terminal codons with linker and C9 codons (GSSGGSSG-GGTETSQVAPA). HiBiT-N was constructed by fusing HiBiT peptide (VSGWRLFKKIS) coding sequences with a linker (GSSGGSSG) to the 5′ end of the N gene, as described in references 42 (link) and 43 (link). The pCMV-LgBiT expression plasmid was purchased from Promega. pDSP_1–7 and pDSP_8–11 plasmid DNAs (73 (link), 74 (link)) were provided by Zene Matsuda (University of Tokyo). pcDNA3.1-hACE2-C9 was obtained from Michael Farzan, Scripps Florida. pCAGGS-hTMPRSS2_FLAG was constructed previously (75 (link)). pcDNA3.1-hACE2-LgBiT was constructed by fusing the coding sequence of LgBiT to the 3′ end of hACE2 gene.

Qing E., Kicmal T., Kumar B., Hawkins G.M., Timm E., Perlman S, & Gallagher T. (2021). Dynamics of SARS-CoV-2 Spike Proteins in Cell Entry: Control Elements in the Amino-Terminal Domains. mBio, 12(4), e01590-21.

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