pBOB-CAG-SARS-CoV2-Spike-HA
(Plasmid #141347)

• Purpose: 3. Generation lentiviral vector expressing the codon optimized SARS-CoV2 Spike Glycoprotein ORF with a C-terminal HA tag generated by Junko Ogawa & Gerald M Pao
• Depositing Lab: Gerald Pao
• Publication: Ogawa et al bioRxiv. 2020 Jul 22. doi: 10.1101/2020.07.21.214932.

Backbone

• Vector backbone: pBOB-CAG
• Backbone manufacturer: Verma Lab (Salk Institute)
• Total vector size (bp): 13475
• Vector type: Lentiviral

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): NEB Stable
• Growth instructions: do not overgrow
• Copy number: High Copy

Gene/Insert

• Gene/Insert name: SARS-CoV2 Spike Glycoprotein
• Alt name: Spike
• Alt name: S
• Species: SARS-CoV2 hCoV19_USA EPI_ISL_414366 (GISAID)
• Insert Size (bp): 3858
• Mutation: resynthesized with human codon optimization nucleotide sequence does not match original sequence.
• Entrez Gene: S (a.k.a. GU280_gp02, spike glycoprotein)
• Promoter: CAG
• Tag / Fusion Protein:
  • HA (C terminal on insert)

Cloning Information

• Cloning method: Gibson Cloning
• 5′ sequencing primer: CAG-FWD AGCCTCTGCTAACCATGTTC
• 3′ sequencing primer: WPRE-REV TCCTCCTCCTCTTGTGCTTC

Resource Information

• Supplemental Documents:
  • Western blot from transient and viral expression in cells
  • Immunofluorescence in infected cells
• Articles Citing this Plasmid:
  • 2 References

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
• Industry Terms:
  • Not Available to Industry

Information for Lentiviral Prep (Catalog # 141347-LV)

Purpose

Ready-to-use Lentiviral Prep particles produced from pBOB-CAG-SARS-CoV2-Spike-HA (#141347). In addition to the viral particles, you will also receive purified pBOB-CAG-SARS-CoV2-Spike-HA plasmid DNA.

Lentiviral particles carrying the SARS CoV2 spike glycoprotein with a C-terminal HA tag.

Delivery

• Volume: 1mL
• Titer: ≥5x10⁵ TU/mL
• Pricing: $150 USD for preparation of 1mL virus + $30 USD for plasmid.
• Storage: Store at -80℃. Thaw just before use and keep on ice.
• Shipment: Viral particles are shipped frozen on dry ice. Plasmid DNA (≥ 200ng) will also be included in the shipment.

Viral Production & Use

• Packaging Plasmids: psPAX2 (plasmid #12260)
• Envelope: pMD2.G (plasmid #12259)
• Buffer: OptiPro

Biosafety

Requestor is responsible for compliance with their institution's biosafety regulations. Lentivirus is generally considered BSL-2. AAV is generally considered BSL-1, but may require BSL-2 handling depending on the insert. Biosafety Guide

Terms and Licenses

• Academic/Nonprofit Terms:
  • Terms of Use for Viral Vectors
• Industry Terms:
  • Not Available to Industry

Viral Quality Control

Titering Method:

• ddPCR assay: 293T cells were transduced with serial dilutions of 141347-LV, harvested several days later, and genomic DNA was isolated. Copies of RRE were measured and normalized to RPP30.

Notes:

• PCR confirmation of insert: PCR was carried out with primers targeting the CoV2 spike gene and the WPRE element to confirm the backbone and the insert. The PCR product was visualized on an agarose gel for size confirmation.
  Forward primer: COV2 Spike FP CGCCGCGACTAAAATGTCTG
  Reverse primer: WPRE Rev CATAGCGTAAAAGGAGCAACA

Visit our viral production page for more information.