pBMN DHFR-HiBiT / Lyt2
(Plasmid #179731)

• Purpose: Expresses human dihydrofolate reductase (DHFR) with C-terminal HiBiT tag in mammalian cells. This retroviral vector has an IRES-Lyt-2 downstream of the cloning sites
• Depositing Lab: James Inglese
• Publication: Rana et al Cell Chem Biol. 2024 Feb 15;31(2):221-233.e14. doi: 10.1016/j.chembiol.2023.09.014. Epub 2023 Oct 23.

Backbone

• Vector backbone: pBMN
• Backbone manufacturer: Prof. G. Nolan, Stanford University, Stanford, CA
• Backbone size w/o insert (bp): 6352
• Total vector size (bp): 6952
• Vector type: Retroviral

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH5alpha
• Copy number: High Copy

Gene/Insert

• Gene/Insert name: human DHFR-HiBiT and IRES-Lyt-2
• Alt name: DHFR-HiBiT
• Alt name: DHFR, DHFRP1, DYR
• Species: H. sapiens (human), M. musculus (mouse)
• Insert Size (bp): 600
• GenBank ID: NM_000791
• Entrez Gene: DHFR (a.k.a. DHFR1, DHFRP1, DYR)
• Promoter: LTR/MMLV
• Tag / Fusion Protein:
  • HiBiT (C terminal on insert)

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ cloning site: BamH1 (not destroyed)
• 3′ cloning site: Not1 (not destroyed)
• 5′ sequencing primer: pBMN 5’
• 3′ sequencing primer: IRES reverse

Terms and Licenses

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

Depositor Comments

The human DHFR-HiBiT fusion protein was constructed to have the HiBiT tag (VSGWRLFKKIS) sequence (Schwinn, MK et al. (2018) ACS Chem. Biol. 13(2): 467–474) located at the C-terminus of DHFR. The human DHFR-HiBiT construct was synthesized (Bio Basic) and cloned into the retroviral vector pBMN IRES-Lyt-2 (G. Nolan, Stanford University, Stanford, CA), which expresses the coding region of mouse CD8a (Lyt-2), using the restriction sites BamH1 / Not1. All constructs were sequenced to validate authenticity. BamH1- GCCACC-ATG-DHFR-HiBiT-Stop –Not1