NL4-3Δ6-drEGFP-IRES-mThy1.2
(Plasmid #207881)

• Purpose: HIV reporter vector expressing dGFP and mThy1.2 from Env locus
• Depositing Lab: Edward Browne
• Publication: Peterson et al Cell Chem Biol. 2023 Dec 21;30(12):1617-1633.e9. doi: 10.1016/j.chembiol.2023.11.009.

Backbone

• Vector backbone: pNL43
• Backbone size w/o insert (bp): 14825
• Total vector size (bp): 16757
• Modifications to backbone: -First cloning step was by the Siliciano lab to make a deletion in the Env ORF and insert EGFP into this locus to create pNL4-3-ΔE-GFP. doi: 10.1128/JVI.78.4.1718-1729.2004 -Next the Siliciano lab introduced mutations into all viral proteins other than Tat and Rev, destabilized EGFP with a C-terminal PEST sequence from pzGreen-DR, and mutated the Env signal peptide to promote cytoplasmic retention, creating the pNL4-3-Δ6-drEGFP vector. doi: 10.1172/JCI39199 -In this study we cloned an IRES-mThy1.2 casette into the NheI site immediately after the drEGFP stop codon and verified orientation by sequencing.
• Vector type: Lentiviral

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): NEB Stable
• Growth instructions: We have had luck growing this plasmid with STBL3 cells at 37deg or STBL2 cells at 30deg. Recombination can occur between the two LTRs without these accomodations. We have not used NEB stable to grow the plasmid but this is likely fine.
• Copy number: High Copy

Gene/Insert

• Gene/Insert name: destabilized EGFP-IRES-mThy1.2
• Alt name: drEGFP-IRES-mCD90.2
• Species: M. musculus (mouse)
• Insert Size (bp): 1931
• GenBank ID: NM_009382.3
• Promoter: 5' lentiviral LTR

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ cloning site: XbaI (not destroyed)
• 3′ cloning site: NheI (not destroyed)
• 5′ sequencing primer: gcggagacagcgacgaag

Resource Information

• A portion of this plasmid was derived from a plasmid made by: We received the pNL4-3-Δ6-drEGFP vector from the Siliciano lab at Johns Hopkins University School of Medicine. We are depositing our version to Addgene after receiving written permission (by email) from Dr. Siliciano.

Terms and Licenses

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

Depositor Comments

Please visit https://doi.org/10.1101/2022.05.09.491199 for bioRxiv preprint.