pET28a 6xHis-HaloTag-TEVc-4xtrTUBEmut
(Plasmid #254155)

• Purpose: Bacterial expression of binding-deficient 4xtrTUBEmut with an N-terminal 6xHis and HaloTag followed by a TEV protease cleavage site
• Depositing Lab: Rahul Samant
• Depositing Organization: Babraham Institute
  Located in United Kingdom of Great Britain and Northern Ireland
• Publication: Johnston et al bioRxiv 2026.02.13.703496

Backbone

• Vector backbone: pET28a 6xHis-HaloTag-TEVc
• Backbone manufacturer: Novagen; MRC-PPU Reagents & Services, UK
• Backbone size w/o insert (bp): 6290
• Total vector size (bp): 7007
• Modifications to backbone: Optimised T7 promoter (from Addgene # 154464); Added 6xHis-HaloTag at N-terminal of insert (from Addgene # 62455)
• Vector type: Bacterial Expression

Growth in Bacteria

• Bacterial Resistance(s): Kanamycin, 50 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH5alpha
• Growth instructions: For expression of 6xHis-HaloTag-TEVc-4xtrTUBEmut, plasmid was transformed into Rosetta 2(DE3) competent E. coli (Novagen/Sigma; Cat# 71397) and cultured at 37 °C for 18 h to OD600 ~0.7, adjusted to 1 mM IPTG (Melford Laboratories, Cat# B1008) to induce expression, and incubated for 24 h at 16 °C, before pelleting, lysis, and purification.
• Copy number: Low Copy

Gene/Insert

• Gene/Insert name: 4xtrTUBEmut
• Alt name: 4 tandem repeats of the trypsin-resistant, binding-deficient UBQLN1 UBA domain
• Alt name: 4xTR-TUBEmut
• Species: H. sapiens (human), Synthetic
• Insert Size (bp): 714
• Mutation: All Arg residues in the UBA domain are mutated to Ala; Leu-Leu to Ala-Ala in UBA domain to reduce Ubiquitin affinity
• GenBank ID: NM_013438.5 NM_013438.5
• Entrez Gene: UBQLN1 (a.k.a. DA41, DSK2, PLIC-1, UBQN, XDRP1)
• Promoter: T7
• Tags / Fusion Proteins:
  • HaloTag (N terminal on backbone)
  • TEV cleavage site (N terminal on backbone)
  • His6 (N terminal on insert)
  • His6 (C terminal on backbone)

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ cloning site: None (destroyed during cloning)
• 3′ cloning site: HindIII (not destroyed)
• 5′ sequencing primer: HaloTag-C-For
• 3′ sequencing primer: T7 Term

Resource Information

• A portion of this plasmid was derived from a plasmid made by: pET28a HaloTag-TEVc-[multiple-cloning-site] (obtained from MRC-PPU Reagents & Services, UK, Cat # DU23222); pET28a T7CONS TIR-2 sfGFP (kind gift from Daniel Daley, Addgene plasmid #154464); pRSET-6x-TR-TUBE (kind gift from Yasushi Saeki, Addgene plasmid #110313); pET28a 6xHis-HaloTag-Nrf2 (kind gift from Yimon Aye, Addgene plasmid #62455); 6xHis-TEVc-HaloTag-4xtrTUBEmut (MRC-PPU Reagents & Services; Cat # DU58829; obtained from Yu-Chiang Lai, U. Birmingham)

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
  • HaloTag Vector Limited Use Label License
• Industry Terms:
  • Not Available to Industry

Depositor Comments

Please visit https://doi.org/10.64898/2026.02.13.703496 for bioRxiv preprint.