pND-Ub17-deltaE2
(Plasmid #83221)

• Purpose: Expresses the nDHFR-link1-His6-Ub and UBA1Ta in E.coli
• Depositing Lab: Gali Prag
• Publication: Levin-Kravets et al Nat Methods. 2016 Nov;13(11):945-952. doi: 10.1038/nmeth.4003. Epub 2016 Oct 3.

Backbone

• Vector backbone: pZE21
• Backbone size w/o insert (bp): 2219
• Total vector size (bp): 5925
• Modifications to backbone: The pND-Ub vector is based on a modified pZE21 vector, contacting the pLtetO-1. cDNAs were amplified by polymerase chain reaction (PCR), digested and sub-cloned into the vectors. The mouse cDNA of the nDHFR fragment (residues 1-108) was PCR-amplified and sub-cloned between the KpnI and PacI sites (the PacI site was inserted into the vector by PCR) into the pZE21 vector. Then a flexible linker (Figure 4) was constructed a using two-step PCR method and digested with PacI and NotI (the NotI site was inserted into the vector by PCR) and cloned into the vector downstream to the pLtetO1-nDHFR. Ubiquitylation apparatus cassettes containing a His6-Ub-E2-E1 were PCR-amplified from pGEN plasmids expressing different E2s or Ub mutants (Keren-Kaplan et al, 2011) and sub-cloned as fusion to the C-terminus of the nDHFR-linker1 into the NotI and AvrII endonuclease recognition sites.
• Vector type: Bacterial Expression

Growth in Bacteria

• Bacterial Resistance(s): Kanamycin, 50 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH5alpha
• Copy number: High Copy

Gene/Insert 1

• Gene/Insert name: Ubiquin
• Species: H. sapiens (human)
• Insert Size (bp): 283
• Entrez Gene: LOC199038
• Tag / Fusion Protein:
  • nDHFR-link1-His6-Ub (N terminal on insert)

Gene/Insert 2

• Gene/Insert name: uba1
• Species: Triticum aestivum

Terms and Licenses

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