pND-Ub16-UBCH5B Hs
(Plasmid
#83220)
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PurposeExpresses the nDHFR-link1-His6-Ub, UBCH5B Hs and UBA1Ta in E.coli
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Depositing Lab
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Sequence Information
Ordering
Item | Catalog # | Description | Quantity | Price (USD) | |
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Plasmid | 83220 | Standard format: Plasmid sent in bacteria as agar stab | 1 | $85 |
Backbone
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Vector backbonepZE21
- Backbone size w/o insert (bp) 2219
- Total vector size (bp) 6465
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Modifications to backboneThe 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.
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Vector typeBacterial Expression
Growth in Bacteria
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Bacterial Resistance(s)Kanamycin, 50 μg/mL
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Growth Temperature37°C
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Growth Strain(s)DH5alpha
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Copy numberHigh Copy
Gene/Insert 1
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Gene/Insert nameUbiquitin
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SpeciesH. sapiens (human)
Gene/Insert 2
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Gene/Insert nameE2 ubiquitin conjugating enzyme
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Alt nameUBCH5b
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SpeciesH. sapiens (human)
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Entrez GeneUBE2D2 (a.k.a. E2(17)KB2, PUBC1, UBC4, UBC4/5, UBCH4, UBCH5B)
Terms and Licenses
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Academic/Nonprofit Terms
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Industry Terms
- Not Available to Industry
Trademarks:
- Zeocin® is an InvivoGen trademark.
These plasmids were created by your colleagues. Please acknowledge the Principal Investigator, cite the article in which the plasmids were described, and include Addgene in the Materials and Methods of your future publications.
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For your Materials & Methods section:
pND-Ub16-UBCH5B Hs was a gift from Gali Prag (Addgene plasmid # 83220) -
For your References section:
A bacterial genetic selection system for ubiquitylation cascade discovery. Levin-Kravets O, Tanner N, Shohat N, Attali I, Keren-Kaplan T, Shusterman A, Artzi S, Varvak A, Reshef Y, Shi X, Zucker O, Baram T, Katina C, Pilzer I, Ben-Aroya S, Prag G. Nat Methods. 2016 Nov;13(11):945-952. doi: 10.1038/nmeth.4003. Epub 2016 Oct 3. 10.1038/nmeth.4003 PubMed 27694912