Ub-G76V-GFP vector (Cat. No.: V000830)
Note: The UbG76V-GFP reporter construct, first reported by Dantuma et al. in Nature Biotechnology in 2000, represents the world’s first fluorescent plasmid tool capable of quantitatively measuring the activity of the ubiquitin-proteasome system (UPS) in living intact cells. Wild-type ubiquitin contains a glycine residue at position 76 (Gly76) on its C-terminus, which serves as the canonical cleavage site recognized by deubiquitinating enzymes (DUBs). When this glycine is mutated to valine, generating the G76V variant, two critical functional alterations occur. First, DUBs lose the ability to cleave the N-terminal fused ubiquitin tag from the GFP moiety, so the fusion protein permanently retains a ubiquitin-derived degradation signal. Second, lysine residues within the mutant ubiquitin segment are continuously modified with K48-linked polyubiquitin chains by cellular E3 ubiquitin ligases. These polyubiquitin conjugates are readily recognized by the 19S regulatory complex of the proteasome, which mediates substrate unfolding and rapid translocation into the 20S catalytic core for complete proteolytic degradation.
- Name:
- Ub-G76V-GFP
- Antibiotic Resistance:
- Kanamycin
- Length:
- 4959 bp
- Type:
- Mammalian Expression
- Replication origin:
- ori
- Selection Marker:
- Neomycin (select with G418)
- Copy Number:
- High Copy
- Cloning Method:
- Restriction Enzyme
- 5' Primer:
- EGFP-N (CGTCGCCGTCCAGCTCGACCAG)
- Growth Strain(s):
- DH10B
Resources
Plasmid Protocol
1. Centrifuge at 5,000×g for 5 min.
2. Carefully open the tube and add sterile water to dissolve the DNA: add 20 μl for 5 μg plasmid, and 100 μl for 100 μg plasmid.
3. Close the tube and incubate for 10 minutes at room temperature.
4. Briefly vortex the tube and then do a quick spin to concentrate the liquid at the bottom. Speed is less than 5000×g.
5. Store the plasmid at -20 ℃.
6. The concentration of plasmid re-measurement sometimes differs from the nominal value, which may be due to the position of the lyophilized plasmid in the tube, the efficiency of the re-dissolution, the measurement bias, and adsorption on the wall of the tube, therefore, it is recommended to transform and extract the plasmid before using it
General Plasmid Transform Protocol
1. Take one 100μl of the competent cells and thaw it on ice for 10min, add 2μl of plasmid, then ice bath for 30min, then heat-shock it at 42℃ for 60s, do not stir, and then ice bath for 2min.
2. Add 900μl of LB liquid medium without antibiotics, and incubate at 37℃ for 45min (30℃ for 1-1.5 hours) with 180rpm shaking.
3. Centrifuge at 6000rpm for 5min, leave only 100μl of supernatant to resuspend the bacterial precipitate and spread it onto the target plasmid-resistant LB plate.
4. Invert the plate and incubate at 37℃ for 14h, or at 30℃ for 20h.
5. Pick a single colony into LB liquid medium, add the corresponding antibiotics, incubate at 220rpm for 14h, and extract the plasmid according to the experimental needs and the instructions of the plasmid extraction kit.
References
- Short-lived green fluorescent proteins for quantifying ubiquitin/proteasome-dependent proteolysis in living cells. Dantuma NP, Lindsten K, Glas R, Jellne M, Masucci MG. Nat Biotechnol. 2000 May. 18(5):538-43. 10.1038/75406 PubMed 10802622
Ub-G76V-GFP vector (Cat. No.: V000830) Sequence
LOCUS 40924_48997 4959 bp DNA circular SYN 13-MAY-2021
DEFINITION synthetic circular DNA.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 4959)
AUTHORS Dantuma NP, Lindsten K, Glas R, Jellne M, Masucci MG
TITLE Short-lived green fluorescent proteins for quantifying
ubiquitin/proteasome-dependent proteolysis in living cells.
JOURNAL Nat Biotechnol. 2000 May . 18(5):538-43.
PUBMED 10802622
REFERENCE 2 (bases 1 to 4959)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 4959)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Nat
Biotechnol. 2000 May . 18(5):538-43."
COMMENT SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..4959
/mol_type="other DNA"
/organism="synthetic DNA construct"
enhancer 119..422
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 423..626
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
CDS 696..920
/codon_start=1
/product="human ubiquitin"
/label=ubiquitin
/translation="MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIF
AGKQLEDGRTLSDYNIQKESTLHLVLRLRG"
CDS 963..1679
/codon_start=1
/label=EGFP
/note="enhanced GFP"
/translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
EFVTAAGITLGMDELYK"
polyA_signal 1805..1926
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
rep_origin complement(1933..2388)
/direction=LEFT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 2415..2519
/label=AmpR promoter
promoter 2521..2878
/label=SV40 promoter
/note="SV40 enhancer and early promoter"
CDS 2913..3704
/codon_start=1
/label=NeoR/KanR
/note="aminoglycoside phosphotransferase"
/translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
primer_bind complement(3895..3914)
/label=TK-pA-R
/note="Thymidine kinase polyA, reverse primer"
polyA_signal 3939..3986
/label=HSV TK poly(A) signal
/note="herpes simplex virus thymidine kinase
polyadenylation signal (Cole and Stacy, 1985)"
rep_origin 4315..4903
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"