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PurposeExpresses Perfringolysin O cholesterol sensor, has N-terminal His6-tag and three point mutations, A71T, C459A, and Y181A. Y181A prevents PFO's lytic activity at 4°C.
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Depositing Lab
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Sequence Information
Ordering
Item | Catalog # | Description | Quantity | Price (USD) | |
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Plasmid | 120400 | Standard format: Plasmid sent in bacteria as agar stab | 1 | $85 |
Backbone
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Vector backbonepRSET-B
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Backbone manufacturerInvitrogen
- Backbone size w/o insert (bp) 2887
- Total vector size (bp) 4417
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Vector typeBacterial Expression
Growth in Bacteria
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Bacterial Resistance(s)Ampicillin, 100 μg/mL
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Growth Temperature37°C
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Growth Strain(s)DH5alpha
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Growth instructionsBL21 (DE3) pLysS E. coli competent cells used for protein production. Growth at 37°C until O.D. of 0.6 to 0.8. Then, expression induced with 1 mM IPTG at 37°C for 4 h.
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Copy numberUnknown
Gene/Insert
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Gene/Insert nameMutant form of Perfringolysin O
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Alt namePFO*
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SpeciesClostridium Perfringens
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Insert Size (bp)1530
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MutationAmino acids 1-27 are deleted, starts at amino acid 28. Additionally, has three mutations: A71T, C459A, Y181A.
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GenBank ID
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Tag
/ Fusion Protein
- His6 tag (N terminal on insert)
Cloning Information
- Cloning method Unknown
- 5′ sequencing primer TAA TAC GAC TCA CTA TAG GG
- 3′ sequencing primer GCT AGT TAT TGC TCA GCG G (Common Sequencing Primers)
Resource Information
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A portion of this plasmid was derived from a plasmid made byParent plasmid was obtained from Arthur Johnson, Texas A&M University. Site-directed mutagenesis was used to introduce 3 mutations.
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:
pPFO* was a gift from Arun Radhakrishnan (Addgene plasmid # 120400 ; http://n2t.net/addgene:120400 ; RRID:Addgene_120400) -
For your References section:
Use of mutant 125I-perfringolysin O to probe transport and organization of cholesterol in membranes of animal cells. Das A, Goldstein JL, Anderson DD, Brown MS, Radhakrishnan A. Proc Natl Acad Sci U S A. 2013 Jun 25;110(26):10580-5. doi: 10.1073/pnas.1309273110. Epub 2013 Jun 10. 10.1073/pnas.1309273110 PubMed 23754385