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Purposefor C-terminal tagging with eDHFR trimethoprim regulated degron with neo selection
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Depositing Lab
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Depositing OrganizationUniversity of Colorado Health Sciences Center (Inactive)
Located in United States of America -
Citations
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Sequence Information
Ordering
| Item | Catalog # | Description | Quantity | Price (USD) | |
|---|---|---|---|---|---|
| Plasmid | 86396 | Standard format: Plasmid sent in bacteria as agar stab | 1 | $94 | |
| DNA | 86396-D50 | 50 μg of DNA in Tris buffer | $495 | ||
| DNA | 86396-D100 | 100 μg of DNA in Tris buffer | $585 | ||
Backbone
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Vector backbonepAc5
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Modifications to backbonesee Gonzalez et al Scientific Reports 1:75, 2011
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Vector typeCRISPR
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Selectable markersNeomycin (select with G418)
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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Copy numberUnknown
Gene/Insert
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Gene/Insert nameHA3 eDHFR T2A neo
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SpeciesE coli
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MutationR12Y, G67S and Y100I mutations in E coli DHFR
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Tag
/ Fusion Protein
- HA3 eDHFR T2A neo (C terminal on insert)
Resource Information
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A portion of this plasmid was derived from a plasmid made byThe mutant E coli DHFR gene used in the two plasmids was derived from Cho et al PlosOne 8: e72393 Addgene plasmid 29325.
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.
Depositor Comments
pAc5 HA3-eDHFR-T2A-neo was derived from pAc5gRNA Cas9 (Bassett, A.R.,et al Biol Open 3:42, 2014) by replacing the EcoRI-Hind III fragment encoding Cas9 with a PCR fragment encoding HA3-eDHFR amplified from pBMN DHFR(DD)-YFP (Iwamoto eet al Chem Biol 17:981, 2010.) (Addgene plasmid #29325) and by replacing the puro gene with neo using the NheI-XhoI sites
This degron has R12Y, G67S and Y100I mutations in eDHFR
DNA (Catalog # 86396-D50)
Purpose
Ready-to-use, high-purity DNA prep. DNA aliquots are suitable for use in mammalian cell transfections and other molecular biology applications.
Delivery
- Amount 50 μg
- Concentration 1 μg/μL
- Pricing $495 USD
- Storage 4 ℃ (short-term) or -20 ℃ (long-term)
Terms and Licenses
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Academic/Nonprofit Terms
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Industry Terms
- Not Available to Industry
Quality Control
Every DNA prep is verified by next-generation sequencing, confirming plasmid identity, sequence integrity, and absence of DNA contaminants. Concentration is confirmed to be within ±2.5% of the stated concentration.
Our production process has been validated to consistently yield endotoxin levels below 1.0 EU/μg. Individual lots are not tested for endotoxin.
DNA (Catalog # 86396-D100)
Purpose
Ready-to-use, high-purity DNA prep. DNA aliquots are suitable for use in mammalian cell transfections and other molecular biology applications.
Delivery
- Amount 100 μg
- Concentration 1 μg/μL
- Pricing $585 USD
- Storage 4 ℃ (short-term) or -20 ℃ (long-term)
Terms and Licenses
-
Academic/Nonprofit Terms
-
Industry Terms
- Not Available to Industry
Quality Control
Every DNA prep is verified by next-generation sequencing, confirming plasmid identity, sequence integrity, and absence of DNA contaminants. Concentration is confirmed to be within ±2.5% of the stated concentration.
Our production process has been validated to consistently yield endotoxin levels below 1.0 EU/μg. Individual lots are not tested for endotoxin.
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:
pAc5 HA3-eDHFR-T2A-neo was a gift from David Bentley (Addgene plasmid # 86396 ; http://n2t.net/addgene:86396 ; RRID:Addgene_86396) -
For your References section:
Selectable one-step PCR-mediated integration of a degron for rapid depletion of endogenous human proteins. Sheridan RM, Bentley DL. Biotechniques. 2016 Feb 1;60(2):69-74. doi: 10.2144/000114378. eCollection 2016 Feb. 10.2144/000114378 PubMed 26842351
Map uploaded by the depositor.
