Purpose(Empty Backbone) contains a C-terminal tandem affinity purification tag (relevant to any system) within a construct designed for genome integration into trypanosomes.
|Item||Catalog #||Description||Quantity||Price (USD)|
|Plasmid||51710||Standard format: Plasmid sent in bacteria as agar stab||1||$85|
This material is available to academics and nonprofits only.
Vector backbonepBluescript KS+
Modifications to backboneThe PTP cassette contains 745 bp of a TbU1-70K C-terminal coding region, a NotI restriction site, the PTP tag coding sequence, the translation stop codon TGA, and 470 bp of 3′ flank from TbRPA1, the gene encoding the largest subunit of RNA polymerase I. The resistance marker cassette contains the neomycin phosphotransferase gene (NEOr) flanked 5′ and 3′ by the intergenic regions of heat shock protein 70 (HSP70) genes 2 and 3 and of β- and α-tubulin genes, respectively. In a further development of the original resistance marker cassette, we separated the HSP70 intergenic region from NEOr by an NdeI restriction site and NEOr from the tubulin flank by BamHI, HpaI, and BstBI restrictions sites.
Vector typeT. brucei genome integration vectors
Selectable markersNeomycin (select with G418)
/ Fusion Protein
- PTP (ProtC-TEV-ProtA) (C terminal on backbone)
Growth in Bacteria
Bacterial Resistance(s)Ampicillin, 100 μg/mL
- Cloning method Restriction Enzyme
- 5′ sequencing primer M13-F20
- 3′ sequencing primer M13R (Common Sequencing Primers)
Terms and Licenses
- Not Available to Industry
- Zeocin® is an InvivoGen trademark.
The PTP tag is a derivative of the original TAP tag and has CBP replaced by ProtC.
Gene flanks providing RNA processing signals for PTP fusion and resistance marker are the 3′ flank of TbRPA1 (RPA1-3′), HSP70 genes 2 and 3 intergenic region (H23), the β-α tubulin intergenic region (T), and the 5′ flank of TbRPA2 (RPA2-5′).
pC-PTP-NEO contains a target sequence (U1-70K) which can be replaced by the gene of interest.
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.
For your Materials & Methods section:pC-PTP-NEO was a gift from Arthur Gunzl (Addgene plasmid # 51710 ; http://n2t.net/addgene:51710 ; RRID:Addgene_51710)
For your References section:Highly efficient tandem affinity purification of trypanosome protein complexes based on a novel epitope combination. Schimanski B, Nguyen TN, Gunzl A. Eukaryot Cell. 2005 Nov;4(11):1942-50. 10.1128/EC.4.11.1942-1950.2005 PubMed 16278461