pC-PTP-NEO
(Plasmid #51710)

• Purpose: (Empty Backbone) contains a C-terminal tandem affinity purification tag (relevant to any system) within a construct designed for genome integration into trypanosomes.
• Depositing Lab: Arthur Gunzl
• Publication: Schimanski et al Eukaryot Cell. 2005 Nov;4(11):1942-50.

Backbone

• Vector backbone: pBluescript KS+
• Modifications to backbone: The 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 type: T. brucei genome integration vectors
• Selectable markers: Neomycin (select with G418)
• Tag / Fusion Protein:
  • PTP (ProtC-TEV-ProtA) (C terminal on backbone)

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH5alpha
• Copy number: Unknown

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ sequencing primer: M13-F20
• 3′ sequencing primer: M13R

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
• Industry Terms:
  • Not Available to Industry

Depositor Comments

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.