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RNAi pGad pDex endoTag-N endoTag-C endoTag-N/C

Vectors

Below are maps and sequences for some of the plasmids for stable transformation of Trypanosoma brucei available from the Wickstead lab or collaborators.

News...news...

I've finally found an Open Source plasmid editor program that is, in my opinion, a reasonable substitute for GCK. M. Wayne Davis's ApE: A Plasmid Editor is free, Open Source, runs on Windows/MacOS-X/Linux, and does all the things you'd expect from a plasmid editor (plus a couple of cool extras, like direct alignment of ABI sequencing files to the plasmid sequence). ApE reads GenBank files (amongst other formats), so I've replaced the text files that used to be on this page with GenBank versions. My thanks to Shvetank Sharma of Meharry Medical College, Nashville, who introduced me to the software.

Still presently included on this site are annotated Gene Construction Kit files for anyone who has this good, but expensive, proprietary software. However, since I won't renew my GCK license, these files will be gradually replaced by ApE-readable GenBank files.

I've written a small perl script that will take annotated ApE files and make a pretty SVG files from them (avaiable here). The maps below were generated with this script.


Plasmids for RNAi

Simple modifications to the popular p2T7 and p2T7TiTA plasmids which result in integration to the minichromosomal repeats, providing a tighter off state for knockdown of sensitive transcripts. These plasmids are described in Wickstead et al. (2002) MBP 125:211-6. My thanks to David Wildridge at the University of Glasgow who put together the Vector NTI versions of the plasmids that appear below.
Plasmid map GenBank GCK VectorNTI Marker
p2T7-177 map icon seq icon GCK icon vectorNTI icon BLE
p2T7-TA177 map icon seq icon GCK icon vectorNTI icon HYG
(Zipped archives of complete sets can be downloaded here)

Plasmids for inducible transgene expression (pGad)

Constructs for inducible expression of transgenes from various genomic locations. The pGad series contain a single 2-gene polycistron (GFP and Drug) under the influence of a tetracycline-inducible promoter (c.f. dual-promoter pDex series). These vectors are described in Wickstead et al. (2003) NAR 31:3993-4000.
Plasmid map GenBank GCK Integration site Inducible promoter Transgene Marker
pGad7 map icon seq icon na - EP1 GFP HYG
pGad8-rDNA map icon seq icon GCK icon rDNA spacer EP1 GFP HYG
pGad8-3x177 map icon seq icon GCK icon 177bp repeat EP1 GFP HYG
pGad8-V4 map icon seq icon GCK icon VSG-G4 EP1 GFP HYG
pGad8-V8 map icon seq icon GCK icon VSG-S8 EP1 GFP HYG
pGad8-Tub map icon seq icon GCK icon TUB none GFP HYG
pGad9-rDNA map icon seq icon GCK icon rDNA spacer T7 GFP HYG
pGad9-177 map icon seq icon GCK icon 177bp repeat T7 GFP HYG
pGad9-V4 map icon seq icon GCK icon VSG-G4 T7 GFP HYG
(Zipped archives of complete sets can be downloaded here)

Plasmids for inducible transgene expression (pDex)

Minichromosomal repeat-integrating constructs for inducible expression of transgenes. pDex177, 277, 377 and 477 are derivatives of pLEW100 with various promoters. pDex577 vectors are highly-modular expression vectors designed and constructed by Steve Kelly. These vectors are described in Kelly et al. (2007) MBP 154:103-9 along with a large number of modified forms of pDex377 produced in the lab of Mark Carrington.
Plasmid map GenBank GCK Constitutive promoter Marker Inducible promoter Transgene
pDex177 map icon seq icon GCK icon T7 BLE EP1 LUC
pDex277 map icon seq icon GCK icon T7 BLE T7 LUC
pDex377 map icon seq icon GCK icon rDNA HYG EP1 LUC
pDex477 map icon seq icon GCK icon rDNA BLE EP1 LUC
pDex477-Y2 map icon seq icon na rDNA BLE EP1 TY-YFP-TY
pDex577-G map icon seq icon GCK icon rDNA BLE T7 TY-GFP-TY
pDex577-Y map icon seq icon GCK icon rDNA BLE T7 TY-YFP-TY
pDex577-C map icon seq icon GCK icon rDNA BLE T7 TY-GFP-TY
(Zipped archives of complete sets can be downloaded here)

Plasmids for endogenous locus tagging: C-terminus

A set of plasmids designed for medium-throughput tagging of trypanosomal proteins at the C-terminus. The plasmids allow for easy incorporation of the endogenous locus 3'-intergenic DNA - to express chimeric proteins at levels similar to wild-type. Currently unpublished.
Plasmid map GenBank GCK Marker Tag
pEnG0 map icon seq icon GCK icon HYG GFP
pEnG0B map icon seq icon GCK icon BLAST GFP
pEnG0P map icon seq icon GCK icon PURO GFP
pEnT0 map icon seq icon GCK icon HYG Myc-TY
pEnTT0 map icon seq icon GCK icon HYG 2xTY
pEnTT0B map icon seq icon GCK icon BLAST 2xTY
pEnTT0P map icon seq icon GCK icon PURO 2xTY
(Zipped archives of complete sets can be downloaded here)

Plasmids for endogenous locus tagging: N-terminus

A set of plasmids designed for tagging of trypanosomal proteins at the N-terminus. Similar in style to the C-terminus tagging constructs above. Currently unpublished.
Plasmid map GenBank GCK Marker Tag
pEnNY0 map icon seq icon GCK icon HYG YFP
pEnNTYY0 map icon seq icon GCK icon HYG TY-YFP
pEnNmSt0-B map icon seq icon n/a BLAST mStrawberry
pEnNmSt0-N map icon seq icon n/a NEO mStrawberry
(Zipped archives of complete sets can be downloaded here)

Plasmids for endogenous locus tagging: either terminus

This set of plasmids were designed and constructed by Steve Kelly as a set of highly modular constructs for easy tagging of endogenous genes at either N- or C-termini. They are described in Kelly et al. (2007) MBP 154:103-9 and are provided here as a service.
Plasmid map GenBank GCK Marker Tag
pEnT5-G map icon seq icon GCK icon HYG TY-GFP-TY
pEnT5-Y map icon seq icon GCK icon HYG TY-YFP-TY
pEnT5-C map icon seq icon GCK icon HYG TY-CFP-TY
pEnT6B-G map icon seq icon GCK icon BLAST TY-GFP-TY
pEnT6B-Y map icon seq icon GCK icon BLAST TY-YFP-TY
pEnT6B-C map icon seq icon GCK icon BLAST TY-CFP-TY
pEnT6P-G map icon seq icon GCK icon PURO TY-GFP-TY
pEnT6P-Y map icon seq icon GCK icon PURO TY-YFP-TY
pEnT6P-C map icon seq icon GCK icon PURO TY-CFP-TY
(Zipped archives of complete sets can be downloaded here)


RNAi pGad pDex endoTag-N endoTag-C endoTag-N/C