Vector sequences

   
Plasmid Name version Genbank Seq Resistance markers Fusion tag Amplicon size (kb)
pPOTv6 blast mNeonGreen 6 pPOTv6 blast mNG N term: BLAST C term: BLAST 3Ty::mNG::3Ty N term: 1.85 C term: 2.0
pPOTv6 blast eYFP 6 pPOTv6-blast-eYFP N term: BLAST C term: BLAST 3Ty::eYFP::3Ty N term: 1.85 C term: 2.0
pPOTv6 blast SNAP 6 pPOTv6-blast-SNAP N term: BLAST C term: BLAST 3Ty::SNAP::3Ty N term: 1.85 C term: 2.0
pPOTv6 blast CLIP 6 genbank sequence N term: BLAST C term: BLAST 3Ty::CLIP::3Ty N term: 1.85 C term: 2.0
pPOTv6 puro mNeonGreen 6 pPOTv4-puro-mNG N term: PURO C term: PURO 3Ty::mNG::3Ty N term: 2.5 C term: 2.7
pPOTv7 blast mNeonGreen 7 pPOTv7-blast-mNG N term: BLAST C term: BLAST mNG N term: 1.8 C term: 2.0
pPOTv7 blast eYFP 7 pPOTv7-blast-eYFP N term: BLAST C term: BLAST eYFP N term: 2.25 C term: 2.4
pPOTv7 blast tagRFPt 7 pPOTv4-blast-tagRFPt N term: BLAST C term: BLAST tagRFPt N term: 2.25 C term: 2.4
pPOTv7 blast 10xTy 7 pPOTv7-blast-Ty(10) N term: BLAST C term: BLAST 10xTy N term: 2.25 C term: 2.4
pPOTv7 hygro mNeonGreen 7 pPOTv7-hygro-mNG N term: hygro C term: hygro mNG N term: 2.43 C term: 2.44
pPOTv7 g418 mNeonGreen 7 pPOTv7-g418-mNG N term: g418 C term: g418 mNG N term: 2.19 C term: 2.21
   

Technical notes

  • v6 will include epitopes on the target protein (currently only Ty tags)
  • v7 uses only the indicated protein tag.
  • See the supplemental information in our Open Biology publication for the precise instructions on how to use these vectors.
  • I have now switched to "Roditi" electroporation buffer and 3x 2000V in 4mm cuvettes - this has increased my transfection efficiency about 3 fold.
  • Primers designed to tag a gene will work regardless of the drug resistance or the genetic tag.
  • When preparing DNA containing drug ORF repeats, use a recombination deficient strain of E. coli such as sure2.
  • Sequence files for older versions of pPOT can be found here. They have been replaced because they had lower transfection efficiency for C terminal tagging.

Notes on fluorescent proteins:

  • mNeonGreen is a step change improvement from the other fluorescent proteins. It is better than eYFP or eGFP for genetic tagging in all respects (brighter, more photostable, increased proportion of 'good localisations').
  • Superfolder GFP is optimised for tagging of membrane proteins. However, I have not personally tested that it works better than the other fluorescent proteins and I suspect that mNeonGreen still outperfoms it.
  • tdTomato is very bright but frequently causes mis-localisation of the tagged protein. For this reason I would recommend tagRFPt as the current best red protein.
  If you use these plasmids or any of their derivatives in your work, please cite:

Dean, S., Sunter, J., Wheeler, R. J., Hodkinson, I., Gluenz, E. Gull, K. A toolkit enabling efficient, scalable and reproducible gene tagging in trypanosomatids. Open Biol 5. 2015 (doi:10.1098/rsob.140197)

Please contact me if you have any problems with the protocol or any of the files.