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| Details of the target gene |
| Gene Model of Rodent Parasite |
PBANKA_0507500
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| Gene Model P. falciparum ortholog |
PF3D7_1023300
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| Gene product | conserved protein, unknown function |
| Gene product: Alternative name | |
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| Details of the genetic modification |
| Name of the tag | mCherry-3xMyc |
| Details of tagging | C-terminal |
| Additional remarks: tagging | |
| Commercial source of tag-antibodies | |
| Type of plasmid/construct | (Linear) plasmid double cross-over |
| PlasmoGEM (Sanger) construct/vector used | No |
| Modified PlasmoGEM construct/vector used | No
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| Plasmid/construct map |
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| Plasmid/construct sequence |
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| Restriction sites to linearize plasmid |
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| Selectable marker used to select the mutant parasite | hdhfr |
| Promoter of the selectable marker | pbdhfr |
| Selection (positive) procedure | pyrimethamine |
| Selection (negative) procedure | No |
| Additional remarks genetic modification | PBANKA_0507500 encoding a hydrophobic polypeptide of 79 amino acid residues, remained refractory to endogenous tagging, most likely due to an interference of the fluorescent tag with essential protein functions
The protein was selected in a screen for putative PV (parasitophorous vacuole) proteins.
Proteins are targeted to the PV by means of default protein secretion, which is initiated by the recognition and cleavage of an amino-terminal signal peptide (SP). To identify novel PV candidates, we searched the Plasmodium genome database (PlasmoDB) for SP-containing proteins and sequentially removed proteins containing predicted transmembrane domains, export motifs, apicoplast targeting peptides, endoplasmic reticulum (ER) retention signals and GPI anchors, which could potentially redirect the protein to other locations. Twelfe proteins were selected. 5 proteins were shown to have a PV location in blood- and liver-stages.
For endogenous tagging of the PV candidates, a GFP(PV) co-localisation plasmid was constructed by fusing the bacterial backbone and adjacent mCherry-3xMyc tag sequence of the pBAT vector with the Plasmodium-specific fraction of the pGFP-PV plasmid using the KpnI and PvuII restriction sites. The sequences directly upstream of the stop codons were amplified from genomic DNA (ranging in size from 758 to 1,628 bp) and inserted into the GFP(PV) co-localisation plasmid using HpaI in combination with EcoRI or SacII. For double homologous recombination, 3′ fragments were amplified and cloned into the intermediate vectors as described for the gene deletion constructs. |
| Additional remarks selection procedure | |
Primer information: Primers used for amplification of the target sequences 
Primer information: Primers used for amplification of the target sequences 
| Sequence Primer 1 | |
| Additional information primer 1 | |
| Sequence Primer 2 | |
| Additional information primer 2 | |
| Sequence Primer 3 | |
| Additional information primer 3 | |
| Sequence Primer 4 | |
| Additional information primer 4 | |
| Sequence Primer 5 | |
| Additional information primer 5 | |
| Sequence Primer 6 | |
| Additional information primer 6 | |
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*RMgm-4525