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| Type and details of transgene |
| Is the transgene Plasmodium derived |
Transgene: not Plasmodium |
| Transgene name | GFP |
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| Details of the genetic modification |
| Inducable system used | No |
| Additional remarks inducable system |
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| 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/yfcu |
| Promoter of the selectable marker | eef1a |
| Selection (positive) procedure | No |
| Selection (negative) procedure | 5-fluorocytosine (5-FC) |
| Additional remarks genetic modification | We replaced the positive-negative SM in the OVA::hep17(hep17) s1-GIMO genome with a GFP-expression cassette by GIMO transfection, using construct pL2354. To generate pL2354 we first removed the SM cassette from pL1928 using restriction enzyme site KpnI, resulting in plasmid 2023. Then we replaced the s1 5’ gene targeting region of pL2023 and added the GFP expression cassette between the s1 5’ gene and 3’ targeting regions. The s1 5’ gene targeting region was PCR amplified using the primer pairs p5/p6 and ligated using ApaI and KpnI restriction enzyme sites resulting in the intermediate plasmid F261. The GFP-expression cassette, including the 5’ promoter and 3’ UTR from hsp70 (PBANKA_0711900), obtained from plasmid pBC-GFP-hDHFR, 5 was then cloned into F261 using XhoI and PstI restriction enzymes generating plasmid pL2354. The construct was linearized using KasI and SacII restriction enzymes outside of the 5’ and 3’ TRs before transfection. The construct was used to transfect parasites of the OVA::hep17(hep17) s1-GIMO line using standard methods of GIMO-transfection 2,3. Transfected parasites were selected in mice by applying negative selection by providing 5-fluorocytosine (5-FC) in the drinking water of mice. Negative selection results in selection of transgenic parasites where the hdhfr::yfcu SM in the s1 locus of OVA::hep17hep17 s1 GIMO line is replaced by the GFP-expression cassette, resulting in line 3284. Transfected parasites of line 3284 were cloned by limiting dilution, resulting in the GFPhsp70-OVA::hep17hep17, line (line 3284cl1). |
| Additional remarks selection procedure | |
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| Other details transgene |
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| Promoter |
| Gene Model of Parasite |
PBANKA_0711900
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| Gene Model P. falciparum ortholog |
PF3D7_0818900
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| Gene product | heat shock protein 70 |
| Gene product: Alternative name | HSP70 |
Primer information details of the primers used for amplification of the promoter sequence  Primer information details of the primers used for amplification of the promoter sequence 
| Sequence Primer 1 | |
| Additional information primer 1 | |
| Sequence Primer 2 | |
| Additional information primer 2 | |
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| 3'-UTR |
| Gene Model of Parasite |
PBANKA_0711900
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| Gene product | heat shock protein 70 |
| Gene product: Alternative name | HSP70 |
Primer information details of the primers used for amplification the 3'-UTR sequences Primer information details of the primers used for amplification the 3'-UTR sequences
| Sequence Primer 1 | |
| Additional information primer 1 | |
| Sequence Primer 2 | |
| Additional information primer 2 | |
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| Insertion/Replacement locus |
| Replacement / Insertion | Replacement locus |
| Gene Model of Parasite |
PBANKA_1206800
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| Gene product | zinc finger (CCCH type) protein, putative |
| Gene product: Alternative name | s1 |
Primer information details of the primers used for amplification of the target sequences
Primer information details of the 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 | |
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*RMgm-4921