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| Details of the target gene |
| Gene Model of Rodent Parasite |
PBANKA_1201400
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| Gene Model P. falciparum ortholog |
PF3D7_1003000
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| Gene product | guanylate cyclase organizer UGO, putative |
| Gene product: Alternative name | UGO |
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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 used | (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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| Partial or complete disruption of the gene | Complete |
| Additional remarks partial/complete disruption |
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| Selectable marker used to select the mutant parasite | hdhfr |
| Promoter of the selectable marker | eef1a |
| Selection (positive) procedure | pyrimethamine |
| Selection (negative) procedure | No |
| Additional remarks genetic modification | The gene deletion–targeting vector for pdeα was constructed using the pAB022 plasmid, which contains polylinker sites flanking an hdhfr expression cassette conferring resistance to pyrimethamine. Polymerase chain reaction (PCR) primers MB763 and MB764 were used to generate a fragment of pdeα 5′ upstream sequence from genomic DNA, which was inserted into Hind III and Pst I restriction sites upstream of the hdhfr cassette. A fragment generated with primers MB765 and MB766 from the 3′ flanking region of pdeα was then inserted downstream of the hdhfr cassette using Kpn I and Not I restriction sites. The linear targeting sequence was released using Hind III and Not I, and the construct was transfected into the ANKA line 2.34 or PDEδ-KO.
3xHA, KO, or AID/HA tagging of GCα, UGO, and PDEγ was generated using phage recombineering in Escherichia coli TSA strain with PlasmoGEM vectors (https://plasmogem.umu.se/pbgem/). For final targeting vectors not available in the PlasmoGEM repository, generation of KO and tagging constructs was performed using sequential recombineering and gateway steps as previously described. For each gene of interest (goi), the Zeocin resistance/Phe sensitivity cassette was introduced using oligonucleotides goi HA-F × goi HA-R and goi KO-F × goi KO-R for 3xHA, AID/HA tagging, and KO targeting vectors. Insertion of the gateway (GW) cassette following gateway reaction was confirmed using primer pairs GW1 × goi QCR1 and GW2 × goi QCR2. The modified library inserts were then released from the plasmid backbone using Not I. The UGO-AID/HA and GCα-AID/HA targeting vectors were transfected into the 615 parasite line, and the PDEγ-KO, UGO-3xHA, and GCα-3xHA vectors into the 2.34 line |
| 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-5390