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Details of the target gene |
Gene Model of Rodent Parasite |
PbANKA_0830200
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Gene Model P. falciparum ortholog |
PF3D7_0929400
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Gene product | high molecular weight rhoptry protein 2 |
Gene product: Alternative name | RhopH2 |
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Details of the genetic modification |
Short description of the mutation | The rhoph2 promoter modified to contain a tet-inducible promoter and TRAD4 activating domain |
Inducable system used | TET-based (TRAD4) |
Short description of the conditional mutagenesis | Downregulation of RhopH2 by addition of the tetracycline derivative anhydrotetracycline(ATc) |
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 |
Promoter of the selectable marker | eef1a |
Selection (positive) procedure | pyrimethamine |
Selection (negative) procedure | No |
Additional remarks genetic modification | To engineer the PbRhopH2 inducible knockdown (iKD) line, the first 1477 bp of the PbRhopH2 coding sequence (PbANKA_0830200) that had been PCR amplified with the primers DO291F and DO67R was cloned into the PstI and NheI sites of the modified pPRF-TRAD4-Tet07583 HAPRF-hDHFR (Pino et al., 2012) described in Elsworth et al (Elsworth et al., 2014). Also cloned into the NheI and BssHII sites of this vector were 1279 bp of the rhoph2 5' UTR sequence immediately upstream of the RhopH2 start codon, which had been PCR amplified using the primers DO62F and DO63R. Before transfection into P. berghei ANKA parasites, pTRAD4-iRhopH2ss was linearized with NheI. |
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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