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Details of the target gene |
Gene Model of Rodent Parasite |
PBANKA_0714000
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Gene Model P. falciparum ortholog |
PF3D7_0816800
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Gene product | meiotic recombination protein DMC1, putative |
Gene product: Alternative name | |
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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 | 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 | Partial |
Additional remarks partial/complete disruption |
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Selectable marker used to select the mutant parasite | tgdhfr |
Promoter of the selectable marker | pbdhfr |
Selection (positive) procedure | pyrimethamine |
Selection (negative) procedure | No |
Additional remarks genetic modification | Initially a plasmid construct was designed containing 5' UTR and 3'UTR sequences of PbDmc1 to delete the entire gene, and after at least three failed attempts to generate transformed parasites, the targeting plasmid construct was revised to disrupt the gene by using other 3' and 5' that are located inside the ORF.
The 5' targeting sequence comprised of exons 1 to 3 and at the end of the exon 3, a stop codon (TAA) was added. The 3' targeting sequence comprised of exons 4 to 6. |
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 | 5'-ggtaccCAGCCAGCAAAGTTGCAT-3' |
Additional information primer 1 | primer 587: 567 bp fragment consisting of the 5' region of PbDmc1, nt position +26 to +593 |
Sequence Primer 2 | 5'-aagcttCTACACTTGAGTTTTTCCGCA-3' |
Additional information primer 2 | primer 588: 567 bp fragment consisting of the 5' region of PbDmc1, nt position +26 to +593 |
Sequence Primer 3 | 5'-gaattcCGAGTAGACTTTAGTGGGC-3' |
Additional information primer 3 | promer 589: 3' targeting sequence 490 bp in size, nt position +980 to nt +1470 (comprising of exons 4 through 6 and the spanning introns) |
Sequence Primer 4 | 5'-gcggccgcCGGGAAGGTTTGGAG-3' |
Additional information primer 4 | promer 590: 3' targeting sequence 490 bp in size, nt position +980 to nt +1470 (comprising of exons 4 through 6 and the spanning introns) |
Sequence Primer 5 | |
Additional information primer 5 | |
Sequence Primer 6 | |
Additional information primer 6 | |
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