top of page |
Details of the target gene |
Gene Model of Rodent Parasite |
PBANKA_1003300
|
Gene Model P. falciparum ortholog |
PF3D7_0405600
|
Gene product | TMEM33 domain-containing protein, putative |
Gene product: Alternative name | TMEM33 |
top of page |
Details of the genetic modification |
Name of the tag | mNeon-Green (containing a twin-strep epitope) |
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
|
Plasmid/construct map |
|
Plasmid/construct sequence |
|
Restriction sites to linearize plasmid |
|
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 generation of Pbtmem33(−) and Pbtmem33-mNG strains was accomplished by double-crossover homologous recombination with modified donor plasmids that contain fluorescent protein and the hDHFR expression cassettes. The fluorescent protein markers are eGFP in the knockout donor plasmid (pAA20), and mNeonGreen (mNG) in the tagging plasmid (pAA32). PCR amplified fragments of 5′ UTR and 3′ UTR as homologous regions of the target loci were cloned into the knockout plasmids flanking the human DHFR cassette and the fluorescence protein marker cassettes to generate Pbp230p and Pbtmem33 constructs by using SacII/BamHI and HindIII/KpnI restriction enzymes, respectively. Similarly, downstream coding sequence and 3′ UTR sequences of TMEM33 were PCR amplified and cloned into the tagging plasmid to generate Pbtmem33-mNG construct by using SacII/EcoRI and HindIII/KpnI restriction enzymes, respectively. Transfection of P. berghei ANKA parasites were performed with SacII/KpnI linearized donor DNA plasmids, and the positive drug selection was done by oral pyrimethamine treatment. |
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 | |
|
|
top of page |