RMgmDB - Rodent Malaria genetically modified Parasites

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Summary

RMgm-936
Malaria parasiteP. yoelii
Genotype
DisruptedGene model (rodent): PY17X_0811300; Gene model (P.falciparum): PF3D7_0317100; Gene product: 6-cysteine protein (B9)
Transgene
Transgene not Plasmodium: GFP-Luciferase
Promoter: Gene model: PBANKA_1133300; Gene model (P.falciparum): PF3D7_1357100; Gene product: elongation factor 1-alpha (eef1a)
3'UTR: Gene model: PBANKA_0719300; Gene product: bifunctional dihydrofolate reductase-thymidylate synthase, putative (dhfr/ts)
Replacement locus: Gene model: PY03857; Gene product: Pfs230 paralog-related (P230p; 230p)
Phenotype Liver stage;
Last modified: 24 December 2014, 10:59
  *RMgm-936
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene disruption, Introduction of a transgene
Reference (PubMed-PMID number) Reference 1 (PMID number) : 24509910
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. yoelii
Parent strain/lineP. y. yoelii 17XNL
Name parent line/clone RMgm-689
Other information parent lineRMgm-689 (1971cl1) is a P.y.yoelii 17XNL mutant expressing the fusion protein GFP-Luciferase under the control of the constitutive P. berghei eef1a promoter. The mutant does not contain a drug-selectable marker.
The mutant parasite was generated by
Name PI/ResearcherT. Annoura; S.M. Khan; C.J. Janse
Name Group/DepartmentLeiden Malaria Research Group
Name InstituteLeiden University Medical Center (LUMC)
CityLeiden
CountryThe Netherlands
Name of the mutant parasite
RMgm numberRMgm-936
Principal name2174cl8cl5
Alternative namePyΔb9
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageNot different from wild type
Gametocyte/GameteNot different from wild type
Fertilization and ookineteNot different from wild type
OocystNot different from wild type
SporozoiteNot different from wild type
Liver stageDevelopment of PyΔb9 was like WT parasites in the blood (as they develop a 0.5-2% parasitemia after 8 days from an infection initiated by a single parasite) and in mosquito-stage development (normal production of oocysts and sporozoites). Liver-stage development was analysed in BALB/c mice after inoculation of 104 or 2x105 sporozoites by in vivo imaging and analysis of subsequent blood-stage infections. In vivo imaging of mice infected with 2x105 PyΔb9 sporozoites showed faint luminescent signals in the liver at 3hpi that were significantly higher than background values of uninfected mice. At 40hpi none of the infected mice showed luminescence signals above background. A blood-stage infection was only detected in 1 out of 8 mice infected with 2x105 sporozoites with a long prepatent period of 10 days. These results indicate that PyΔb9 parasites, like P. berghei Δb9, are severely compromised in liver stage development.
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of B9 (PY17X_0811300; PY00153) and expresses the fusion gene GFP-Luciferase under control of the P. berghei eef1a promoter

Protein (function)
The B9 protein contains a 4-cysteine domain with a structure that is highly similar to the structure of 4-cysteine domains in the known proteins of the 6-Cys family and is identified as a 6-Cys-related protein. B9 is predicted to be glycosylphophatidylinositol (GPI) anchored. B9 transcripts are found in sporozoites. Evidence is presented for translational repression of b9 transcripts in P. berghei sporozoites. The B9 protein is found in (young) liver stages. For P. falciparum B9 evidence is presented for a location at the plasmalemma membrane of liver stages. P. falciparum and P. berghei mutants lacking expression of B9 abort development during development of young liver stages.

Phenotype
See RMgm-932, RMgm-933 and RMgm-934 for RMgm-P. berghei mutants lacking expression of B9. These mutants have been analysed in more detail than the P. yoelii mutant described here.
Development of PyΔb9 was like WT parasites in the blood (as they develop a 0.5-2% parasitemia after 8 days from an infection initiated by a single parasite) and in mosquito-stage development (normal production of oocysts and sporozoites). Liver-stage development was analysed in BALB/c mice after inoculation of 104 or 2x105 sporozoites by in vivo imaging and analysis of subsequent blood-stage infections. In vivo imaging of mice infected with 2x105 PyΔb9 sporozoites showed faint luminescent signals in the liver at 3hpi that were significantly higher than background values of uninfected mice. At 40hpi none of the infected mice showed luminescence signals above background. A blood-stage infection was only detected in 1 out of 8 mice infected with 2x105 sporozoites with a long prepatent period of 10 days. These results indicate that PyΔb9 parasites, like P. berghei Δb9, are severely compromised in liver stage development.

Additional information



Supporting Figure S8
Generation of P. yoelii mutants lacking expression of B9 (PyΔb9)

A. Schematic representation of the construct pL1438 targeting b9 for gene deletion by double cross-over homologous recombination at the target regions (grey boxes), and the locus before and after disruption. The construct contains the hdhfr::yfcu selection marker (SM). Primer positions for diagnostic PCRs and amplicon sizes are shown (see Table S1 for primer sequences). B. Diagnostic PCRs (left) and Southern analyses of separated chromosomes (right) confirm the correct integration of the construct in Δb9. Primer pairs and amplicon sizes are shown in A. Selectable marker (M; primers 4698/4699); 3’-integration event (3’; primers 4239/7260); ORF (O; primers 7261/7262). See Table S1 for the sequence of the primers. For Southern analyses, pulsed field gel-separated chromosomes were hybridized with a 3’UTR pbdhfr probe. PyΔb9 mutant has been generated in the reference PyGFP-Luccon which has a gfp-luciferase gene integrated into the silent 230p locus (PY03857 and PY04774) on chromosome 3. Hybridization with the 3’UTR dhfr-ts probe recognizes the integrated construct on chromosome 8, the reporter GFP-Luccon construct on chromosome 3, and the endogenous dhfr-ts gene located on chromosome 2.

Other mutants
RMgm-932: P. berghei mutant lacking expression of B9 in the WT background. B9 was disrupted by double cross-over homologous recombination using a plasmid DNA construct.
RMgm-933: P. berghei mutant lacking expression of B9 in the GFP-Luciferase expressing background. B9 was disrupted by double cross-over homologous recombination using a linear construct generated by the 'Anchor-tagging' PCR-based method.
RMgm-934: P. berghei mutant lacking expression of B9. The mutant does not contain a selectable marker.
RMgm-935: P. berghei triple knock-out mutant lacking expression of B9, P52 and P36


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PY17X_0811300
Gene Model P. falciparum ortholog PF3D7_0317100
Gene product6-cysteine protein
Gene product: Alternative nameB9
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct used(Linear) plasmid double cross-over
PlasmoGEM (Sanger) construct/vector usedNo
Modified PlasmoGEM construct/vector usedNo
Plasmid/construct map
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Plasmid/construct sequence
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AATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTT
AATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACC
GATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGCGCCTGATGCGGTATTTT
CTCCTTACGCATCTGTGCGGTATTTCACACCGCATATGGTGCACTCTCAGTACAATCTGC
TCTGATGCCGCATAGTTAAGCCAGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGA
CGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGC
ATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGACGAAAGGGCCTCGTGATA
CGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACT
TTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATG
TATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGT
ATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCT
GTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCA
CGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCC
GAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCC
CGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTG
GTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTA
TGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATC
GGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTT
GATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATG
CCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCT
TCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGC
TCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCT
CGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTAC
ACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCC
TCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGAT
TTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATG
ACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATC
AAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAA
CCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAG
GTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTA
GGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTA
CCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAG
TTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTG
GAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCATTGAGAAAGCGCCACG
CTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAG
CGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGC
CACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAA
AACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATG
TTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCT
GATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAA
GAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGG
CACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAG
CTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGA
ATTGTGAGCGGTAAACAATTTCACACAGGAAACAGCTTGACCATGATTACGCCAAGCTTG
GGCCCTTTCCACATGTATGTGCAGGTGAATTTTGTTAATATAAAAATATAGAGATATATA
AGCTATAATTATTTATTTCACCTAAAATGTTTAAATTAAAATTTAGTATGTACTTATTTA
CTTTTTTTTTCATATTATTTTTTCGAAAAAAAGAAAAAAAAATATATATGTAACTATTTG
TATGATAGGCTTATTCATGTTTTGAAGTAAAATAAATTGGATATGTTTTGGGAAATAAAA
TAATCATAGTTCAAAATAGTAACAAGCTGACTACAAATTCACAGACACGTCCACATATAT
TATTTATATAATTTTTAAGCATAAAAAAATAAAAAGCAATGAAAAAATGGAGATAAAATA
AGCAAGCATAAATATGTACATGTACATGTATATTATTGTTGTATAGGCATTTGTTTGGTA
ATATTTTTATATGCACTTTTTCAAATTACTGTAATGATTTTTTTATTTAAAAATGCATCT
CATAAAATTTGGTAAATATAAAAAAGGGAAAAAAAACTTTTGTATATTTTTGGAAATATT
TTCATTTCACAATTAGGGGGTCATTAAAAATTATATTATAAAACTCTCTCCTTATTTTCA
GCAATTTTAAAGCTGAATTATTTTTTTGGTGGTAGTTTCATAAAAAAATAAAAAAATAAA
TAATGAATAGTGAACGAAATTGTGAATGAATGTGAAACGGCGGTTTATAGAATAAATCTT
TAGAAAATTAAAAAACTGCATGTATTCAAAGGTCTCTAAAATAATGGAAATAAATTAGAT
ATTGTTTCTGTCTTTTGTGCATGTAATTATTTTTCACTTTTATTAATCATGGTTATTTTG
GCAATAATAAAATATTCTCCAAATTGGTGGTTTTATATATTTGATATATACACATATCTA
TATATGAATATAAATAAATAATTTTCGGTAATTGCAAAAAGATTGTCTTTTTCTCAAATG
TATATAAGCATTTTTTTTTTTTTTTTTTTTTTTACAATTAACAATTTGGTTATATAAAAT
TTAACAAAGCATGCATAAATGGTTGCACATTTTTATTCTCTTTTTATTCGAAAAATATGA
AAAAATATGAGATAAATGAGAAAAAGTTTGCTTATTTATTAAAAAAAAGGAAAAAAATAC
ATATAATATATATTCAAAGTTATATTGGCCATTCTATATGCCAAAGGGTACACCAAAGTT
TGGAGGCGGATCAAATCAAAAAATATCTTACGATTTTGCGACAGAATAAAAAAGCGAGGA
AACAATTTCAGAACGTGGTTATGACTTGTTCTTAAGTTTACAATTTAATATTCATACTTT
AAGTATTTTTTGTAGTATCCTAGATATTGTGCTTTAAATGCTCACCCCTCAAAGCACCAG
TAATATTTTCATCCACTGAAATACCATTAAATTTTCAAAAAAATACTATGCATATAATGT
TATACATATAAACATAAAACGCCATGTAAATCAAAAAATATATAAAAATATGTATAAAAA
TAAATATGCACTAAATATAAGCTAATTATGCATAAAAATTAAAGTGCCCTTTATTAACTA
GTCGTAATTATTTATATTTCTATGTTATAAAAAAATCCTCATATAATAATATAATTAATA
TATGTAATGTTTTTTTTATTTTATAATTTTAATATAAAATAATATGTAAATTAATTCAAA
AAATAAATATAATTGTTGTGAAACAAAAAACGTAATTTTTTCATTTGCCTTCAAAATTTA
AATTTATTTTAATATTTCCTAAAATATATATACTTTGTGTATAAATATATAAAAATATAT
ATTTGCTTATAAATAAATAAAAAATTTTATAAAACATAGGGGGATCCATGGTTGGTTCGC
TAAACTGCATCGTCGCTGTGTCCCAGAACATGGGCATCGGCAAGAACGGGGACCTGCCCT
GGCCACCGCTCAGGAACGAATTTAGATATTTCCAGAGAATGACCACAACCTCTTCAGTAG
AAGGTAAACAGAATCTGGTGATTATGGGTAAGAAGACCTGGTTCTCCATTCCTGAGAAGA
ATCGACCTTTAAAGGGTAGAATTAATTTAGTTCTCAGCAGAGAACTCAAGGAACCTCCAC
AAGGAGCTCATTTTCTTTCCAGAAGTCTAGATGATGCCTTAAAACTTACTGAACAACCAG
AATTAGCAAATAAAGTAGACATGGTCTGGATAGTTGGTGGCAGTTCTGTTTATAAGGAAG
CCATGAATCACCCAGGCCATCTTAAACTATTTGTGACAAGGATCATGCAAGACTTTGAAA
GTGACACGTTTTTTCCAGAAATTGATTTGGAGAAATATAAACTTCTGCCAGAATACCCAG
GTGTTCTCTCTGATGTCCAGGAGGAGAAAGGCATTAAGTACAAATTTGAAGTATATGAGA
AGAATGATGCTAGCGGAGGAGGTGGATCTGGTGGAGGTGGAAGTGCTAGCGTGACAGGGG
GAATGGCAAGCAAGTGGGATCAGAAGGGTATGGACATTGCCTATGAGGAGGCGGCCTTAG
GTTACAAAGAGGGTGGTGTTCCTATTGGCGGATGTCTTATCAATAACAAAGACGGAAGTG
TTCTCGGTCGTGGTCACAACATGAGATTTCAAAAGGGATCCGCCACACTACATGGTGAGA
TCTCCACTTTGGAAAACTGTGGGAGATTAGAGGGCAAAGTGTACAAAGATACCACTTTGT
ATACGACGCTGTCTCCATGCGACATGTGTACAGGTGCCATCATCATGTATGGTATTCCAC
GCTGTGTTGTCGGTGAGAACGTTAATTTCAAAAGTAAGGGCGAGAAATATTTACAAACTA
GAGGTCACGAGGTTGTTGTTGTTGACGATGAGAGGTGTAAAAAGATCATGAAACAATTTA
TCGATGAAAGACCTCAGGATTGGTTTGAAGATATTGGTGAGGCTTCGGAACCATTTAAGA
ACGTCTACTTGCTACCTCAAACAAACCAATTGCTGGGTTTGTACACCATCATCAGAAATA
AGAATACAACTAGACCTGATTTCATTTTCTACTCCGATAGAATCATCAGATTGTTGGTTG
AAGAAGGTTTGAACCATCTACCTGTGCAAAAGCAAATTGTGGAAACTGACACCAACGAAA
ACTTCGAAGGTGTCTCATTCATGGGTAAAATCTGTGGTGTTTCCATTGTCAGAGCTGGTG
AATCGATGGAGCAAGGATTAAGAGACTGTTGTAGGTCTGTGCGTATCGGTAAAATTTTAA
TTCAAAGGGACGAGGAGACTGCTTTACCAAAGTTATTCTACGAAAAATTACCAGAGGATA
TATCTGAAAGGTATGTCTTCCTATTAGACCCAATGCTGGCCACCGGTGGTAGTGCTATCA
TGGCTACAGAAGTCTTGATTAAGAGAGGTGTTAAGCCAGAGAGAATTTACTTCTTAAACC
TAATCTGTAGTAAGGAAGGGATTGAAAAATACCATGCCGCCTTCCCAGAGGTCAGAATTG
TTACTGGTGCCCTCGACAGAGGTCTAGATGAAAACAAGTATCTAGTTCCAGGGTTGGGTG
ACTTTGGTGACAGATACTACTGTGTTTAACTCGATCCCGTTTTTCTTACTTATATATTTA
TACCAATTGATTGTATTTATAACTGTAAAAATGTGTATGTTGTGTGCATATTTTTTTTTG
TGCATGCACATGCATGTAAATAGCTAAAATTATGAACATTTTATTTTTTGTTCAGAAAAA
AAAAACTTTACACACATAAAATGGCTAGTATGAATAGCCATATTTTATATAAATTAAATC
CTATGAATTTATGACCATATTAAAAATTTAGATATTTATGGAACATAATATGTTTGAAAC
AATAAGACAAAATTATTATTATTATTATTATTTTTACTGTTATAATTATGTTGTCTCTTC
AATGATTCATAAATAGTTGGACTTGATTTTTAAAATGTTTATAATATGATTAGCATAGTT
AAATAAAAAAAGTTGAAAAATTAAAAAAAAACATATAAACACAAATGATGTTTTTTCCTT
CAATTTCGGGTACCCTCGAGGCTAGCGATATCTGATCACCCGGGGCGGCCGCGGACCAAG
TAATGAAACCCGATATGTTTATATACGATTACAAAAAACTTATAAACCTATAAAAAAAGT
TTTGGGAGTAAATTTTTCAGATACTTTTGATATATATTATAAATATTATAAATTTGATCA
AGAAAATGTTAAATTTTTAATAAATGATTTTTCAGAAACTAATTACATTGGTATGATATG
TCAAATAAATAAAGAAACAGATGTAAAACCATGTAGTTTTACTTTTATAAGTGATCCGAA
TCAAAATACATCTATATCAAGTTCATCTCACATAAAAAAACCACCTTTTTTATCTTATTT
TAATAAAAATAGAATACAAAATCAAAATAGTGTTGAATATGTAAGTGAGACACGTTTTAT
TTTTCCGAAAAATTTTAACGAAACTTTACAAGAAAAAGGAATCAAAGATATACATTTTAG
ATGTATTTGTTCTACTCAAAATGGGAAAAATAATGTTGATACTCATAATGAAATACAATA
TTTTATAACAACTGGAAATATATCAAATGATGATATGCTTGAAATTCCTACACATATTAC
AAATAACAATTTTTCTACACCTAATCTAGATTCAAAAACAAACAATAGAAATCATAAATT
ATCAAAAAGAAATCGTTATTTACCTAATAATATGAATGAAGAAAATACAAAATATCATTA
TAATACTGCTTCAGTTACTCAATGCAAATTTAATTTTTTTCAGGTTTTTATAGGTATAAC
TATATTCCATATTTTGATATCATTTTTATAGAAATTATAATAATATAAAACAATGTGAAA
TTTGAAAAAAAACAAATATAAATATATCTAGACATGAATAAAATGTTATTCCTTTCTCTA
ACAATTATGATACTAGTTTCTTATGTACACCCCTTACATTTTATAATCCTTTAATTATGT
AAAAAATAAAACACTTTCAAAATAACAATGAGAATGCACACAATAATTCAATAAAATTAT
TTAATTCCGTTGATGCATTCGTTTTTTTTCAATCTTTGTCATTTTCTCTTAAATGCGACA
TTATTTGCAG
Restriction sites to linearize plasmid
Partial or complete disruption of the geneComplete
Additional remarks partial/complete disruption
Selectable marker used to select the mutant parasitehdhfr/yfcu
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modification
Additional remarks selection procedure
Primer information: Primers used for amplification of the target sequences  Click to view information
Primer information: Primers used for amplification of the target sequences  Click to hide information
Sequence Primer 1CATGGGCCCTTTCCACATGTATGTGCAGGTG
Additional information primer 17209 (ApaI); ∆Pyb9 5' target F
Sequence Primer 2GCGCCTTAAGAACAAGTCATAACCACGTTCTG
Additional information primer 27210 (AflII); ∆Pyb9 5' target R
Sequence Primer 3ATAGTTTAGCGGCCGCGGACCAAGTAATGAAACCCG
Additional information primer 37211 (NotI); ∆Pyb9 3' target F
Sequence Primer 4GGAATTCTGCAAATAATGTCGCATTTAAGAG
Additional information primer 47212 (EcoRI); ∆Pyb9 3' target R
Sequence Primer 5
Additional information primer 5
Sequence Primer 6
Additional information primer 6

  Transgene: Mutant parasite expressing a transgene
Type and details of transgene
Is the transgene Plasmodium derived Transgene: not Plasmodium
Transgene nameGFP-Luciferase
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct(Linear) plasmid double cross-over
PlasmoGEM (Sanger) construct/vector usedNo
Modified PlasmoGEM construct/vector usedNo
Plasmid/construct map
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Plasmid/construct sequence
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AGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGC
ACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGC
TCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAA
TTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCTTG
GTACCGAGCTCGGATCCACTAGTAACGGCCGCCAGTGTGCTGGAATTCGCCCTTGAACTC
GTACTCCTTGGTGACGGGTACCGTGATGGAATGGCAACATCTGATCACATTTTAAAAATA
GTTGATGATGATAGTAAAACGATTAAATATTTTAACGATATACCTTATCAGATGTGTAAT
TTTGATTATAATTTAAGCAAATTAAGTGAAATACAAATATGTGAAAAAACAATAAATGAA
TTTAGTTTATTCATGTATAATTGTGAGCAAATAACAGATAATAGAATCGTATATGGTAAA
GAACCTATTAATACCATAAAATATTTAAGTAATGTATTTCCTATAAATAAATTTACAGAT
TTATTTTTTAATACAAAAGATATAGATATACCAGAAATAAATGAACAATTTAAAGGTTTT
AAATTTTTTATGACCTCATTTATAAATCATGGATCATATCCACTAACCATAGAATGTGGT
GTAACAAATGGTGGGACTGATTATAGAAGAGCAATTATTTTATTACATGTTCGAACAGAT
TTAAAAGATAGACCAGTTTCATTTTGTGATTTTAGAAAAGGTGAATTATATAATTATTTA
AATGCTTATAGTGAAGGAGATCTATGTATAATAATTTCCAAATCAAATACAAGTTTTGGT
TTTAGATGCCCCGAAAATACAAAAAAAATGCCAGAAAAATGTTTTACTCAAGTATATGAA
AAAGGCTATCTACATGATTCCTATAAAATTAATACTAAAAATATTATTAACTATTCATTT
GAAAATCCAGAATATGCATTAGCTGGTTTTAATTATACATTAACAAAATCATATCAATTT
GAATGTCATTGTGTAGATAAAGAAACAGAACAAATTGTAAAAACTGTTTTAGTAAAATAT
GTAAATGAAGATGAAATATATGATTATAATGATTTACCATTGGTTAATCATAAATCTATT
GTTGCACATCCAAATAAAACACATCTATGTGACTTTATGACATCTGATAATATGTTATCA
CCTAAAAAAGAAGATTCAGTAAATTATGTTTGTAATGTATTTCCAAAACCATTAGAATAT
GTAGCATTACATTGTCCAACCGAGGTCGACGATGCTTGTAGATGCCCGGGGCCCAGCTTA
ATTCTTTTCGAGCTCTTTATGCTTAAGTTTACAATTTAATATTCATACTTTAAGTATTTT
TTGTAGTATCCTAGATATTGTGCTTTAAATGCTCACCCCTCAAAGCACCAGTAATATTTT
CATCCACTGAAATACCATTAAATTTTCAAAAAAATACTATGCATATAATGTTATACATAT
AAACATAAAACGCCATGTAAATCAAAAAATATATAAAAATATGTATAAAAATAAATATGC
ACTAAATATAAGCTAATTATGCATAAAAATTAAAGTGCCCTTTATTAACTAGTCGTAATT
ATTTATATTTCTATGTTATAAAAAAATCCTCATATAATAATATAATTAATATATGTAATG
TTTTTTTTATTTTATAATTTTAATATAAAATAATATGTAAATTAATTCAAAAAATAAATA
TAATTGTTGTGAAACAAAAAACGTAATTTTTTCATTTGCCTTCAAAATTTAAATTTATTT
TAATATTTCCTAAAATATATATACTTTGTGTATAAATATATAAAAATATATATTTGCTTA
TAAATAAATAAAAAATTTTATAAAACATAGGGGGATCTATGAGTAAAGGAGAAGAACTTT
TCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTT
CTGTCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTTAAATTTATTT
GCACTACTGGAAAACTACCTGTTCCATGGCCAACACTTGTCACTACTTTCGGTTATGGTG
TTCAATGCTTTGCGAGATACCCAGATCATATGAAACAGCATGACTTTTTCAAGAGTGCCA
TGCCCGAAGGTTATGTACAGGAAAGAACTATATTTTTCAAAGATGACGGGAACTACAAGA
CACGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATAGAATCGAGTTAAAAGGTA
TTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAATTGGAATACAACTATAACTCAC
ACAATGTATACATCATGGCAGACAAACAAAAGAATGGAATCAAAGTTAACTTCAAAATTA
GACACAACATTGAAGATGGAAGCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAA
TTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACCTGTCCACACAATCTGCCCTTT
CGAAAGATCCCAACGAAAAGAGAGACCACATGGTCCTTCTTGAGTTTGTAACAGCTGCTG
GGATTACACATGGCATGGATGAACTATACAAAGGGATCCTGGCTAGCCAGTCGACCTGCA
GGCATGCAAGCTTGCGGCCGATCCAAATGGAAGACGCCAAAAACATAAAGAAAGGCCCGG
CGCCATTCTATCCGCTGGAAGATGGAACCGCTGGAGAGCAACTGCATAAGGCTATGAAGA
GATACGCCCTGGTTCCTGGAACAATTGCTTTTACAGATGCACATATCGAGGTGAACATCA
CGTACGCGGAATACTTCGAAATGTCCGTTCGGTTGGCAGAAGCTATGAAACGATATGGGC
TGAATACAAATCACAGAATCGTCGTATGCAGTGAAAACTCTCTTCAATTCTTTATGCCGG
TGTTGGGCGCGTTATTTATCGGAGTTGCAGTTGCGCCCGCGAACGACATTTATAATGAAC
GTGAATTGCTCAACAGTATGAACATTTCGCAGCCTACCGTAGTGTTTGTTTCCAAAAAGG
GGTTGCAAAAAATTTTGAACGTGCAAAAAAAATTACCAATAATCCAGAAAATTATTATCA
TGGATTCTAAAACGGATTACCAGGGATTTCAGTCGATGTACACGTTCGTCACATCTCATC
TACCTCCCGGTTTTAATGAATACGATTTTGTACCAGAGTCCTTTGATCGTGACAAAACAA
TTGCACTGATAATGAATTCCTCTGGATCTACTGGGTTACCTAAGGGTGTGGCCCTTCCGC
ATAGAACTGCCTGCGTCAGATTCTCGCATGCCAGAGATCCTATTTTTGGCAATCAAATCA
TTCCGGATACTGCGATTTTAAGTGTTGTTCCATTCCATCACGGTTTTGGAATGTTTACTA
CACTCGGATATTTGATATGTGGATTTCGAGTCGTCTTAATGTATAGATTTGAAGAAGAGC
TGTTTTTACGATCCCTTCAGGATTACAAAATTCAAAGTGCGTTGCTAGTACCAACCCTAT
TTTCATTCTTCGCCAAAAGCACTCTGATTGACAAATACGATTTATCTAATTTACACGAAA
TTGCTTCTGGGGGCGCACCTCTTTCGAAAGAAGTCGGGGAAGCGGTTGCAAAACGCTTCC
ATCTTCCAGGGATACGACAAGGATATGGGCTCACTGAGACTACATCAGCTATTCTGATTA
CACCCGAGGGGGATGATAAACCGGGCGCGGTCGGTAAAGTTGTTCCATTTTTTGAAGCGA
AGGTTGTGGATCTGGATACCGGGAAAACGCTGGGCGTTAATCAGAGAGGCGAATTATGTG
TCAGAGGACCTATGATTATGTCCGGTTATGTAAACAATCCGGAAGCGACCAACGCCTTGA
TTGACAAGGATGGATGGCTACATTCTGGAGACATAGCTTACTGGGACGAAGACGAACACT
TCTTCATAGTTGACCGCTTGAAGTCTTTAATTAAATACAAAGGATATCAGGTGGCCCCCG
CTGAATTGGAATCGATATTGTTACAACACCCCAACATCTTCGACGCGGGCGTGGCAGGTC
TTCCCGACGATGACGCCGGTGAACTTCCCGCCGCCGTTGTTGTTTTGGAGCACGGAAAGA
CGATGACGGAAAAAGAGATCGTGGATTACGTCGCCAGTCAAGTAACAACCGCGAAAAAGT
TGCGCGGAGGAGTTGTGTTTGTGGACGAAGTACCGAAAGGTCTTACCGGAAAACTCGACG
CAAGAAAAATCAGAGAGATCCTCATAAAGGCCAAGAAGGGCGGAAAGATCGCCGTGTAAT
TCTAGAAGATCCCGTTTTTCTTACTTATATATTTATACCAATTGATTGTATTTATAACTG
TAAAAATGTGTATGTTGTGTGCATATTTTTTTTTGTGCATGCACATGCATGTAAATAGCT
AAAATTATGAACATTTTATTTTTTGTTCAGAAAAAAAAAACTTTACACACATAAAATGGC
TAGTATGAATAGCCATATTTTATATAAATTAAATCCTATGAATTTATGACCATATTAAAA
ATTTAGATATTTATGGAACATAATATGTTTGAAACAATAAGACAAAATTATTATTATTAT
TATTATTTTTACTGTTATAATTATGTTGTCTCTTCAATGATTCATAAATAGTTGGACTTG
ATTTTTAAAATGTTTATAATATGATTAGCATAGTTAAATAAAAAAAGTTGAAAAATTAAA
AAAAAACATATAAACACAAATGATGTTTTTTCCTTCAACCCGGGCCTTCAATTTCGGATC
CACTAGAAGTAAAAGGGGTAAGACAGCAATTGTTAAAATTAATGTTCATCCAAATTCTTT
AAAGATTATGGGATGTGATTTTGTCGGAGCATATTCTTCTTATTTTATATTTAGTAAAAA
ATGGGATCAAATAACTCCAAATTATGTTTGTGAAATTAATGTAGAAGATGATTCTATTAT
TGGATTGGCTTGTCCTTATAATACAAAAATATATCCTTCTGATTGTTTTGAAAGTGTTAT
AAAAAATAATAAAGTTTACAAAAGAGATACCTTAATTGAATATAAAAATACATTTTTTTA
TCAAAAAAATGGAAAACCTACATTATCTTTTATACAAATTAAAAAAGTTTATTCTGACCA
TCTTACTTGTAAGTGCTTTGAAAATAATAATGGAAATTACAAAGAAGTTACTATCAAATT
AATTTATAAATCATATATATTAGGAACACCAAAAATGACTTTAAATAAACCATTTATGAA
ATATAAAAGTGTTAATTTTTTGGATTATCTGATTGGGAAGAAATTCTAATTTTTATAGTA
TCCCCAGCCCCTTTTGATTCAATATAGTTTTAGCTTGCCGATTTCTTTGGGTTCGTGTTT
TGCGATTTCTTTGGGTTCGTGTTTTGCGATTTCTTTGGGTTCGTGTTTTGCGATTTGTTT
GGGTTCGTGTTTTGCGATTTGTTTGGGTTCGTGTTTTGTGATTTCTTTGCGATTTTTGTG
TGTTTAGTGATTTTTGTGTGTGTCGATTGTGCGTTTTTTATTATATTATTGTTATCATAT
TTGATTTACCTTTTGATATTAGGACATATATATATGCATATATTTTTTTGTATTTCCTGA
TTTGTTTGTGATCATAATTTTCGATAAAATGAGTTTTTATTATTGATGCTTTAAACATTC
CAGTTGGTTCCAGATCTCTTAGTACTGCTGAGTGTCAATGACCAACCTAAGGGCGAATTC
TGCAGATATCCATCACACTGGCGGCCGCTCGAGCATGCATCTAGAGGGCCCAATTCGCCC
TATAGTGAGTCGTATTACAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAAC
CCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAAT
AGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGG
ACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCG
CTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCA
CGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTA
GTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGC
CATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTG
GACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTAT
AAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTA
ACGCGAATTTTAACAAAATTCAGGGCGCAAGGGCTGCTAAAGGAAGCGGAACACGTAGAA
AGCCAGTCCGCAGAAACGGTGCTGACCCCGGATGAATGTCAGCTACTGGGCTATCTGGAC
AAGGGAAAACGCAAGCGCAAAGAGAAAGCAGGTAGCTTGCAGTGGGCTTACATGGCGATA
GCTAGACTGGGCGGTTTTATGGACAGCAAGCGAACCGGAATTGCCAGCTGGGGCGCCCTC
TGGTAAGGTTGGGAAGCCCTGCAAAGTAAACTGGATGGCTTTCTTGCCGCCAAGGATCTG
ATGGCGCAGGGGATCAAGATCTGATCAAGAGACAGGATGAGGATCGTTTCGCATGATTGA
ACAAGATGGATTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGA
CTGGGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCAGGG
GCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTGCAGGACGA
GGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCTCGACGT
TGTCACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGATCTCCT
GTCATCCCACCTTGCTCCTGCCGAGAAAGTATCCATCATGGCTGATGCAATGCGGCGGCT
GCATACGCTTGATCCGGCTACCTGCCCATTCGACCACCAAGCGAAACATCGCATCGAGCG
AGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTGGACGAAGAGCATCA
GGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGCGCATGCCCGACGGCGAGGA
TCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATATCATGGTGGAAAATGGCCGCTT
TTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGACCGCTATCAGGACATAGCGTT
GGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCTGACCGCTTCCTCGTGCT
TTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGTT
CTTCTGAATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCC
TTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAA
GATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGT
AAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTT
CTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGC
ATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACG
GATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCG
GCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAAC
ATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCA
AACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTA
ACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGAT
AAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAA
TCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAG
CCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAAT
AGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTT
TACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTG
AAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGA
GCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTA
ATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAA
GAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACT
GTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACA
TACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTT
ACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGG
GGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAG
CGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTA
AGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTAT
CTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCG
TCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCC
TTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAAC
CGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGC
GAGTCAGTGAGCGAGGAAGCGGAAG
Restriction sites to linearize plasmid
Selectable marker used to select the mutant parasitehdhfr/yfcu
Promoter of the selectable markereef1a
Selection (positive) procedureNo
Selection (negative) procedure5-fluorocytosine (5-FC)
Additional remarks genetic modification
Additional remarks selection procedure
Other details transgene
Promoter
Gene Model of Parasite PBANKA_1133300
Gene Model P. falciparum ortholog PF3D7_1357100
Gene productelongation factor 1-alpha
Gene product: Alternative nameeef1a
Primer information details of the primers used for amplification of the promoter sequence  Click to view information
Primer information details of the primers used for amplification of the promoter sequence  Click to hide information
Sequence Primer 1
Additional information primer 1
Sequence Primer 2
Additional information primer 2
3'-UTR
Gene Model of Parasite PBANKA_0719300
Gene productbifunctional dihydrofolate reductase-thymidylate synthase, putative
Gene product: Alternative namedhfr/ts
Primer information details of the primers used for amplification the 3'-UTR sequences  Click to view information
Primer information details of the primers used for amplification the 3'-UTR sequences  Click to hide information
Sequence Primer 1
Additional information primer 1
Sequence Primer 2
Additional information primer 2
Insertion/Replacement locus
Replacement / InsertionReplacement locus
Gene Model of Parasite PY03857
Gene productPfs230 paralog-related
Gene product: Alternative nameP230p; 230p
Primer information details of the primers used for amplification of the target sequences  Click to view information
Primer information details of the primers used for amplification of the target sequences  Click to hide information
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