RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-42
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_1002200; Gene model (P.falciparum): PF3D7_0404500; Gene product: 6-cysteine protein (P36p; Pb36p; P52)
DisruptedGene model (rodent): PBANKA_1002100; Gene model (P.falciparum): PF3D7_0404400; Gene product: 6-cysteine protein (P36)
Transgene
Transgene not Plasmodium: GFP (gfp-mu3)
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: PBANKA_0306000; Gene product: 6-cysteine protein (230p)
Phenotype Liver stage;
Last modified: 27 February 2012, 14:40
  *RMgm-42
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene disruption, Gene disruption, Introduction of a transgene
Reference (PubMed-PMID number) Reference 1 (PMID number) : 22342550
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone P. berghei ANKA 507cl1 (RMgm-7)
Other information parent lineP.berghei ANKA 507cl1 (RMgm-7) is a reference ANKA mutant line which expresses GFP under control of a constitutive promoter (PubMed: PMID: 16242190).
The mutant parasite was generated by
Name PI/ResearcherT. Annoura; C.J. Janse; S.M. Khan
Name Group/DepartmentLeiden Malaria Research Group
Name InstituteLeiden University Medical Center
CityLeiden
CountryThe Netherlands
Name of the mutant parasite
RMgm numberRMgm-42
Principal name795cl1
Alternative namep36p-;p36-
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 stageGliding motility, hepatocyte traversal and hepatocyte invasion in vitro (HepG2) of mutant sporozoites is similar to wild type sporozoites. Liver stage development is strongly impaired and parasites do not develop into the schizont stage and most invaded parasites cannot be detected anymore at 24 hours after infection. Inside the hepatocytes the formation of a parasitophourous vacuole is not detected at 15 and 24h after infections as shown by staining with antibodies against the parasithophorous vacuole membrane proteins.
Infection of mice (C57BL/6) by bite of infected mosquitoes did not result in blood stage infection. Infection of BALB/c mice by intravenous injection of 50.000 sporozoites did not result in blood stage infection. Only intravenous inoculation of 50.000 sporozoites in C57BL/6 mice resulted in blood stage infection in a low percentage (10-20%) of mice.
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of the P52 (P36p) and P36 protein and expresses the reporter protein GFP. Both genes have been disrupted  using a single DNA construct (double knock-out mutant). The genes have been disrupted in a reporter mutant (507cl1; RMgm-7) that expresses GFP constitutively throughout the life cycle

Protein (function)
P52 (P36p) and P36 are members of a small family of proteins, the 6-cysteine (cys) family of (surface) proteins (Thompson J. et al., Mol. Biochem. Parasitol. (2001)118, 147-54). The proteins are characterised by domains of roughly 120 amino acids in size that contain six positionally conserved cysteines (6-cys). Although some species of Plasmodium (may) contain unique members of the 6-cys family, ten members have been identified that are conserved both in structure as well as in genome organisation throughout the genus. Some of the conserved 6-cys proteins are encoded by genes that form paralogous gene-pairs which are closely linked in the genome separated by less then 2 kb of intergenic region. Most members have a GPI anchor and are predicted membrane surface proteins whereas others appear to be secreted and most members are expressed in a discrete stage-specific manner in gametocytes, sporozoites or merozoites.

Phenotype
P36 and p52  (p36p) are paralogous genes, located in tandem next to each other. Both genes have been disrupted  using a single DNA construct (double knock-out mutant).
Gliding motility, hepatocyte traversal and hepatocyte invasion in vitro (HepG2) of mutant sporozoites is similar to wild type sporozoites. Liver stage development is strongly impaired and parasites do not develop into the schizont stage and most invaded parasites cannot be detected anymore at 24 hours after infection. Inside the hepatocytes the formation of a parasitophourous vacuole is not detected at 15 and 24h after infections as shown by staining with antibodies against the parasithophorous vacuole membrane proteins.
The early arrest of Δp52+p36 parasites after hepatocyte invasion is similar to the phenotype reported for P. berghei mutants lacking expression of only P52 and P. yoelii mutants lacking both P52 and P36 (see below).
Infection of mice (C57BL/6) by bite of infected mosquitoes did not result in blood stage infection. Infection of BALB/c mice by intravenous injection of 50.000 sporozoites did not result in blood stage infection. Only intravenous inoculation of 50.000 sporozoites in C57BL/6 mice resulted in blood stage infection in a low percentage (10-20%) of mice.

Additional information

Other mutants

An independent P. berghei mutant lacking the expression of both P52 (P36p) and P36 (RMgm-597)
P. berghei mutants lacking expression of P52 (P36p) (RMgm-40, RMgm-41, RMgm-44).
P. berghei mutants lacking expression of P36 (RMgm-45; RMgm-353).
A P. yoelii mutant (RMgm-43) lacking the expression of both P52 (P36p) and P36.

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  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1002200
Gene Model P. falciparum ortholog PF3D7_0404500
Gene product6-cysteine protein
Gene product: Alternative nameP36p; Pb36p; P52
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct usedPlasmid 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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"GGGCCCCCCCTCGAGGTCGACGGTATCGATGAATAATAGTAAATGATGAAGCGTCGGAGA
ATTTTCATGTACTACTGTTTCTGCTTCTTATTGAAATATGTAGCCTTTAGCAATGTGACA
AATCCTAATACGACCTTAGGACACTTTGAAATTTGTAAAATAAATATATATTCAGGCGAT
GCCGAAGAATGTGTTTTAGAAAATGAATTTGGGAAAATGTTTTTATTTATTTGTGATATT
GATTATAATGAGATGTCAAAAAATATAGTGCTTCCATCAGAATGTGCTAAAAAAACATAT
ATAGATCATGTAAACCCAAATGGAACATCGCCAGAAGTTAATACTTATGATATATTTCCA
GATTTGATCGGAGCAAATGAATCCAAATTTCGTGATAAATTTTATTTTTATGGTACCCCA
TATTCATCTAAAGACATTGATTTTATATGTTTATGCTTTTCTGAAGTAAAACCAGATATA
AAACATATAATAAAAATGAGTTTTAAAAAAATGACAAAAAAAATAAAAGGATGTGATTTT
GGAGATAATGTGCCAACGAAAAAGGATTTAACAAATGGGAAAGCACTATATGAAAATTCT
AGTTGTCATATATATGCATATCCAGGGGACGTAATTAAGCTTGCATGCCTGCAGGTCAAC
AATAAATAATAAATAAATATTGTGGAAATAAAATAACATATAATTATTTTTAATACATTG
ATTTCCCTTTTATTTTTTTAAATTTCATTGATATAAAAATATATAATAATAACATATATG
ATTTCAAATTAATCTTTTCAAAAATGGTGTTTATTTTTGTATGTTTGTGTATGAATTAAT
CACATAACACATCTATTAAATTGAGTTGGTAATATAGACACAAATAAATATATATATTTT
TATAGCTTAAAAGTGTGTTATGAATATTTTAAGCATATTTTCTTTTTCTTTGGATTGTGT
AAAATGAACTCATATAATGCGTTTTTTTGTTTTTGTTATTTTGTCATTTTGTTATTTTGC
TATTTTATGGATTAATTTTTGTTTATAAAATGGGAAATAATTTTAACATATTTAAATAAA
TGGAGAAAAAATATAAAATAATTATAAAAAAAAGTTAATACACATTTTTTCCTGTTATAG
ACCTTATATTTATTTATCCATATATATATATATATATATATATATATATATATACATACC
AAGTGAATTAAGAGGAAAGCTAATTTATTATTCAGAATAATATATGAACTATATATAATT
TTTATTATTTTGGTGTATATTAATCTGTCTATATGCATACATGCAATAATTTATCGACTT
ATATATCAAATAACATAAAATAGAAGTGTTTTAAATTATGGATATATGCTCAATATTCAT
TTTTTTTAATAAGTTAGCTATATTTAAATTATACATTTTATATATGGTCTCTTTTTTTTT
TAAATATTATTTAAGTGATCATGAAAATATAAATAATTTTTTTTTATTTAATATCCTTTT
GCTTGCATGTGGTAAATGGAAATTTGGATGTGTTTTGAARGTTCGGATATAGTTGTATGG
ACATATAATATATTTTGTGAAAAATTGGTTTTATGTTTATACTTATGCCAATACTTTTTG
AGTAAAACAAAGCAAGTGCTTATAAATAATTAAAGCCAATTTTATAATATATATTTTTTT
ATTTAATTTGAATTTAGTAGTATAATTTTTTATGGTAAGTGCTCAAAGAGAGTTGCTTAT
AAAGTATGGTTTGTTTCTTTTTCGCCATTTTGAATTACACATTAAAAATATATAGATACA
TATATTATAATATGAAATCATTAATAATTTAGGGAAATTCTACAAATTTAAAAACGAATA
AAATAATTGTTTTTCATCATGCCATAACACAATATTGATATATACATGTACAAACATTTT
TTTTATTTGGAAAATATAAATTATATAAAAAAAAATGTATAGTATACAAAATGAGCATAT
TCACACGGGGTGGACGTTCATTTTTTCATTTTTCCCCTGTTTTTTATGAGTATATGATAA
AATTTTATGAACATTTACACAAAATGAAAATGGATATATAGGAAAAATGGAGCGGTATTT
CATTTATCTTTGATTGTCATTTGGATATTATATTACCYTGGGTAGGCAATTAAAAATGTT
AAATAACAATTTAAGGAAATTATATTTTATATATTAAAATTAACACTGTATTATATGATT
CGCTTATAAAAGCCACTCTTTCCCCATGCAAAGCTGTTTAATATCAATTTTAACAAATTA
CACACATGTTAATATATTTATATATATAATTTATATATTTATAATTTATATATTTATATT
TTTATTATTTATATATTTATTATTTATTGTGTGTGTCAATTCGGGTAGGATATACCTCTT
TTTTATTGTTTAAAGCGATTTGTATTCTAAAATATAAAGRATTTGAAAAAGAGAAAGATA
GAATATGATCCCATCATATATAGCCCTATAATTTTTATTTAGCAGCGAATTAATTTTTCT
ATTAAGTTTATGTGTAATTAAAATAACGGAATATATATAATACAATAAAAAAGTGCATAA
ATTAAAATTTTTTCAATTAAATTTTTTTTTTTAAGGGGTTATATAATATTAAATATATAA
AATACGATTATATATTTTTGCTACAATTTTTTATATTAAGATATAAATAGTAAATAAATG
GTATTATATGGCATGTAATATATAAATTTTTTCCAATTTTTATTTTATATACACTTTTCC
TTTTTTTGTCATAAAACTTAAACAATTTACACATTCATTTTAAAAATTGACTATTTGTTT
CAACATTTTTTGAGTTTCCGTTTTATAATAGTATTTTCATTTGTATATTGCTTATATATA
TAAATACACACCTAAATGTTACAAAGGATCAATGCATAAACCGGTGTGTCTGGTCGTCGC
GATGACCCCCAAGAGGGGCATCGGCATCAACAACGGCCTCCCGTGGCCCCACTTGACCAC
AGATTTCAAACACTTTTCTCGTGTGACAAAAACGACGCCCGAAGAAGCCAGTCGCCTGAA
CGGGTGGCTTCCCAGGAAATTTGCAAAGACGGGCGACTCTGGACTTCCCTCTCCATCAGT
CGGCAAGAGATTCAACGCCGTTGTCATGGGACGGAAAACCTGGGAAAGCATGCCTCGAAA
GTTTAGACCCCTCGTGGACAGATTGAACATCGTCGTTTCCTCTTCCCTCAAAGAAGAAGA
CATTGCGGCGGAGAAGCCTCAAGCTGAAGGCCAGCAGCGCGTCCGAGTCTGTGCTTCACT
CCCAGCAGCTCTCAGCCTTCTGGAGGAAGAGTACAAGGATTCTGTCGACCAGATTTTTGT
CGTGGGAGGAGCGGGACTGTACGAGGCAGCGCTGTCTCTGGGCGTTGCCTCTCACCTGTA
CATCACGCGTGTAGCCCGCGAGTTTCCGTGCGACGTTTTCTTCCCTGCGTTCCCCGGAGA
TGACATTCTTTCAAACAAATCAACTGCTGCGCAGGCTGCAGCTCCTGCCGAGTCTGTGTT
CGTTCCCTTTTGTCCGGAGCTCGGAAGAGAGAAGGACAATGAAGCGACGTATCGACCCAT
CTTCATTTCCAAGACCTTCTCAGACAACGGGGTTCCCTACGACTTTGTGGTTCTCGAGAA
GAGAAGGAAGACTGACGACGCAGCCACTGCGGAACCGAGCAACGCAATGAGCTCCTTGAC
GTCCACGAGGGAGACAACTCCCGTGCACGGGTTGCAGGCTCCTTCTTCGGCCGCAGCCAT
TGCCCCGGTGTTGGCGTGGATGGACGAAGAAGACCGGAAAAAACGCGAGCAAAAGGAACT
GATTCGGGCCGTTCCGCATGTTCACTTTAGAGGCCATGAAGAGTTCCAGTACCTTGATCT
CATTGCCGACATTATTAACAATGGAAGGACAATGGATGACCGAACGGGCGTTGGTGTCAT
CTCCAAATTCGGCTGCACTATGCGCTACTCGCTGGATCAGGCCTTTCCACTTCTCACCAC
AAAGCGTGTGTTCTGGAAAGGGGTCCTCGAAGAGTTGCTGTGGTTCATTCGCGGCGACAC
GAACGCAAACCATCTTTCTGAGAAGGGCGTGAAGATCTGGGACAAGAATGTGACACGCGA
GTTCCTCGATTCGCGCAATCTCCCCCACCGAGAGGTCGGAGACATCGGCCCGGGCTACGG
CTTCCAGTGGAGACACTTCGGCGCGGCATACAAAGACATGCACACAGACTACACAGGGCA
GGGCGTCGACCAGCTGAAGAATGTGATCCAGATGCTGAGAACGAATCCAACAGATCGTCG
CATGCTCATGACTGCCTGGAATCCTGCAGCGCTGGACGAAATGGCGCTGCCGCCTTGTCA
CTTGTTGTGCCAGTTCTACGTGAACGACCAGAAGGAGCTGTCGTGCATCATGTATCAGCG
GTCGTGCGATGTCGGCCTCGGCGTCCCCTTCAACATCGCTTCCTATTCGCTTTTGACGCT
CATGGTTGCACACGTCTGCAACCTAAAACCTAAGGAGTTCATTCACTTCATGGGGAACAC
GCATGTCTACACGAACCATGTCGAGGCTTTAAAAGAGCAGCTGCGGAGAGAACCGAGACC
GTTCCCCATTGTGAACATCCTCAACAAGGAACGCATCAAGGAAATCGACGATTTCACCGC
CGAGGATTTTGAGGTCGTGGGCTACGTCCCGCACGGACGAATCCAGATGGAGATGGCTGT
CTAGCGGAAATACAGAAGCTAGCTTTGATCCCGTTTTTCTTACTTATATATTTATACCAA
TTGATTGTATTTATAACTGTAAAAATGTGTATGTTGTGTGCATATTTTTTTTTGTGCATG
CACATGCATGTAAATAGCTAAAATTATGAACATTTTATTTTTTGTTCAGAAAAAAAAAAC
TTTACACACATAAAATGGCTAGTATGAATAGCCATATTTTATATAAATTAAATCCTATGA
ATTTATGACCATATTAAAAATTTAGATATTTATGGAACATAATATGTTTGAAACAATAAG
ACAAAATTATTATTATTATTATTATTTTTACTGTTATAATTATGTTGTCTCTTCAATGAT
TCATAAATAGTTGGACTTGATTTTTAAAATGTTTATAATATGATTAGCATAGTTAAATAA
AAAAAGTTGAAAAATTAAAAAAAAACATATAAACACAAATGATGTTTTTTCCTTCAATTT
CGATATATCCCGGATATCCGATTTAGCATCTCATCATGGTAATCAAAATTCTAAAATGTT
CTTAACGGCATACTTATTGATACCTAATGAGATAGAAAGGGACATTTTAATATATTGTAC
TTGTTCATATAATGGGAACAAAGGACTAGCAATATATCACATTTTTTCCACAAAAGAAGA
TTAATATGTGATGTGTGTAGAAGAGTGAGGGAGTTGAAATATTGAATCCAGCATTTCTAA
CGCAAGTCATACAATTTTGTTTATGTTTTAATGACACATAATAGTGCAATTTGTTTAACT
TTTTTAAATATGTTATATTCGCACTTTTTATATTTTTTATAAGTTCAATCAATATGCGTT
CTTATTAGTTTTCAATTAAAGTACTTAATTTGTATTTTTCCTTTTTTTTAACATCGTTTT
TAAATTAAAAGTAAAAATCATAATAAAAAATTATAAATCGGTTGATTATTAAAAATAAGT
ATGCATGTGCATAATTTATAAACAATAGCATTTTAATACATATATACCACAAATTTTAGT
TTGCTACTGTTTTCTATTTGAAGTGTCTGTTTAATTATTCCATATGATAGAATCTTAATT
GTACTATAGCAAATATGTGTATGAACAAATATACTTGTATTCATTTGTAGAATAATAGTA
ATGTAGACATTATATTTTTTTATTAGTATGAAACAAAACAATTAACAAAATTTACATTAT
TAAATGAGTTAAATGTAATAAATTACAAGCACAAATTAATTATGTAACTCGTTTGGGGTC
AAATTATGTTTTAAAACTTGAAAAAATTGCAATGTAAAGGTTATTCGTGCTGATCTTATG
TTTATTTTTTTTTTTTACAATTCGTTCTAGAGAAATTTCAAGGTGTGATTTTCGCAGTAC
CAGGATCCACTAGTTCTAGAGCGGCCGCCACCGCGGTGGAGCTCCAATTCGCCCTATAGT
GAGTCGTATTACGCGCGCTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCT
GGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGC
GAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGGAC
GCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCT
ACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACG
TTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGT
GCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCA
TCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGA
CTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAA
GGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAAC
GCGAATTTTAACAAAATATTAACGCTTACAATTTAGGTGGCACTTTTCGGGGAAATGTGC
GCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGAC
AATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATT
TCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAG
AAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCG
AACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAA
TGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGC
AAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAG
TCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAA
CCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGC
TAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGG
AGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAA
CAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAA
TAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTG
GCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAG
CACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGG
CAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATT
GGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTT
AATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAAC
GTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAG
ATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGG
TGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCA
GAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGA
ACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCA
GTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGC
AGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACA
CCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAA
AGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTC
CAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGC
GTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGG
CCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTAT
CCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCA
GCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCA
AACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCG
ACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCAC
CCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAAC
AATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCTCGAAATTAACCCTCACT
AAAGGGAACAAAAGCTGGTACC
"
Restriction sites to linearize plasmid
Partial or complete disruption of the genePartial
Additional remarks partial/complete disruption The 5'UTR and 590pb of Pb36p's 1415bp coding region remain intact
Selectable marker used to select the mutant parasitetgdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationIn this mutant line the genes encoding P36 (PB000892.00.0) and P36p (PB000891.00.0) have been disrupted (double knock-out mutant) using a single DNA construct.
The genes have been disrupted in a reporter mutant that expresses GFP constitutively throughout the life cycle (507cl1; RMgm-7).
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 1CGATCGATGAATAATAGTAAATGATGAAACGTCG
Additional information primer 1L903 (ClaI); Pb36p 5'target sense
Sequence Primer 2CCCAAGCTTAATTACGTCCCCTGGATATGC
Additional information primer 2L904 (HindIII); Pb36p 5'target a-sense
Sequence Primer 3GGATATCCGATTTAGCATCTCATCATGG
Additional information primer 3L864 (EcoRV); Pbs36F 3'ko
Sequence Primer 4CGGGGTACCTGGTACTGCGAAAATCACACC
Additional information primer 4L865 (KpnI); Pbs36R 3'ko
Sequence Primer 5
Additional information primer 5
Sequence Primer 6
Additional information primer 6

  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1002100
Gene Model P. falciparum ortholog PF3D7_0404400
Gene product6-cysteine protein
Gene product: Alternative nameP36
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct usedPlasmid 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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"GGGCCCCCCCTCGAGGTCGACGGTATCGATGAATAATAGTAAATGATGAAGCGTCGGAGA
ATTTTCATGTACTACTGTTTCTGCTTCTTATTGAAATATGTAGCCTTTAGCAATGTGACA
AATCCTAATACGACCTTAGGACACTTTGAAATTTGTAAAATAAATATATATTCAGGCGAT
GCCGAAGAATGTGTTTTAGAAAATGAATTTGGGAAAATGTTTTTATTTATTTGTGATATT
GATTATAATGAGATGTCAAAAAATATAGTGCTTCCATCAGAATGTGCTAAAAAAACATAT
ATAGATCATGTAAACCCAAATGGAACATCGCCAGAAGTTAATACTTATGATATATTTCCA
GATTTGATCGGAGCAAATGAATCCAAATTTCGTGATAAATTTTATTTTTATGGTACCCCA
TATTCATCTAAAGACATTGATTTTATATGTTTATGCTTTTCTGAAGTAAAACCAGATATA
AAACATATAATAAAAATGAGTTTTAAAAAAATGACAAAAAAAATAAAAGGATGTGATTTT
GGAGATAATGTGCCAACGAAAAAGGATTTAACAAATGGGAAAGCACTATATGAAAATTCT
AGTTGTCATATATATGCATATCCAGGGGACGTAATTAAGCTTGCATGCCTGCAGGTCAAC
AATAAATAATAAATAAATATTGTGGAAATAAAATAACATATAATTATTTTTAATACATTG
ATTTCCCTTTTATTTTTTTAAATTTCATTGATATAAAAATATATAATAATAACATATATG
ATTTCAAATTAATCTTTTCAAAAATGGTGTTTATTTTTGTATGTTTGTGTATGAATTAAT
CACATAACACATCTATTAAATTGAGTTGGTAATATAGACACAAATAAATATATATATTTT
TATAGCTTAAAAGTGTGTTATGAATATTTTAAGCATATTTTCTTTTTCTTTGGATTGTGT
AAAATGAACTCATATAATGCGTTTTTTTGTTTTTGTTATTTTGTCATTTTGTTATTTTGC
TATTTTATGGATTAATTTTTGTTTATAAAATGGGAAATAATTTTAACATATTTAAATAAA
TGGAGAAAAAATATAAAATAATTATAAAAAAAAGTTAATACACATTTTTTCCTGTTATAG
ACCTTATATTTATTTATCCATATATATATATATATATATATATATATATATATACATACC
AAGTGAATTAAGAGGAAAGCTAATTTATTATTCAGAATAATATATGAACTATATATAATT
TTTATTATTTTGGTGTATATTAATCTGTCTATATGCATACATGCAATAATTTATCGACTT
ATATATCAAATAACATAAAATAGAAGTGTTTTAAATTATGGATATATGCTCAATATTCAT
TTTTTTTAATAAGTTAGCTATATTTAAATTATACATTTTATATATGGTCTCTTTTTTTTT
TAAATATTATTTAAGTGATCATGAAAATATAAATAATTTTTTTTTATTTAATATCCTTTT
GCTTGCATGTGGTAAATGGAAATTTGGATGTGTTTTGAARGTTCGGATATAGTTGTATGG
ACATATAATATATTTTGTGAAAAATTGGTTTTATGTTTATACTTATGCCAATACTTTTTG
AGTAAAACAAAGCAAGTGCTTATAAATAATTAAAGCCAATTTTATAATATATATTTTTTT
ATTTAATTTGAATTTAGTAGTATAATTTTTTATGGTAAGTGCTCAAAGAGAGTTGCTTAT
AAAGTATGGTTTGTTTCTTTTTCGCCATTTTGAATTACACATTAAAAATATATAGATACA
TATATTATAATATGAAATCATTAATAATTTAGGGAAATTCTACAAATTTAAAAACGAATA
AAATAATTGTTTTTCATCATGCCATAACACAATATTGATATATACATGTACAAACATTTT
TTTTATTTGGAAAATATAAATTATATAAAAAAAAATGTATAGTATACAAAATGAGCATAT
TCACACGGGGTGGACGTTCATTTTTTCATTTTTCCCCTGTTTTTTATGAGTATATGATAA
AATTTTATGAACATTTACACAAAATGAAAATGGATATATAGGAAAAATGGAGCGGTATTT
CATTTATCTTTGATTGTCATTTGGATATTATATTACCYTGGGTAGGCAATTAAAAATGTT
AAATAACAATTTAAGGAAATTATATTTTATATATTAAAATTAACACTGTATTATATGATT
CGCTTATAAAAGCCACTCTTTCCCCATGCAAAGCTGTTTAATATCAATTTTAACAAATTA
CACACATGTTAATATATTTATATATATAATTTATATATTTATAATTTATATATTTATATT
TTTATTATTTATATATTTATTATTTATTGTGTGTGTCAATTCGGGTAGGATATACCTCTT
TTTTATTGTTTAAAGCGATTTGTATTCTAAAATATAAAGRATTTGAAAAAGAGAAAGATA
GAATATGATCCCATCATATATAGCCCTATAATTTTTATTTAGCAGCGAATTAATTTTTCT
ATTAAGTTTATGTGTAATTAAAATAACGGAATATATATAATACAATAAAAAAGTGCATAA
ATTAAAATTTTTTCAATTAAATTTTTTTTTTTAAGGGGTTATATAATATTAAATATATAA
AATACGATTATATATTTTTGCTACAATTTTTTATATTAAGATATAAATAGTAAATAAATG
GTATTATATGGCATGTAATATATAAATTTTTTCCAATTTTTATTTTATATACACTTTTCC
TTTTTTTGTCATAAAACTTAAACAATTTACACATTCATTTTAAAAATTGACTATTTGTTT
CAACATTTTTTGAGTTTCCGTTTTATAATAGTATTTTCATTTGTATATTGCTTATATATA
TAAATACACACCTAAATGTTACAAAGGATCAATGCATAAACCGGTGTGTCTGGTCGTCGC
GATGACCCCCAAGAGGGGCATCGGCATCAACAACGGCCTCCCGTGGCCCCACTTGACCAC
AGATTTCAAACACTTTTCTCGTGTGACAAAAACGACGCCCGAAGAAGCCAGTCGCCTGAA
CGGGTGGCTTCCCAGGAAATTTGCAAAGACGGGCGACTCTGGACTTCCCTCTCCATCAGT
CGGCAAGAGATTCAACGCCGTTGTCATGGGACGGAAAACCTGGGAAAGCATGCCTCGAAA
GTTTAGACCCCTCGTGGACAGATTGAACATCGTCGTTTCCTCTTCCCTCAAAGAAGAAGA
CATTGCGGCGGAGAAGCCTCAAGCTGAAGGCCAGCAGCGCGTCCGAGTCTGTGCTTCACT
CCCAGCAGCTCTCAGCCTTCTGGAGGAAGAGTACAAGGATTCTGTCGACCAGATTTTTGT
CGTGGGAGGAGCGGGACTGTACGAGGCAGCGCTGTCTCTGGGCGTTGCCTCTCACCTGTA
CATCACGCGTGTAGCCCGCGAGTTTCCGTGCGACGTTTTCTTCCCTGCGTTCCCCGGAGA
TGACATTCTTTCAAACAAATCAACTGCTGCGCAGGCTGCAGCTCCTGCCGAGTCTGTGTT
CGTTCCCTTTTGTCCGGAGCTCGGAAGAGAGAAGGACAATGAAGCGACGTATCGACCCAT
CTTCATTTCCAAGACCTTCTCAGACAACGGGGTTCCCTACGACTTTGTGGTTCTCGAGAA
GAGAAGGAAGACTGACGACGCAGCCACTGCGGAACCGAGCAACGCAATGAGCTCCTTGAC
GTCCACGAGGGAGACAACTCCCGTGCACGGGTTGCAGGCTCCTTCTTCGGCCGCAGCCAT
TGCCCCGGTGTTGGCGTGGATGGACGAAGAAGACCGGAAAAAACGCGAGCAAAAGGAACT
GATTCGGGCCGTTCCGCATGTTCACTTTAGAGGCCATGAAGAGTTCCAGTACCTTGATCT
CATTGCCGACATTATTAACAATGGAAGGACAATGGATGACCGAACGGGCGTTGGTGTCAT
CTCCAAATTCGGCTGCACTATGCGCTACTCGCTGGATCAGGCCTTTCCACTTCTCACCAC
AAAGCGTGTGTTCTGGAAAGGGGTCCTCGAAGAGTTGCTGTGGTTCATTCGCGGCGACAC
GAACGCAAACCATCTTTCTGAGAAGGGCGTGAAGATCTGGGACAAGAATGTGACACGCGA
GTTCCTCGATTCGCGCAATCTCCCCCACCGAGAGGTCGGAGACATCGGCCCGGGCTACGG
CTTCCAGTGGAGACACTTCGGCGCGGCATACAAAGACATGCACACAGACTACACAGGGCA
GGGCGTCGACCAGCTGAAGAATGTGATCCAGATGCTGAGAACGAATCCAACAGATCGTCG
CATGCTCATGACTGCCTGGAATCCTGCAGCGCTGGACGAAATGGCGCTGCCGCCTTGTCA
CTTGTTGTGCCAGTTCTACGTGAACGACCAGAAGGAGCTGTCGTGCATCATGTATCAGCG
GTCGTGCGATGTCGGCCTCGGCGTCCCCTTCAACATCGCTTCCTATTCGCTTTTGACGCT
CATGGTTGCACACGTCTGCAACCTAAAACCTAAGGAGTTCATTCACTTCATGGGGAACAC
GCATGTCTACACGAACCATGTCGAGGCTTTAAAAGAGCAGCTGCGGAGAGAACCGAGACC
GTTCCCCATTGTGAACATCCTCAACAAGGAACGCATCAAGGAAATCGACGATTTCACCGC
CGAGGATTTTGAGGTCGTGGGCTACGTCCCGCACGGACGAATCCAGATGGAGATGGCTGT
CTAGCGGAAATACAGAAGCTAGCTTTGATCCCGTTTTTCTTACTTATATATTTATACCAA
TTGATTGTATTTATAACTGTAAAAATGTGTATGTTGTGTGCATATTTTTTTTTGTGCATG
CACATGCATGTAAATAGCTAAAATTATGAACATTTTATTTTTTGTTCAGAAAAAAAAAAC
TTTACACACATAAAATGGCTAGTATGAATAGCCATATTTTATATAAATTAAATCCTATGA
ATTTATGACCATATTAAAAATTTAGATATTTATGGAACATAATATGTTTGAAACAATAAG
ACAAAATTATTATTATTATTATTATTTTTACTGTTATAATTATGTTGTCTCTTCAATGAT
TCATAAATAGTTGGACTTGATTTTTAAAATGTTTATAATATGATTAGCATAGTTAAATAA
AAAAAGTTGAAAAATTAAAAAAAAACATATAAACACAAATGATGTTTTTTCCTTCAATTT
CGATATATCCCGGATATCCGATTTAGCATCTCATCATGGTAATCAAAATTCTAAAATGTT
CTTAACGGCATACTTATTGATACCTAATGAGATAGAAAGGGACATTTTAATATATTGTAC
TTGTTCATATAATGGGAACAAAGGACTAGCAATATATCACATTTTTTCCACAAAAGAAGA
TTAATATGTGATGTGTGTAGAAGAGTGAGGGAGTTGAAATATTGAATCCAGCATTTCTAA
CGCAAGTCATACAATTTTGTTTATGTTTTAATGACACATAATAGTGCAATTTGTTTAACT
TTTTTAAATATGTTATATTCGCACTTTTTATATTTTTTATAAGTTCAATCAATATGCGTT
CTTATTAGTTTTCAATTAAAGTACTTAATTTGTATTTTTCCTTTTTTTTAACATCGTTTT
TAAATTAAAAGTAAAAATCATAATAAAAAATTATAAATCGGTTGATTATTAAAAATAAGT
ATGCATGTGCATAATTTATAAACAATAGCATTTTAATACATATATACCACAAATTTTAGT
TTGCTACTGTTTTCTATTTGAAGTGTCTGTTTAATTATTCCATATGATAGAATCTTAATT
GTACTATAGCAAATATGTGTATGAACAAATATACTTGTATTCATTTGTAGAATAATAGTA
ATGTAGACATTATATTTTTTTATTAGTATGAAACAAAACAATTAACAAAATTTACATTAT
TAAATGAGTTAAATGTAATAAATTACAAGCACAAATTAATTATGTAACTCGTTTGGGGTC
AAATTATGTTTTAAAACTTGAAAAAATTGCAATGTAAAGGTTATTCGTGCTGATCTTATG
TTTATTTTTTTTTTTTACAATTCGTTCTAGAGAAATTTCAAGGTGTGATTTTCGCAGTAC
CAGGATCCACTAGTTCTAGAGCGGCCGCCACCGCGGTGGAGCTCCAATTCGCCCTATAGT
GAGTCGTATTACGCGCGCTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCT
GGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGC
GAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGGAC
GCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCT
ACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACG
TTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGT
GCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCA
TCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGA
CTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAA
GGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAAC
GCGAATTTTAACAAAATATTAACGCTTACAATTTAGGTGGCACTTTTCGGGGAAATGTGC
GCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGAC
AATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATT
TCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAG
AAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCG
AACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAA
TGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGC
AAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAG
TCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAA
CCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGC
TAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGG
AGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAA
CAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAA
TAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTG
GCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAG
CACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGG
CAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATT
GGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTT
AATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAAC
GTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAG
ATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGG
TGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCA
GAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGA
ACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCA
GTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGC
AGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACA
CCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAA
AGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTC
CAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGC
GTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGG
CCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTAT
CCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCA
GCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCA
AACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCG
ACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCAC
CCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAAC
AATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCTCGAAATTAACCCTCACT
AAAGGGAACAAAAGCTGGTACC
"
Restriction sites to linearize plasmid
Partial or complete disruption of the geneComplete
Additional remarks partial/complete disruption
Selectable marker used to select the mutant parasitetgdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationIn this mutant line the genes encoding P36 (PB000892.00.0) and P36p (PB000891.00.0) have been disrupted (double knock-out mutant) using a single DNA construct.
The genes have been disrupted in a reporter mutant that expresses GFP constitutively throughout the life cycle (507cl1; RMgm-7).
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 1CGATCGATGAATAATAGTAAATGATGAAACGTCG
Additional information primer 1L903 (ClaI); Pb36p 5'target sense
Sequence Primer 2CCCAAGCTTAATTACGTCCCCTGGATATGC
Additional information primer 2L904 (HindIII); Pb36p 5'target a-sense
Sequence Primer 3GGATATCCGATTTAGCATCTCATCATGG
Additional information primer 3L864 (EcoRV); Pbs36F 3'ko
Sequence Primer 4CGGGGTACCTGGTACTGCGAAAATCACACC
Additional information primer 4L865 (KpnI); Pbs36R 3'ko
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 (gfp-mu3)
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/constructPlasmid 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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1 aattcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt
51 tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta
101 atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg
151 aatggcgaat ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg
201 tatttcacac cgcatatggt gcactctcag tacaatctgc tctgatgccg
251 catagttaag ccagccccga cacccgccaa cacccgctga cgcgccctga
301 cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc
351 cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga
401 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata
451 ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg
501 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
551 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt
601 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt
651 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
701 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac
751 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat
801 gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg
851 ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg
901 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt
951 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
1001 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg
1051 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct
1101 gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa
1151 tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct
1201 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc
1251 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
1301 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat
1351 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac
1401 tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta
1451 agcattggta actgtcagac caagtttact catatatact ttagattgat
1501 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
1551 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
1601 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
1651 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
1701 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
1751 tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta
1801 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
1851 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
1901 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
1951 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
2001 actgagatac ctacagcgtg agcattgaga aagcgccacg cttcccgaag
2051 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
2101 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
2151 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
2201 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
2251 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
2301 tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc
2351 gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa
2401 gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta
2451 atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca
2501 acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac
2551 tttatgcttc cggctcgtat gttgtgtgga attgtgagcg gataacaatt
2601 tcacacagga aacagctatg accatgatta cgccaagctt ccgcgggtat
2651 atggtaaaga acctactaac acaataaaat atttaaataa tgtatttcct
2701 ataaataaat ttacagattt attttttaat acaaaagata tagatatacc
2751 agaaataaat gatcagttta aaggttttaa attctttatg acatcattta
2801 taaatcatgg atcatatcca ctaacaatag aatgtggtgt aacaaatggt
2851 ggaactagtt ataaaagagc aattatttta ttgcatgttc gaactgattt
2901 aaaagataga ccagtttcat tttgtgattt tcgaaaagga gaattatata
2951 attatttgaa tgcttatact gaaggggatg tatgcataat aatttccaaa
3001 tcaaatacaa gttttggttt tagatgccca gtaaatacaa aaaaaatgcc
3051 aaaaaattgt tttacgcaag tatatgaaaa agggtatcta aatgacgcca
3101 ataaaattaa tactaaaaat gttattaact attcatttga aaatccagaa
3151 tatgcgctag ctggttytaa ttatacatta acaaaatcgt atcaatttga
3201 atgtcattgt gtagataaag aaacagaaca aattgtaaaa acggttttag
3251 tcaaatatgt aaatgaagat gaaatatatg attataatga ttttccaatg
3301 gtgaatcaca aacctattat tgcacatcca aataaaacac atcaagcttg
3351 catgcctgca gcccagctta attcttttcg agctctttat gcttaagttt
3401 acaatttaat attcatactt taagtatttt ttgtagtatc ctagatattg
3451 tgctttaaat gctcacccct caaagcacca gtaatatttt catccactga
3501 aataccatta aattttcaaa aaaatactat gcatataatg ttatacatat
3551 aaacataaaa cgccatgtaa atcaaaaaat atataaaaat atgtataaaa
3601 ataaatatgc actaaatata agctaattat gcataaaaat taaagtgccc
3651 tttattaact agtcgtaatt atttatattt ctatgttata aaaaaatcct
3701 catataataa tataattaat atatgtaatg ttttttttat tttataattt
3751 taatataaaa taatatgtaa attaattcaa aaaataaata taattgttgt
3801 gaaacaaaaa acgtaatttt ttcatttgcc ttcaaaattt aaatttattt
3851 taatatttcc taaaatatat atactttgtg tataaatata taaaaatata
3901 tatttgctta taaataaata aaaaatttta taaaacatag ggggatccat
3951 gagtaaagga gaagaacttt tcactggagt tgtcccaatt cttgttgaat
4001 tagatggtga tgttaatggg cacaaatttt ctgtcagtgg agagggtgaa
4051 ggtgatgcaa catacggaaa acttaccctt aaatttattt gcactactgg
4101 aaaactacct gttccatggc caacacttgt cactactttc ggttatggtg
4151 ttcaatgctt tgcgagatac ccagatcata tgaaacagca tgactttttc
4201 aagagtgcca tgcccgaagg ttatgtacag gaaagaacta tatttttcaa
4251 agatgacggg aactacaaga cacgtgctga agtcaagttt gaaggtgata
4301 cccttgttaa tagaatcgag ttaaaaggta ttgattttaa agaagatgga
4351 aacattcttg gacacaaatt ggaatacaac tataactcac acaatgtata
4401 catcatggca gacaaacaaa agaatggaat caaagttaac ttcaaaatta
4451 gacacaacat tgaagatgga agcgttcaac tagcagacca ttatcaacaa
4501 aatactccaa ttggcgatgg ccctgtcctt ttaccagaca accattacct
4551 gtccacacaa tctgcccttt cgaaagatcc caacgaaaag agagaccaca
4601 tggtccttct tgagtttgta acagctgctg ggattacaca tggcatggat
4651 gaactataca aataaatgga tcccgttttt cttacttata tatttatacc
4701 aattgattgt atttataact gtaaaaatgt gtatgttgtg tgcatatttt
4751 tttttgtgca tgcacatgca tgtaaatagc taaaattatg aacattttat
4801 tttttgttca gaaaaaaaaa actttacaca cataaaatgg ctagtatgaa
4851 tagccatatt ttatataaat taaatcctat gaatttatga ccatattaaa
4901 aatttagata tttatggaac ataatatgtt tgaaacaata agacaaaatt
4951 attattatta ttattatttt tactgttata attatgttgt ctcttcaatg
5001 attcataaat agttggactt gatttttaaa atgtttataa tatgattagc
5051 atagttaaat aaaaaaagtt gaaaaattaa aaaaaaacat ataaacacaa
5101 atgatgtttt ttccttcaat ttcgggtacc gagctcgaat tctcttgagc
5151 ccgttaatga aatagataca attcattcat gttatataca tctagaacat
5201 aatctgaata tggttcaagt taaatgtcca aaaattataa aaagtgatga
5251 tatttttgat ggtaatacca taatagacac caaggtaaca tcacgaagta
5301 gtcaacaaaa taatttttat ttagaaaata cagatgttga accagaagaa
5351 atagagaaat ataaaaatat agaatacata ccagaaaacg atgaagtaat
5401 gcatctagac aaaaaagaaa agctagatga tatattacca ggtgttatca
5451 tattagataa aaataaaatg ttcaaagaaa aaggacattt cacttttgtt
5501 actccattaa ttgtagaaaa ggtattaata ttaaaaatat attgtgataa
5551 tactaaaaca ataattaata atatgaaagg gaaaaaaggt attacagtaa
5601 taaggatttc tcaaaataca acaaaaaata aattttatgg atgtgacttt
5651 tcaggtaatt ctaaaaaaac attttactat tccaatgttt atgatttaga
5701 aaaaaaaaat gagttttgtg aaatagaatt aaaagaaaat atagtagtta
5751 gcttaaattg tccaactggt aaaattaatc caaaaaattg ttttagaaat
5801 gtatatataa aaagtaatat gaatgaacaa acaaccgaaa atatagaaaa
5851 tatatttaac gaaataaaag ttatagatgc agattatttt ataaataatt
5901 catcaacctt tttgatgatt tccaaaatta caaaaaaaga gtttgatttt
5951 tattgtacat gtgaagatta taaaaccaaa aatataggaa caatatatat
6001 taaaaattat gaatatctag attcaaaacc taaatataaa aataaacaaa
6051 tttcctatat agatgtagtt ccatacccgc ggggaaaggg cg"
Restriction sites to linearize plasmid KspI
Selectable marker used to select the mutant parasitegfp (FACS)
Promoter of the selectable markereef1a
Selection (positive) procedureFACS (flowsorting)
Selection (negative) procedureNo
Additional remarks genetic modificationThe GFP gene (1 copy) has been inserted into the 230p locus (PB000214.00.0) by double cross-over integration.
Additional remarks selection procedureThe gfp gene is introduced into the genome using a DNA construct without a drug-selectable marker. This reporter mutant (507cl1; RMgm-7) has been selected by FACS sorting after transfection based on GFP fluorescence.
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 PBANKA_0306000
Gene product6-cysteine protein
Gene product: Alternative name230p
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 15'-cccaagcttccgcgggtatatggtaaagaacctactaacac
Additional information primer 1primer L1345: 0.7kb 5'region of PB000214.00.0 (230p gene)
Sequence Primer 25'-cccaagcttgatgtgttttatttggatgtgc
Additional information primer 2primer L1346: 0.7kb 5'region of PB000214.00.0 (230p gene)
Sequence Primer 35'-ccggaattctcttgagcccgttaatg
Additional information primer 3primer L1347: 1kb 3'region of PB000214.00.0 (230p gene)
Sequence Primer 45'-tccccgcgggtatggaactacatctatatagg
Additional information primer 4primer L1348: 1kb 3'region of PB000214.00.0 (230p gene)