Summary

RMgm-662
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_0100600; Gene model (P.falciparum): Not available; Gene product: schizont membrane associated cytoadherence protein (Schizont Membrane Associated Cytoadherence protein; SMAC.)
Transgene
Transgene not Plasmodium: A fusion of GFP (gfp-mu3) and Luciferase Firefly (LucIAV)
Promoter: Gene model: PBANKA_0915000; Gene model (P.falciparum): PF3D7_1133400; Gene product: apical membrane antigen 1
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 Asexual bloodstage;
Last modified: 4 October 2017, 12:36
  *RMgm-662
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) : 22184632
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 1037m1f1mocl1 (RMgm-32)
Other information parent lineP. berghei ANKA 1037m1f1mocl1 (1037cl1; RMgm-32) is a reference ANKA mutant line which expresses GFP-luciferase under control of a schizont-specific promoter. This reference line does not contain a drug-selectable marker (PubMed: PMID: 20019192).
The mutant parasite was generated by
Name PI/ResearcherJ. Fonager; B. Franke-Fayard; C.J. Janse
Name Group/DepartmentLeiden Malaria Research Group
Name InstituteLeiden University Medical Center
CityLeiden
CountryThe Netherlands
Name of the mutant parasite
RMgm numberRMgm-662
Principal name1242cl5m2cl1
Alternative nameΔsmac3(-sm)
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageAsexual blood stages show a reduced growth rate in mice (see 'Additional remarks phenotype')
Gametocyte/GameteNot tested
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of Schizont Membrane Associated Cytoadherence protein (SMAC). The mutant does not contain a drug selectable marker which has been removed by the negative selection procedure using the negative selectable marker yfcu (yeast cytosine deaminase and uridyl-phosphoribosyltransferase) and the drug 5-fluorocytosine (5-FC)( (Braks et al., 2006, Nucleic Acids Res 34, e39).

Protein (function)
The protein was detected in proteome analyses of mebranes of infected red blood cells. It is a predicted 19 kDa protein, it contains a signal sequence, a PEXEL motif (RFLVE) and a transmembrane domain. Orthologs are only found in the murine Plasmodium species. It is exported into the cytoplasm of the host erythrocyte (see below)

Phenotype
Asexual blood stages show a reduced growth rate in mice (see 'Additional information')

Additional information
Phenotype analyses indicate that that the reduced growth rate of blood stages in mice is not the result of a prolonged cell cycle (schizogony) or a reduced number of merozoites produced per schizont.
Evidence is provided that parasites lacking SMAC-expression have a reduced capacity to adhere to the host receptor CD36 resulting in a strongly reduced CD36-mediated sequestration phenotype. The non-sequestering parasites have a reduced growth and evidence is provided that in addition to avoiding spleen removal other factors related to CD36-mediated sequestration are beneficial for parasite growth.

The mutant parasites cause cerebral complications in C57Bl/6 mice (with around one day delay)(unpublished observations).

Analyses of a mutant expressing a mCherry-tagged version of SMAC (SMAC::mCherry; RMgm-663) showed export of SMAC into the cytoplasm of the host erythrocyte. Staining of  live SMAC::mCherry and wild type infected erythrocytes with anti-mCherry or anti-SMAC antibodies did not provide evidence for exposure of SMAC on the surface of infected erythrocytes.

Other mutants
RMgm-661: An independent mutant lacking expression of SMAC
RMgm-663: A mutant expressing a mCherry-tagged version of SMAC


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0100600
Gene Model P. falciparum ortholog Not available
Gene productschizont membrane associated cytoadherence protein
Gene product: Alternative nameSchizont Membrane Associated Cytoadherence protein; SMAC.
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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ATCGAATTCCTAAGATCATTATGGGATCATCTGCAGCGATATTTTTTCTTGTCGAAGATT
TAGGAATTAGAGCTGTGCTATTTTTAATATTTTCTGCTGCTGTACTCGCTTATTTCACTT
CCATATAAATTAAAAGAACAAACACATATTTATATATTAAAATAATAATAATATTCTTTT
ACTATTATTAATGAGATAAATATGAAATTATGCATATTTAACTAATAAAAAATACACAAT
GTGCATTATAAATATAGCAATCGTTACAATATATTTACATTATTAATATATTTTCATTAT
TTGTGAATGTTAATAATTAAGTGTTATGGATATGGTAGATTATTTTATACTTGAGACAGT
GTGCTTATGCATCTATGTATTAATTGTTATTATTAAAATTTATAATAAAGTGCAATAAGC
TATATTTTGAAATATTTCCTATATAAATATATACATTTTATGAGTTTATAATTCTATATA
CATTCTTGTATTAATATTAAATGAATTTTTTTTTGTGAAAAAACATAAATAATATATAGT
GATAATACACAAATGATTGAAACGTGAGGTGAAACATCTTTTCATTTATGAATACAACTC
TTTAAGTATGTTTTTTTTTCAGTGAAAACGCAAACTTGTATAGAATATATTTTTTTTATT
AGTTCTCCATCATTATTTATATAGTTTTATCTCCCCATAAAATATTAATAATACAACCCT
CTAGAGCGGCCGCCACCGCGGTGGAGCTCCAATTCGCCCTATAGTGAGTCGTATTACAAT
TCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAAT
CGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGAT
CGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGGACGCGCCCTGTAGCGGCGCA
TTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTA
GCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGT
CAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGAC
CCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTT
TTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGA
ACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCG
GCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATA
TTAACGCTTACAATTTAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTT
TATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGC
TTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTC
CCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAA
AAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCG
GTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAG
TTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCC
GCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTA
CGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTG
CGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACA
ACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATAC
CAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTAT
TAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGG
ATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATA
AATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTA
AGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAA
ATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAG
TTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGG
TGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACT
GAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCG
TAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATC
AAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATA
CTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTA
CATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTC
TTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGG
GGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTAC
AGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGG
TAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGT
ATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCT
CGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGG
CCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATA
ACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCA
GCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGC
GTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTG
AGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTA
TGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACA
GCTATGACCATGATTACGCCAAGCTCGAAATTAACCCTCACTAAAGGGAACAAAAGCTGG
TACCGTGTATATATAATCAAATACCAAGGATAATATCTATATATTTATATTTTCTAAAAG
AGACATTTTAGATATAATATAAACTTTATTGTAATTATTACATTTCACGTAAAAATATAT
AATTTTTTTCGAGGAAGTTTTAAATAAAATATTTATATGATATATTTATTAATTTTTTAT
TTAATTAGTAATTTGTTACATTTATATATTGAACCCATTTTAAAAATATACATATTAAAT
AGCTTTAGAATTATGACAATTAATTTTTATAATAATAAAAAGTTCCATTTATTTGTTTTC
TGAATCGGTAAAGAGTATTAATAAAAACCCAATTGTAAACGGTCCCAATATATTTTTTAA
AGAATGTGTTACTATGTATTTAAATTACCCAAATTAAACAAGTAAAAAATATCACAATAT
ATATTATTTTCCCCGAGATGCTATAAATAATTATTTTTGATAGATTGACAAATATATATT
GTTCTTTTTTGACGTAATGCATTATATATCATATATGTAGCACTTTTTAGTTTTAAAATA
ATAATTTAATATAATACACAGTGCATGCACCATTTTACATGAATCTGATATAATACTTAT
TTATCATTTTATTTATTAGCCATTTATTATAAACAAACTATATATTATTATCTTTTACAT
ACATCCTTAACTCTTTTAACAACCAGTACACATAATATTTGCAACATTGTGAATATAAAA
TGAAATATTTAACGCACATATTTTTTTTTATTATACTTTTTCAATTTATAGTTGATAAAT
ACGATAACAATGGAAAATCATTGGACAAGAAGCTTGGGCCCCCGCGGTGGCGGCCGCTCT
AGCTTTGATCCCGTTTTTCTTACTTATATATTTATACCAATTGATTGTATTTATAACTGT
AAAAATGTGTATGTTGTGTGCATATTTTTTTTTGTGCATGCACATGCATGTAAATAGCTA
AAATTATGAACATTTTATTTTTTGTTCAGAAAAAAAAAACTTTACACACATAAAATGGCT
AGTATGAATAGCCATATTTTATATAAATTAAATCCTATGAATTTATGACCATATTAAAAA
TTTAGATATTTATGGAACATAATATGTTTGAAACAATAAGACAAAATTATTATTATTATT
ATTATTTTTACTGTTATAATTATGTTGTCTCTTCAATGATTCATAAATAGTTGGACTTGA
TTTTTAAAATGTTTATAATATGATTAGCATAGTTAAATAAAAAAAGTTGAAAAATTAAAA
AAAAACATATAAACACAAATGATGTTTTTTCCTTCAATTTCGATTGATAATTCCTGCAGC
CCAGCTTAATTCTTTTCGAGCTCTTTATGCTTAAGTTTACAATTTAATATTCATACTTTA
AGTATTTTTTGTAGTATCCTAGATATTGTGCTTTAAATGCTCACCCCTCAAAGCACCAGT
AATATTTTCATCCACTGAAATACCATTAAATTTTCAAAAAAATACTATGCATATAATGTT
ATACATATAAACATAAAACGCCATGTAAATCAAAAAATATATAAAAATATGTATAAAAAT
AAATATGCACTAAATATAAGCTAATTATGCATAAAAATTAAAGTGCCCTTTATTAACTAG
AACTAGTCGTAATTATTTATATTTCTATGTTATAAAAAAATCCTCATATAATAATATAAT
TAATATATGTAATGTTTTTTTTATTTTATAATTTTAATATAAAATAATATGTAAATTAAT
TCAAAAAATAAATATAATTGTTGTGAAACAAAAAACGTAATTTTTTCATTTGCCTTCAAA
ATTTAAATTTATTTTAATATTTCCTAAAATATATATACTTTGTGTATAAATATATAAAAA
TATATATTTGCTTATAAATAAATAAAAAATTTTATAAAACATAGGGGGATCCATGGTTGG
TTCGCTAAACTGCATCGTCGCTGTGTCCCAGAACATGGGCATCGGCAAGAACGGGGACCT
GCCCTGGCCACCGCTCAGGAACGAATTTAGATATTTCCAGAGAATGACCACAACCTCTTC
AGTAGAAGGTAAACAGAATCTGGTGATTATGGGTAAGAAGACCTGGTTCTCCATTCCTGA
GAAGAATCGACCTTTAAAGGGTAGAATTAATTTAGTTCTCAGCAGAGAACTCAAGGAACC
TCCACAAGGAGCTCATTTTCTTTCCAGAAGTCTAGATGATGCCTTAAAACTTACTGAACA
ACCAGAATTAGCAAATAAAGTAGACATGGTCTGGATAGTTGGTGGCAGTTCTGTTTATAA
GGAAGCCATGAATCACCCAGGCCATCTTAAACTATTTGTGACAAGGATCATGCAAGACTT
TGAAAGTGACACGTTTTTTCCAGAAATTGATTTGGAGAAATATAAACTTCTGCCAGAATA
CCCAGGTGTTCTCTCTGATGTCCAGGAGGAGAAAGGCATTAAGTACAAATTTGAAGTATA
TGAGAAGAATGATGCTAGCGGAGGAGGTGGATCTGGTGGAGGTGGAAGTGCTAGCGTGAC
AGGGGGAATGGCAAGCAAGTGGGATCAGAAGGGTATGGACATTGCCTATGAGGAGGCGGC
CTTAGGTTACAAAGAGGGTGGTGTTCCTATTGGCGGATGTCTTATCAATAACAAAGACGG
AAGTGTTCTCGGTCGTGGTCACAACATGAGATTTCAAAAGGGATCCGCCACACTACATGG
TGAGATCTCCACTTTGGAAAACTGTGGGAGATTAGAGGGCAAAGTGTACAAAGATACCAC
TTTGTATACGACGCTGTCTCCATGCGACATGTGTACAGGTGCCATCATCATGTATGGTAT
TCCACGCTGTGTTGTCGGTGAGAACGTTAATTTCAAAAGTAAGGGCGAGAAATATTTACA
AACTAGAGGTCACGAGGTTGTTGTTGTTGACGATGAGAGGTGTAAAAAGATCATGAAACA
ATTTATCGATGAAAGACCTCAGGATTGGTTTGAAGATATTGGTGAGGCTTCGGAACCATT
TAAGAACGTCTACTTGCTACCTCAAACAAACCAATTGCTGGGTTTGTACACCATCATCAG
AAATAAGAATACAACTAGACCTGATTTCATTTTCTACTCCGATAGAATCATCAGATTGTT
GGTTGAAGAAGGTTTGAACCATCTACCTGTGCAAAAGCAAATTGTGGAAACTGACACCAA
CGAAAACTTCGAAGGTGTCTCATTCATGGGTAAAATCTGTGGTGTTTCCATTGTCAGAGC
TGGTGAATCGATGGAGCAAGGATTAAGAGACTGTTGTAGGTCTGTGCGTATCGGTAAAAT
TTTAATTCAAAGGGACGAGGAGACTGCTTTACCAAAGTTATTCTACGAAAAATTACCAGA
GGATATATCTGAAAGGTATGTCTTCCTATTAGACCCAATGCTGGCCACCGGTGGTAGTGC
TATCATGGCTACAGAAGTCTTGATTAAGAGAGGTGTTAAGCCAGAGAGAATTTACTTCTT
AAACCTAATCTGTAGTAAGGAAGGGATTGAAAAATACCATGCCGCCTTCCCAGAGGTCAG
AATTGTTACTGGTGCCCTCGACAGAGGTCTAGATGAAAACAAGTATCTAGTTCCAGGGTT
GGGTGACTTTGGTGACAGATACTACTGTGTTTAACTCGATCCCGTTTTTCTTACTTATAT
ATTTATACCAATTGATTGTATTTATAACTGTAAAAATGTGTATGTTGTGTGCATATTTTT
TTTTGTGCATGCACATGCATGTAAATAGCTAAAATTATGAACATTTTATTTTTTGTTCAG
AAAAAAAAAACTTTACACACATAAAATGGCTAGTATGAATAGCCATATTTTATATAAATT
AAATCCTATGAATTTATGACCATATTAAAAATTTAGATATTTATGGAACATAATATGTTT
GAAACAATAAGACAAAATTATTATTATTATTATTATTTTTACTGTTATAATTATGTTGTC
TCTTCAATGATTCATAAATAGTTGGACTTGATTTTTAAAATGTTTATAATATGATTAGCA
TAGTTAAATAAAAAAAGTTGAAAAATTAAAAAAAAACATATAAACACAAATGATGTTTTT
TCCTTCAATTTCGGGTACGTACCCTCGAGGCTAGCGAT
Restriction sites to linearize plasmid Asp718, XbaI, ScaI
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 markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedure5-fluorocytosine (5-FC)
Additional remarks genetic modificationTwo general gene targeting DNA constructs pL0001 or pL0037 were used to generate constructs to disrupt the genes selected from the proteome analyses. These constructs are aimed at targeted gene disruption by double cross-over homologous recombination. Sequences of the open reading frame (ORF) and UTR regions of the selected genes were retrieved from PlasmoDB and GeneDB (www.plasmodb.org; www.genedb.org). To replace the ORF of the target genes with the selection cassette containing the pyrimethamine-resistant dhfr/ts of Toxoplasma gondii, the 5′ and 3′ flanking regions of the ORF were cloned up- and down-stream of the selection cassette of pL0001 or pL0037.
To obtain a drug-selectable marker free Δsmac parasites line, the positive T. gondii dhfr/ts selectable marker of pL1351 was replaced with the drug selection cassette of pL0037 (HindIII/EcoRI fragment) that contains a fusion of the positive selectable marker, hdhfr, and the negative selectable marker yfcu (Braks et al., 2006). This construct, pL1386, was used to transfect parasites of reference line wt-GFP-Lucschiz and positive selection with pyrimethamine followed by cloning of parasites resulted in the generation of line 1242cl5 (Δsmac3). To select for parasites which had removed the selectable marker cassette we applied negative selection with 5-fluorocytosine (5-FC)
Additional remarks selection procedureThe mutant does not contain a drug selectable marker which has been removed by the negative selection procedure using the negative selectable marker yfcu (yeast cytosine deaminase and uridyl-phosphoribosyltransferase) and the drug 5-fluorocytosine (5-FC)( (Braks et al., 2006, Nucleic Acids Res 34, e39).
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 1GCCGGTACCGTGTATATATAATCAAATACCAAGG
Additional information primer 13423: 5'forward (Asp718)
Sequence Primer 2GCCAAGCTTCTTGTCCAATGATTTTCCATTG
Additional information primer 23424: 5'reverse (HindIII)
Sequence Primer 3GCCGAATTCCTAAGATCATTATGGGATCATCTG
Additional information primer 33425: 3'forward (EcoRI)
Sequence Primer 4GCCTCTAGAGGGTTGTATTATTAATATTTTATGGG
Additional information primer 43426: 3'reverse (XbaI)
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 nameA fusion of GFP (gfp-mu3) and Luciferase Firefly (LucIAV)
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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1 aattcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc
121 gatcgccctt cccaacagtt gcgcagcctg aatggcgaat ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatatggt gcactctcag tacaatctgc
241 tctgatgccg catagttaag ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc
361 atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact
481 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt
601 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca
721 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc
841 cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta
961 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt
1081 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct
1201 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct
1321 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc
1441 tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg
1561 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
1681 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta
1801 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
1921 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agcattgaga aagcgccacg
2041 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
2161 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
2281 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa
2401 gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtgagttag
2521 ctcactcatt aggcacccca ggctttacac tttatgcttc cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg accatgatta cgccaagctt
2641 ccgcgggtat atggtaaaga acctactaac acaataaaat atttaaataa tgtatttcct ataaataaat ttacagattt attttttaat acaaaagata tagatatacc agaaataaat
2761 gatcagttta aaggttttaa attctttatg acatcattta taaatcatgg atcatatcca ctaacaatag aatgtggtgt aacaaatggt ggaactagtt ataaaagagc aattatttta
2881 ttgcatgttc gaactgattt aaaagataga ccagtttcat tttgtgattt tcgaaaagga gaattatata attatttgaa tgcttatact gaaggggatg tatgcataat aatttccaaa
3001 tcaaatacaa gttttggttt tagatgccca gtaaatacaa aaaaaatgcc aaaaaattgt tttacgcaag tatatgaaaa agggtatcta aatgacgcca ataaaattaa tactaaaaat
3121 gttattaact attcatttga aaatccagaa tatgcgctag ctggttytaa ttatacatta acaaaatcgt atcaatttga atgtcattgt gtagataaag aaacagaaca aattgtaaaa
3241 acggttttag tcaaatatgt aaatgaagat gaaatatatg attataatga ttttccaatg gtgaatcaca aacctattat tgcacatcca aataaaacac atcaagcttg catgcctgca
3361 ggaattcgat atcgaattcc catggaacca aaataacata ttaatgaatt ttcttttatt gtgaaaatgt tcttatttgt tttaaaaaat caaagtaaat ttaattcctt taattgtgct
3481 aaatattgtg tttgtataac tataatgaca tttcctatgc ttttttaata ttatatataa ttttttgtgt tttttcaaaa aaaacgtgta tttgttttta tttgagtgta caatttgcat
3601 agtgagttta aagtgtttga aaattattta atttataatt ttcataaaac gtttaatcat ttatcaaaat tgcggttgca atatagacag aaaatattaa cttaattttc attgttttgt
3721 aaaacatttt cattattttt tttaacaaaa ttaaagtaaa tgagatggta aattatttta attataacca tttgtttaat tttttttatt ttattactat tatttttttt cttttgtgct
3841 gcgcaattaa aaagagaatt aagggtgcac caatatattt aagcaatctt ttgcgacaca caagaaaaaa tataacatca acatatttaa aataatggaa attttttaat aatttattct
3961 atatatattt aatatatgta aaaaaactaa tattgtaata taattaaata aaacttgaaa ttgatataat atgtatatat attgcatgtt tagctttaaa taatatttaa gtattgattt
4081 tctttacaca caaaaacatt tgccttttta tgtgaggatt attgtttctt tatttatatt gctatgaatt tattcttagt ataaatttat tttttttcgg ctttgaaaat aattttatat
4201 atataaaaag aaaaaatatg gaaaataaaa ttcataaatc atatttgaca catttcaatt tttttttctt atttcacagc tagccatata ataatatata tgtgtctatg ggttcaaata
4321 gcttaatttc aatgtgcgtg attttttttt ataaaaaaac taattttatg tttggttatg catgcaactg ttataaaata aaaattgtaa aatttgtttt tttgtacaca tatttgtact
4441 ttttttaatg cacatgaaaa aagaaaggaa aaaaaagggg gaaaaaaaag gaaaaaaaaa gggaaaaaag ggaaaaaacg tacatctacg cattgttatt tagcaataaa atagagaaca
4561 aatctataaa tatataatgt gtacaacaaa ttaatttaat ttatattttt actgtaattt cataataatt ttcattttta cattttaatg tttttttaat tggaatataa ttcaaaatga
4681 ttttgacaca ttttacttat agaagaaggc tatataatta tataagtgtg tatatataag tattgtatgg taattgtttt ataaattaaa tttttaaaaa acattattct attttataat
4801 ttgtaaatta aaaatattaa taaaataaaa cgatataaaa ggatccatga gtaaaggaga agaacttttc actggagttg tcccaattct tgttgaatta gatggtgatg ttaatgggca
4921 caaattttct gtcagtggag agggtgaagg tgatgcaaca tacggaaaac ttacccttaa atttatttgc actactggaa aactacctgt tccatggcca acacttgtca ctactttcgg
5041 ttatggtgtt caatgctttg cgagataccc agatcatatg aaacagcatg actttttcaa gagtgccatg cccgaaggtt atgtacagga aagaactata tttttcaaag atgacgggaa
5161 ctacaagaca cgtgctgaag tcaagtttga aggtgatacc cttgttaata gaatcgagtt aaaaggtatt gattttaaag aagatggaaa cattcttgga cacaaattgg aatacaacta
5281 taactcacac aatgtataca tcatggcaga caaacaaaag aatggaatca aagttaactt caaaattaga cacaacattg aagatggaag cgttcaacta gcagaccatt atcaacaaaa
5401 tactccaatt ggcgatggcc ctgtcctttt accagacaac cattacctgt ccacacaatc tgccctttcg aaagatccca acgaaaagag agaccacatg gtccttcttg agtttgtaac
5521 agctgctggg attacacatg gcatggatga actatacaaa gggatcctgg ctagccagtc gacctgcagg catgcaagct tgcggccgat ccaaatggaa gacgccaaaa acataaagaa
5641 aggcccggcg ccattctatc cgctggaaga tggaaccgct ggagagcaac tgcataaggc tatgaagaga tacgccctgg ttcctggaac aattgctttt acagatgcac atatcgaggt
5761 gaacatcacg tacgcggaat acttcgaaat gtccgttcgg ttggcagaag ctatgaaacg atatgggctg aatacaaatc acagaatcgt cgtatgcagt gaaaactctc ttcaattctt
5881 tatgccggtg ttgggcgcgt tatttatcgg agttgcagtt gcgcccgcga acgacattta taatgaacgt gaattgctca acagtatgaa catttcgcag cctaccgtag tgtttgtttc
6001 caaaaagggg ttgcaaaaaa ttttgaacgt gcaaaaaaaa ttaccaataa tccagaaaat tattatcatg gattctaaaa cggattacca gggatttcag tcgatgtaca cgttcgtcac
6121 atctcatcta cctcccggtt ttaatgaata cgattttgta ccagagtcct ttgatcgtga caaaacaatt gcactgataa tgaattcctc tggatctact gggttaccta agggtgtggc
6241 ccttccgcat agaactgcct gcgtcagatt ctcgcatgcc agagatccta tttttggcaa tcaaatcatt ccggatactg cgattttaag tgttgttcca ttccatcacg gttttggaat
6361 gtttactaca ctcggatatt tgatatgtgg atttcgagtc gtcttaatgt atagatttga agaagagctg tttttacgat cccttcagga ttacaaaatt caaagtgcgt tgctagtacc
6481 aaccctattt tcattcttcg ccaaaagcac tctgattgac aaatacgatt tatctaattt acacgaaatt gcttctgggg gcgcacctct ttcgaaagaa gtcggggaag cggttgcaaa
6601 acgcttccat cttccaggga tacgacaagg atatgggctc actgagacta catcagctat tctgattaca cccgaggggg atgataaacc gggcgcggtc ggtaaagttg ttccattttt
6721 tgaagcgaag gttgtggatc tggataccgg gaaaacgctg ggcgttaatc agagaggcga attatgtgtc agaggaccta tgattatgtc cggttatgta aacaatccgg aagcgaccaa
6841 cgccttgatt gacaaggatg gatggctaca ttctggagac atagcttact gggacgaaga cgaacacttc ttcatagttg accgcttgaa gtctttaatt aaatacaaag gatatcaggt
6961 ggcccccgct gaattggaat cgatattgtt acaacacccc aacatcttcg acgcgggcgt ggcaggtctt cccgacgatg acgccggtga acttcccgcc gccgttgttg ttttggagca
7081 cggaaagacg atgacggaaa aagagatcgt ggattacgtc gccagtcaag taacaaccgc gaaaaagttg cgcggaggag ttgtgtttgt ggacgaagta ccgaaaggtc ttaccggaaa
7201 actcgacgca agaaaaatca gagagatcct cataaaggcc aagaagggcg gaaagatcgc cgtgtaattc tagaagatcc cgtttttctt acttatatat ttataccaat tgattgtatt
7321 tataactgta aaaatgtgta tgttgtgtgc atattttttt ttgtgcatgc acatgcatgt aaatagctaa aattatgaac attttatttt ttgttcagaa aaaaaaaact ttacacacat
7441 aaaatggcta gtatgaatag ccatatttta tataaattaa atcctatgaa tttatgacca tattaaaaat ttagatattt atggaacata atatgtttga aacaataaga caaaattatt
7561 attattatta ttatttttac tgttataatt atgttgtctc ttcaatgatt cataaatagt tggacttgat ttttaaaatg tttataatat gattagcata gttaaataaa aaaagttgaa
7681 aaattaaaaa aaaacatata aacacaaatg atgttttttc cttcaatttc gggtaccgag ctcgaattct cttgagcccg ttaatgaaat agatacaatt cattcatgtt atatacatct
7801 agaacataat ctgaatatgg ttcaagttaa atgtccaaaa attataaaaa gtgatgatat ttttgatggt aataccataa tagacaccaa ggtaacatca cgaagtagtc aacaaaataa
7921 tttttattta gaaaatacag atgttgaacc agaagaaata gagaaatata aaaatataga atacatacca gaaaacgatg aagtaatgca tctagacaaa aaagaaaagc tagatgatat
8041 attaccaggt gttatcatat tagataaaaa taaaatgttc aaagaaaaag gacatttcac ttttgttact ccattaattg tagaaaaggt attaatatta aaaatatatt gtgataatac
8161 taaaacaata attaataata tgaaagggaa aaaaggtatt acagtaataa ggatttctca aaatacaaca aaaaataaat tttatggatg tgacttttca ggtaattcta aaaaaacatt
8281 ttactattcc aatgtttatg atttagaaaa aaaaaatgag ttttgtgaaa tagaattaaa agaaaatata gtagttagct taaattgtcc aactggtaaa attaatccaa aaaattgttt
8401 tagaaatgta tatataaaaa gtaatatgaa tgaacaaaca accgaaaata tagaaaatat atttaacgaa ataaaagtta tagatgcaga ttattttata aataattcat caaccttttt
8521 gatgatttcc aaaattacaa aaaaagagtt tgatttttat tgtacatgtg aagattataa aaccaaaaat ataggaacaa tatatattaa aaattatgaa tatctagatt caaaacctaa
8641 atataaaaat aaacaaattt cctatataga tgtagttcca tacccgcggg gaaagggcg
Restriction sites to linearize plasmid KspI (SacII)
Selectable marker used to select the mutant parasitegfp (FACS)
Promoter of the selectable markerama-1
Selection (positive) procedureFACS (flowsorting)
Selection (negative) procedureNo
Additional remarks genetic modificationThe GFP-Luciferase gene (1 copy) has been inserted into the 230p locus (PB000214.00.0) by double cross-over integration.
Additional remarks selection procedureThis reporter mutant expressing GFP-Luciferase does not contain a drug-selectable marker. This mutant has been selected by FACS sorting after transfection based on GFP fluorescence.
Other details transgene
Promoter
Gene Model of Parasite PBANKA_0915000
Gene Model P. falciparum ortholog PF3D7_1133400
Gene productapical membrane antigen 1
Gene product: Alternative name
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 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