Summary

RMgm-129
Malaria parasiteP. berghei
Genotype
Transgene
Transgene not Plasmodium: GFP (gfp-mut3)
Promoter: Gene model: PBANKA_0616700; Gene model (P.falciparum): PF3D7_0719200; Gene product: NIMA related kinase 4
3'UTR: Gene model: PBANKA_0719300; Gene product: bifunctional dihydrofolate reductase-thymidylate synthase, putative (dhfr/ts)
Insertion locus: Gene model: Not available; Gene product: Not available (small subunit ribosomal rna gene (c- or d-type unit))
Phenotype Gametocyte/Gamete;
Last modified: 7 June 2009, 14:03
  *RMgm-129
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Introduction of a transgene
Reference (PubMed-PMID number) Reference 1 (PMID number) : 15935755
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 cl15cy1
Other information parent lineA reference wild type clone from the ANKA strain of P. berghei (PubMed: PMID: 17406255).
The mutant parasite was generated by
Name PI/ResearcherS.M. Khan, G.R. Mair, C.J. Janse, A.P. Waters
Name Group/DepartmentLeiden Malaria Research Group
Name InstituteLeiden University Medical Center (LUMC)
CityLeiden
CountryThe Netherlands
Name of the mutant parasite
RMgm numberRMgm-129
Principal name501
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationNo
Phenotype
Asexual blood stageNot different from wild type
Gametocyte/GameteGFP expression in female gametocytes. No expression could be detected in asexual blood stages and male gametocytes.
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant expresses GFP under the control of the promoter of the gene encoding NEK4 (serine/threonine protein kinase, NEK4, MAL7P1.100).

Protein (function)
NEK4 is a serine/threonine NIMA-related kinase; these kinases are mostly described as being involved in controlling/coordinating signalling processes in mitosis. See also the phenotype description of mutants lacking expression of NEK4 (RMgm-59, RMgm-60).

The NIMA-related protein kinases (Neks) constitute an extended family of eukaryotic mitotic serine/threonine kinases. The best characterized members of the Nek family include NIMA (never in mitosis/Aspergillus), the founding member from the fungus Aspergillus nidulans, and its closest homologue in mammals, Nek2. Initially identified as a kinase essential for mitotic entry in Aspergillus, NIMA has been also shown to participate in nuclear membrane fission. Eleven members of the NIMA kinase family (Nek1-11) have now been identified in various human tissues, and together fulfil a number of cell cycle-related functions in centrosome separation, mitosis, meiosis and checkpoint control. The P. falciparum kinome includes four NIMA-related serine/threonine kinases. Pfnek-1 (PlasmoDB identifier PFL1370w) clusters within the Aspergillus NIMA/human Nek2 branch in phylogenetic trees, while clear orthology to mammalian or yeast Neks could not be assigned for the three other P. falciparum sequences (Pfnek-2, -3 and -4, PlasmoDB identifiers PFE1290w, PFL0080c and MAL7P1.100,respectively).

Phenotype
Phenotype analyses indicate absence of promoter activity in asexual blood stages and male gametocytes and active transcription in female gametocytes. The absence in male gametocytes and prescence in females is in agreement with proteome analysis of males and females (Khan, S.M. et al., 2005, Cell, 121, 675-87). See further 'Additional information.

Additional information
The construct GFPcon (RMgm-5) containing GFP under the control of the eef1a promoter formed the basis for the construct driving expression of GFP under control of the nek4 promoter. This constructs contains a c/d-ssurrna target fragment for integration into the c/d-rrna genomic locus. GFP is flanked by the 3'-UTR sequence of the dhfr gene of P. berghei. The eef1a promoter region is replaced by a 678 bp fragment (EcoRV/BamHI) of the promoter region of nek4.
Because it has been shown that 3'-UTR regions can influence expression of genes in Plasmodium, care has to be taken in interpretation of expression of transgenes when 3'-UTR sequences of heterologous genes are used.

Other mutants


  Transgene: Mutant parasite expressing a transgene
Type and details of transgene
Is the transgene Plasmodium derived Transgene: not Plasmodium
Transgene nameGFP (gfp-mut3)
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/constructPlasmid single 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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ATCAAGAGTCTTTTGAAAGCCGAGCAAAAGTGTTAAAATTGATTAAATATATTTTTTAAA
AAAGTGTAAAAAATATATGGGGGTAAGCAAAAGGGAATATACATAATATAAATATAGAAA
CAACATTTGTGAAATGTAACAACCAATAAGTATATTAAGAAATACTCATTAATAAGAATA
AAAAAAAATTTTAGTTCAAAAAAAAAAAATGAAAGATATTTTTTAGTATATACACCTAAT
ATTAGGAAACGCCATAAACTTTATCATCAAATCGCTACACCGCTTTTTAGGAAAAAAAAA
TATAATAAATAGAAAGTATGGAAAATGGACAAAAAAAAAAAATCATAAACTTAATATTAT
GACTGAAAAATATTTTTTAATAAATTTATATACAAATATGTATACTAATTTTTTTGTATT
ATACAATAATTAATTTTTACTAAACACAATCTATTTAATCTACAAAATGTAGTATTATTT
TTTATTCACCATATTCATTAATTTTCTCTCTCTATATATATGTAACTATAATATACACAC
AAAAAAAACAGTATATTTCAACATTGTATTTTTATTCCTTGGATTGTGTATTGCTCATTC
TTTATTATACATATTCTCACAAAATTTTTTTTTATTAACTAATATAATTTCCTGTAACCT
TACAATTTGAAATTCCTAAAGGGATCCATGAGTAAAGGAGAAGAACTTTTCACTGGAGTT
GTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGA
GAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTTAAATTTATTTGCACTACTGGA
AAACTACCTGTTCCATGGCCAACACTTGTCACTACTTTCGGTTATGGTGTTCAATGCTTT
GCGAGATACCCAGATCATATGAAACAGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGT
TATGTACAGGAAAGAACTATATTTTTCAAAGATGACGGGAACTACAAGACACGTGCTGAA
GTCAAGTTTGAAGGTGATACCCTTGTTAATAGAATCGAGTTAAAAGGTATTGATTTTAAA
GAAGATGGAAACATTCTTGGACACAAATTGGAATACAACTATAACTCACACAATGTATAC
ATCATGGCAGACAAACAAAAGAATGGAATCAAAGTTAACTTCAAAATTAGACACAACATT
GAAGATGGAAGCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGC
CCTGTCCTTTTACCAGACAACCATTACCTGTCCACACAATCTGCCCTTTCGAAAGATCCC
AACGAAAAGAGAGACCACATGGTCCTTCTTGAGTTTGTAACAGCTGCTGGGATTACACAT
GGCATGGATGAACTATACAAATAAATTCTAGAAGATCCCGTTTTTCTTACTTATATATTT
ATACCAATTGATTGTATTTATAACTGTAAAAATGTGTATGTTGTGTGCATATTTTTTTTT
GTGCATGCACATGCATGTAAATAGCTAAAATTATGAACATTTTATTTTTTGTTCAGAAAA
AAAAAACTTTACACACATAAAATGGCTAGTATGAATAGCCATATTTTATATAAATTAAAT
CCTATGAATTTATGACCATATTAAAAATTTAGATATTTATGGAACATAATATGTTTGAAA
CAATAAGACAAAATTATTATTATTATTATTATTTTTACTGTTATAATTATGTTGTCTCTT
CAATGATTCATAAATAGTTGGACTTGATTTTTAAAATGTTTATAATATGATTAGCATAGT
TAAATAAAAAAAGTTGAAAAATTAAAAAAAAACATATAAACACAAATGATGTTTTTTCCT
TCAATTTCGGGTACCGAGCTCGAATTCAACCTGGTTGATCTTGCCAGTAGTCATATGCTT
GTCTCAAAGATTAAGCCATGCAAGTGAAAGTATATGCACATTTATTGCAGAAACTGCGAA
CGGCTCATTAAAACAGTTATAATCTACTTGACATTTTATTATAAGGATAACTACGGAAAA
GCTGTAGCTAATACTTGTTAAGTACTTTTACTCCCCGGAGTAATTGTATGTATTTGTTAA
GACCCCTAAGAAAAAATGATATTAAAGGAATTATAACAAAGAAGCAACACATAATATAAT
TATTCAGTGTGTATCAATCGAGTTTCTGACCTATCAGCTTTTGATGTTAGGGTATTGACC
TAACATGGCTTTGACGGGTAACGGGGAATTAGAGTTCGATTCCGGAGAGGGAGCCTGAGA
AATAGCTACCACATCTAAGGAAGGCAGCAGGCGCGTAAATTACCCAATTCTAAATAAGAG
AGGTAGTGACAAGAAATAACAATATAAGGCCAAATTTTGGTTTTATAATTGGAATGATGG
GAATTTAAAACCTTCCCAAAAATCAATTGGAGGGCAAGTCTGGTGCCAGCAGCCGCGGTA
ATTCCAGCTCCAATAGCGTATATTAAAATTGTTGCAGTTAAAACGCTCGTAGTTGAACTT
CAAGGGTATAATTATTTTAAGCAACTCACTTGGAAAGAATCATGACTTCTGTCACTGCTT
TTATCCTTGTTGCAGTTCTTTTAATACAGGGCCCTTTGAGAGCCCATTAATTTATGACTG
GGTTTCTCGTTACTTTGAGTAAATTAGAGTGTTTAAAGCAAACAGATAAAGCGTATTTTA
CTGTGTTTGAATACTATAGCATGGAATAACAACATTGAATAGGTCAAAAGTTTTTGAAAA
ATTTTTCTTATTTTGGCTTAGATACAGTTAATAGGAGTAGCTTGGGGGCATTTGTATTCA
GATGTCAGAGGTGAAATTCTTAGATTTTCTGGAGACAAACAACTGCGAAAGCATTTGCCT
AAAATACTTCCATTAATCAAGAACGAAAGTTAAGGGAGTGAAGACGATCAGATACCGTCG
TAATCTTAACCATAAACTATGCCGACTAAGTGTTGGATGAAAATTTATAAATAAAACTAT
CTTCTTTAAAGGAGTAGTTTTTTAGATGCTTCCTTCAGTACCTTATGAGAAATCAAAGTC
TTTGGGTTCTGGGGCGAGTATTCGCGCAAGCGAGAAAGTTAAAAGAATTGACGGAAGGGC
ACCACCAGGCGTGGAGCTTGCGGCTTAATTTGACTCAACACGGGGAAACTCACTAGTTTA
AGACAAGAGTAGGATTGACAGATTAATAGCTCTTTCTTGATTTCTTGGATGGTGATGCAT
GGCCGTTTTTAGTTCGTGAATATGATTTGTCTGGTTAATTCCGATAACGAACGAGATCTT
AACCTGCTAATTAGCGGTGGATATGTGATATTCTTCGAAGGTGGACTAACTATAGCGTTT
TCGAAGGTATGTTGCATAATCAAATTGGTTTACCCTTTGTTTTTTTGTAGCATATTCTTT
TATTTCGTTGGGTTTTTTCCCTAGTAAGGATGTATCTGCTTTATTTAATGCTTCTTAGAG
GAACGATGTGTGTCTAACACAAGGAAGTTTAAGGCAACAACAGGTCTGTGATGTCCTTAG
ATATACTAGGCTGCACGCGTGCTACACTGATATGTAAAACGAGTATTTAAAATTATATCT
GTATGGTAGATAATTTAATTTCTACGTATTATCAGCATATACTTTTCCTACACTGAAATA
GTGAAGGTAATCTTTATCAATACATATCGTGATGGGGATAGATTATTGCAATTATTAATC
TTGAACGAGGAATGCCTAGTAAGCATGATTCATCAGATTGTGCTGACTACGTCCCTGCCC
TTTGTACACACCGCCCGTCGCTCCTACCGATTGAAAGATATGATGAATTGTTTGGACAAG
AAAATAGAAATTTTATTTTTATTTTTTTTGGAAGGACCGTAAATCCTATCTTTTAAAGGA
AGGAGAAGTCGTAACAAGGTTTCCGTAGGTGAATTCACTGGCCGTCGTTTTACAACGTCG
TGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGC
CAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCT
GAATGGCGAATGGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACA
CCGCATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGCCCCG
ACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTA
CAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACC
GAAACGCGCGAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGAT
AATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTAT
TTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATA
AATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCT
TATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAA
AGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAA
CAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTT
TAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGG
TCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCA
TCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAA
CACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTT
GCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGC
CATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAA
ACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGA
GGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGC
TGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGA
TGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGA
ACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGA
CCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGAT
CTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTT
CCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCT
GCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCC
GGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACC
AAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACC
GCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTC
GTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTG
AACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATA
CCTACAGCGTGAGCATTGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTA
TCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGC
CTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTG
ATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTT
CCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGT
GGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGA
GCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCC
CGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGG
CAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACA
CTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGG
AAACAGCTATGACCATGATTACGCCAAGCTTGCATGCCTGCAGGTCAACAATAAATAATA
AATAAATATTGTGGAAATAAAATAACATATAATTATTTTTAATACATTGATTTCCCTTTT
ATTTTTTTAAATTTCATTGATATAAAAATATATAATAATAACATATATGATTTCAAATTA
ATCTTTTCAAAAATGGTGTTTATTTTTGTATGTTTGTGTATGAATTAATCACATAACACA
TCTATTAAATTGAGTTGGTAATATAGACACAAATAAATATATATATTTTTATAGCTTAAA
AGTGTGTTATGAATATTTTAAGCATATTTTCTTTTTCTTTGGATTGTGTAAAATGAACTC
ATATAATGCGTTTTTTTGTTTTTGTTATTTTGTCATTTTGTTATTTTGCTATTTTATGGA
TTAATTTTTGTTTATAAAATGGGAAATAATTTTAACATATTTAAATAAATGGAGAAAAAA
TATAAAATAATTATAAAAAAAAGTTAATACACATTTTTTCCTGTTATAGACCTTATATTT
ATTTATCCATATATATATATATATATATATATATATATATATACATACCAAGTGAATTAA
GAGGAAAGCTAATTTATTATTCAGAATAATATATGAACTATATATAATTTTTATTATTTT
GGTGTATATTAATCTGTCTATATGCATACATGCAATAATTTATCGACTTATATATCAAAT
AACATAAAATAGAAGTGTTTTAAATTATGGATATATGCTCAATATTCATTTTTTTTAATA
AGTTAGCTATATTTAAATTATACATTTTATATATGGTCTCTTTTTTTTTTAAATATTATT
TAAGTGATCATGAAAATATAAATAATTTTTTTTTATTTAATATCCTTTTGCTTGCATGTG
GTAAATGGAAATTTGGATGTGTTTTGAARGTTCGGATATAGTTGTATGGACATATAATAT
ATTTTGTGAAAAATTGGTTTTATGTTTATACTTATGCCAATACTTTTTGAGTAAAACAAA
GCAAGTGCTTATAAATAATTAAAGCCAATTTTATAATATATATTTTTTTATTTAATTTGA
ATTTAGTAGTATAATTTTTTATGGTAAGTGCTCAAAGAGAGTTGCTTATAAAGTATGGTT
TGTTTCTTTTTCGCCATTTTGAATTACACATTAAAAATATATAGATACATATATTATAAT
ATGAAATCATTAATAATTTAGGGAAATTCTACAAATTTAAAAACGAATAAAATAATTGTT
TTTCATCATGCCATAACACAATATTGATATATACATGTACAAACATTTTTTTTATTTGGA
AAATATAAATTATATAAAAAAAAATGTATAGTATACAAAATGAGCATATTCACACGGGGT
GGACGTTCATTTTTTCATTTTTCCCCTGTTTTTTATGAGTATATGATAAAATTTTATGAA
CATTTACACAAAATGAAAATGGATATATAGGAAAAATGGAGCGGTATTTCATTTATCTTT
GATTGTCATTTGGATATTATATTACCYTGGGTAGGCAATTAAAAATGTTAAATAACAATT
TAAGGAAATTATATTTTATATATTAAAATTAACACTGTATTATATGATTCGCTTATAAAA
GCCACTCTTTCCCCATGCAAAGCTGTTTAATATCAATTTTAACAAATTACACACATGTTA
ATATATTTATATATATAATTTATATATTTATAATTTATATATTTATATTTTTATTATTTA
TATATTTATTATTTATTGTGTGTGTCAATTCGGGTAGGATATACCTCTTTTTTATTGTTT
AAAGCGATTTGTATTCTAAAATATAAAGRATTTGAAAAAGAGAAAGATAGAATATGATCC
CATCATATATAGCCCTATAATTTTTATTTAGCAGCGAATTAATTTTTCTATTAAGTTTAT
GTGTAATTAAAATAACGGAATATATATAATACAATAAAAAAGTGCATAAATTAAAATTTT
TTCAATTAAATTTTTTTTTTTAAGGGGTTATATAATATTAAATATATAAAATACGATTAT
ATATTTTTGCTACAATTTTTTATATTAAGATATAAATAGTAAATAAATGGTATTATATGG
CATGTAATATATAAATTTTTTCCAATTTTTATTTTATATACACTTTTCCTTTTTTTGTCA
TAAAACTTAAACAATTTACACATTCATTTTAAAAATTGACTATTTGTTTCAACATTTTTT
GAGTTTCCGTTTTATAATAGTATTTTCATTTGTATATTGCTTATATATATAAATACACAC
CTAAATGTTACAAAGGATCAATGCATAAACCGGTGTGTCTGGTCGTCGCGATGACCCCCA
AGAGGGGCATCGGCATCAACAACGGCCTCCCGTGGCCCCACTTGACCACAGATTTCAAAC
ACTTTTCTCGTGTGACAAAAACGACGCCCGAAGAAGCCAGTCGCCTGAACGGGTGGCTTC
CCAGGAAATTTGCAAAGACGGGCGACTCTGGACTTCCCTCTCCATCAGTCGGCAAGAGAT
TCAACGCCGTTGTCATGGGACGGAAAACCTGGGAAAGCATGCCTCGAAAGTTTAGACCCC
TCGTGGACAGATTGAACATCGTCGTTTCCTCTTCCCTCAAAGAAGAAGACATTGCGGCGG
AGAAGCCTCAAGCTGAAGGCCAGCAGCGCGTCCGAGTCTGTGCTTCACTCCCAGCAGCTC
TCAGCCTTCTGGAGGAAGAGTACAAGGATTCTGTCGACCAGATTTTTGTCGTGGGAGGAG
CGGGACTGTACGAGGCAGCGCTGTCTCTGGGCGTTGCCTCTCACCTGTACATCACGCGTG
TAGCCCGCGAGTTTCCGTGCGACGTTTTCTTCCCTGCGTTCCCCGGAGATGACATTCTTT
CAAACAAATCAACTGCTGCGCAGGCTGCAGCTCCTGCCGAGTCTGTGTTCGTTCCCTTTT
GTCCGGAGCTCGGAAGAGAGAAGGACAATGAAGCGACGTATCGACCCATCTTCATTTCCA
AGACCTTCTCAGACAACGGGGTTCCCTACGACTTTGTGGTTCTCGAGAAGAGAAGGAAGA
CTGACGACGCAGCCACTGCGGAACCGAGCAACGCAATGAGCTCCTTGACGTCCACGAGGG
AGACAACTCCCGTGCACGGGTTGCAGGCTCCTTCTTCGGCCGCAGCCATTGCCCCGGTGT
TGGCGTGGATGGACGAAGAAGACCGGAAAAAACGCGAGCAAAAGGAACTGATTCGGGCCG
TTCCGCATGTTCACTTTAGAGGCCATGAAGAGTTCCAGTACCTTGATCTCATTGCCGACA
TTATTAACAATGGAAGGACAATGGATGACCGAACGGGCGTTGGTGTCATCTCCAAATTCG
GCTGCACTATGCGCTACTCGCTGGATCAGGCCTTTCCACTTCTCACCACAAAGCGTGTGT
TCTGGAAAGGGGTCCTCGAAGAGTTGCTGTGGTTCATTCGCGGCGACACGAACGCAAACC
ATCTTTCTGAGAAGGGCGTGAAGATCTGGGACAAGAATGTGACACGCGAGTTCCTCGATT
CGCGCAATCTCCCCCACCGAGAGGTCGGAGACATCGGCCCGGGCTACGGCTTCCAGTGGA
GACACTTCGGCGCGGCATACAAAGACATGCACACAGACTACACAGGGCAGGGCGTCGACC
AGCTGAAGAATGTGATCCAGATGCTGAGAACGAATCCAACAGATCGTCGCATGCTCATGA
CTGCCTGGAATCCTGCAGCGCTGGACGAAATGGCGCTGCCGCCTTGTCACTTGTTGTGCC
AGTTCTACGTGAACGACCAGAAGGAGCTGTCGTGCATCATGTATCAGCGGTCGTGCGATG
TCGGCCTCGGCGTCCCCTTCAACATCGCTTCCTATTCGCTTTTGACGCTCATGGTTGCAC
ACGTCTGCAACCTAAAACCTAAGGAGTTCATTCACTTCATGGGGAACACGCATGTCTACA
CGAACCATGTCGAGGCTTTAAAAGAGCAGCTGCGGAGAGAACCGAGACCGTTCCCCATTG
TGAACATCCTCAACAAGGAACGCATCAAGGAAATCGACGATTTCACCGCCGAGGATTTTG
AGGTCGTGGGCTACGTCCCGCACGGACGAATCCAGATGGAGATGGCTGTCTAGCGGAAAT
ACAGAAGCTAGCTTTGATCCCGTTTTTCTTACTTATATATTTATACCAATTGATTGTATT
TATAACTGTAAAAATGTGTATGTTGTGTGCATATTTTTTTTTGTGCATGCACATGCATGT
AAATAGCTAAAATTATGAACATTTTATTTTTTGTTCAGAAAAAAAAAACTTTACACACAT
AAAATGGCTAGTATGAATAGCCATATTTTATATAAATTAAATCCTATGAATTTATGACCA
TATTAAAAATTTAGATATTTATGGAACATAATATGTTTGAAACAATAAGACAAAATTATT
ATTATTATTATTATTTTTACTGTTATAATTATGTTGTCTCTTCAATGATTCATAAATAGT
TGGACTTGATTTTTAAAATGTTTATAATATGATTAGCATAGTTAAATAAAAAAAGTTGAA
AAATTAAAAAAAAACATATAAACACAAATGATGTTTTTTCCTTCAATTTCGAT
Restriction sites to linearize plasmid
Selectable marker used to select the mutant parasitetgdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationThe construct GFPcon (RMgm-5) containing GFP under the control of the eef1a promoter formed the basis for the construct driving expression of GFP under control of the nek4 promoter.
This constructs contains a c/d-ssurrna target fragment for integration into the c/d-rrna genomic locus. GFP is flanked by the 3'-UTR sequence of the dhfr gene of P. berghei. The eef1a promoter region is replaced by a 678 bp fragment (EcoRV/BamHI) of the promoter region of nek4.

Because it has been shown that 3'-UTR regions can influence expression of genes in Plasmodium, care has to be taken in interpretation of expression of transgenes when 3'-UTR sequences of heterologous genes are used.

The gfp gene has been integrated into the genome by single cross-over integration. Therefore the possibility exists of reversion to the wild-type genotype by removal of the integrated DNA construct.
Additional remarks selection procedure
Other details transgene
Promoter
Gene Model of Parasite PBANKA_0616700
Gene Model P. falciparum ortholog PF3D7_0719200
Gene productNIMA related kinase 4
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 1aaGATATCaagagtcttttgaaagccgagc
Additional information primer 1forward
Sequence Primer 2aaGGATCCctttaggaatttcaaattgtaagg
Additional information primer 2reverse
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 / InsertionInsertion locus
Gene Model of Parasite Not available
Gene productNot available
Gene product: Alternative namesmall subunit ribosomal rna gene (c- or d-type unit)
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