Summary

RMgm-663
Malaria parasiteP. berghei
Genotype
TaggedGene model (rodent): PBANKA_0100600; Gene model (P.falciparum): Not available; Gene product: schizont membrane associated cytoadherence protein (Schizont Membrane Associated Cytoadherence protein; SMAC)
Name tag: mCherry
Phenotype Asexual bloodstage;
Last modified: 21 December 2011, 10:00
  *RMgm-663
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene tagging
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 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/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-663
Principal name1262cl2
Alternative namesmac::mCherry
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stagesmac::mCherry signals detected in all blood stages, both in the cytoplasm of the parasite and in the cytoplasm of the infected erythrocyte (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  expresses a (C-terminal) mCherry-tagged version of Schizont Membrane Associated Cytoadherence protein (SMAC). 

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
The blood stages of the smac::mCherry parasites showed strong mCherry-fluorescence in the cytoplasm of the host erythrocyte demonstrating export of SMAC into the host erythrocyte. In synchronized blood stages weak SMAC::mCherry was detected in the cytoplasm of developing trophozoites (5 to 12 h). Fluorescence increased in maturing trophozoites and schizonts with a patchy fluorescent signal in the cytoplasm of red blood cells infected with immature and mature schizonts. SMAC::mCherry was not observed at or close to the irbc surface membrane. 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.

Phenotype analyses of mutants lacking expression of SMAC (RMgm-661, RMgm-662) indicate that asexual blood stages show a reduced growth rate in mice (see 'Additional information').

Additional information
Phenotype analyses of mutants lacking SMAC-expression (RMgm-661, RMgm-662) 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.

Other mutants
RMgm-661: A mutant lacking expression of SMAC
RMgm-662: An independent mutant lacking expression of SMAC


  Tagged: Mutant parasite with a tagged 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
Name of the tagmCherry
Details of taggingC-terminal
Additional remarks: tagging
Commercial source of tag-antibodiesSanta Cruz Biotechnology
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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CTAAATTGTAAGCGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTC
ATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGACCGA
GATAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGTCCACTATTAAAGAACGTGGACTC
CAACGTCAAAGGGCGAAAAACCGTCTATCAGGGCGATGGCCCACTACGTGAACCATCACC
CTAATCAAGTTTTTTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAG
CCCCCGATTTAGAGCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAA
AGCGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCAC
CACACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCCCATTCGCCATTCAGGCTGCG
CAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGG
GGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTG
TAAAACGACGGCCAGTGAGCGCGCGTAATACGACTCACTATAGGGCGAATTGGAGCTCCA
CCGCGGTGGCGGCCGCTCTAGAACTAGTGTTACATTTATATATTGAACCCATTTTAAAAA
TATACATATTAAATAGCTTTAGAATTATGACAATTAATTTTTATAATAATAAAAAGTTCC
ATTTATTTGTTTTCTGAATCGGTAAAGAGTATTAATAAAAACCCAATTGTAAACGGTCCC
AATATATTTTTTAAAGAATGTGTTACTATGTATTTAAATTACCCAAATTAAACAAGTAAA
AAATATCACAATATATATTATTTTCCCCGAGATGCTATAAATAATTATTTTTGATAGATT
GACAAATATATATTGTTCTTTTTTGACGTAATGCATTATATATCATATATGTAGCACTTT
TTAGTTTTAAAATAATAATTTAATATAATACACAGTGCATGCACCATTTTACATGAATCT
GATATAATACTTATTTATCATTTTATTTATTAGCCATTTATTATAAACAAACTATATATT
ATTATCTTTTACATACATCCTTAACTCTTTTAACAACCAGTACACATAATATTTGCAACA
TTGTGAATATAAAATGAAATATTTAACGCACATATTTTTTTTTATTATACTTTTTCAATT
TATAGTTGATAAATACGATAACAATGGAAAATCATTGGACAAGTTTTTAAATATACGAAA
TGAGAGATTTCTAGTAGAATATTATGACGCTAACATTGATCAATATAATGGCAATCAATC
AAAAAAAAAAAAAAATATATATACATATGAATCAAATGGTATTTCACAAAACACTACTAA
TACCAAAAACCAATCAGCAAGTTTTTATGCACAACCTACCCACGGCCAATATAAATACCA
TCGCAATAATGTATATACTCCCTCTGAAGAGTTCAATTTTTATAATAGTACGTTCATGGA
AAGGCTAAGACATAATTCTAAGATCATTATGGGATCATCTGCAGCGATATTTTTTCTTGT
CGAAGATTTAGGAATTAGAGCTGTGCTATTTTTAATATTTTCTGCTGCTGTACTCGCTTA
TTTCACTTCCATAGGATCCGGTGGAGGTGGAAGATCCATGGTGAGCAAGGGCGAGGAGGA
TAACATGGCCATCATCAAGGAGTTCATGCGCTTCAAGGTGCACATGGAGGGCTCCGTGAA
CGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCCGCCCCTACGAGGGCACCCAGAC
CGCCAAGCTGAAGGTGACCAAGGGTGGCCCCCTGCCCTTCGCCTGGGACATCCTGTCCCC
TCAGTTCATGTACGGCTCCAAGGCCTACGTGAAGCACCCCGCCGACATCCCCGACTACTT
GAAGCTGTCCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTGATGAACTTCGAGGACGGCGG
CGTGGTGACCGTGACCCAGGACTCCTCCCTGCAGGACGGCGAGTTCATCTACAAGGTGAA
GCTGCGCGGCACCAACTTCCCCTCCGACGGCCCCGTAATGCAGAAGAAGACCATGGGCTG
GGAGGCCTCCTCCGAGCGGATGTACCCCGAGGACGGCGCCCTGAAGGGCGAGATCAAGCA
GAGGCTGAAGCTGAAGGACGGCGGCCACTACGACGCTGAGGTCAAGACCACCTACAAGGC
CAAGAAGCCCGTGCAGCTGCCCGGCGCCTACAACGTCAACATCAAGTTGGACATCACCTC
CCACAACGAGGACTACACCATCGTGGAACAGTACGAACGCGCCGAGGGCCGCCACTCCAC
CGGCGGCATGGACGAGCTGTACAAGTAATCTAGAATTATTAATATATATGAATATATATA
CATCGTTGTATGCGTAATATACGTGTAACTGTTTTTTTTAATTTTTATATTAACTTTTCC
TTATACATTTTAATGCCATTAACTAATAATATTTTACTTGAAAAAATTCTAAATTTTTAT
AACTCTAATATAAAATCTGTGAATAATAAAAAATTAGCATAAAGTACGAATTAAACATCG
AATATTTGTTTTAAAGGGGCACACATTATGTGCATATAATTTTATTCTTTGAATTTAATT
TTTTGCTTATAATTAGATTAAATTAATATAATTTTTTTATTGAAAATAAAAACTTATTTT
AAAAAATTAACAAATGGAAGTTAATGAAAAATACGAAAAAAGTAAAGGGTTAATTCTTAT
ATGGTCGGTACCGAGCTCGAATTCGATATCGAAATTGAAGGAAAAAACATCATTTGTGTT
TATATGTTTTTTTTTAATTTTTCAACTTTTTTTATTTAACTATGCTAATCATATTATAAA
CATTTTAAAAATCAAGTCCAACTATTTATGAATCATTGAAGAGACAACATAATTATAACA
GTAAAAATAATAATAATAATAATAATTTTGTCTTATTGTTTCAAACATATTATGTTCCAT
AAATATCTAAATTTTTAATATGGTCATAAATTCATAGGATTTAATTTATATAAAATATGG
CTATTCATACTAGCCATTTTATGTGTGTAAAGTTTTTTTTTTCTGAACAAAAAATAAAAT
GTTCATAATTTTAGCTATTTACATGCATGTGCATGCACAAAAAAAAATATGCACACAACA
TACACATTTTTACAGTTATAAATACAATCAATTGGTATAAATATATAAGTAAGAAAAACG
GGATCAAAGCTAGCTTCTGTATTTCCGCTAGACAGCCATCTCCATCTGGATTCGTCCGTG
CGGGACGTAGCCCACGACCTCAAAATCCTCGGCGGTGAAATCGTCGATTTCCTTGATGCG
TTCCTTGTTGAGGATGTTCACAATGGGGAACGGTCTCGGTTCTCTCCGCAGCTGCTCTTT
TAAAGCCTCGACATGGTTCGTGTAGACATGCGTGTTCCCCATGAAGTGAATGAACTCCTT
AGGTTTTAGGTTGCAGACGTGTGCAACCATGAGCGTCAAAAGCGAATAGGAAGCGATGTT
GAAGGGGACGCCGAGGCCGACATCGCACGACCGCTGATACATGATGCACGACAGCTCCTT
CTGGTCGTTCACGTAGAACTGGCACAACAAGTGACAAGGCGGCAGCGCCATTTCGTCCAG
CGCTGCAGGATTCCAGGCAGTCATGAGCATGCGACGATCTGTTGGATTCGTTCTCAGCAT
CTGGATCACATTCTTCAGCTGGTCGACGCCCTGCCCTGTGTAGTCTGTGTGCATGTCTTT
GTATGCCGCGCCGAAGTGTCTCCACTGGAAGCCGTAGCCCGGGCCGATGTCTCCGACCTC
TCGGTGGGGGAGATTGCGCGAATCGAGGAACTCGCGTGTCACATTCTTGTCCCAGATCTT
CACGCCCTTCTCAGAAAGATGGTTTGCGTTCGTGTCGCCGCGAATGAACCACAGCAACTC
TTCGAGGACCCCTTTCCAGAACACACGCTTTGTGGTGAGAAGTGGAAAGGCCTGATCCAG
CGAGTAGCGCATAGTGCAGCCGAATTTGGAGATGACACCAACGCCCGTTCGGTCATCCAT
TGTCCTTCCATTGTTAATAATGTCGGCAATGAGATCAAGGTACTGGAACTCTTCATGGCC
TCTAAAGTGAACATGCGGAACGGCCCGAATCAGTTCCTTTTGCTCGCGTTTTTTCCGGTC
TTCTTCGTCCATCCACGCCAACACCGGGGCAATGGCTGCGGCCGAAGAAGGAGCCTGCAA
CCCGTGCACGGGAGTTGTCTCCCTCGTGGACGTCAAGGAGCTCATTGCGTTGCTCGGTTC
CGCAGTGGCTGCGTCGTCAGTCTTCCTTCTCTTCTCGAGAACCACAAAGTCGTAGGGAAC
CCCGTTGTCTGAGAAGGTCTTGGAAATGAAGATGGGTCGATACGTCGCTTCATTGTCCTT
CTCTCTTCCGAGCTCCGGACAAAAGGGAACGAACACAGACTCGGCAGGAGCTGCAGCCTG
CGCAGCAGTTGATTTGTTTGAAAGAATGTCATCTCCGGGGAACGCAGGGAAGAAAACGTC
GCACGGAAACTCGCGGGCTACACGCGTGATGTACAGGTGAGAGGCAACGCCCAGAGACAG
CGCTGCCTCGTACAGTCCCGCTCCTCCCACGACAAAAATCTGGTCGACAGAATCCTTGTA
CTCTTCCTCCAGAAGGCTGAGAGCTGCTGGGAGTGAAGCACAGACTCGGACGCGCTGCTG
GCCTTCAGCTTGAGGCTTCTCCGCCGCAATGTCTTCTTCTTTGAGGGAAGAGGAAACGAC
GATGTTCAATCTGTCCACGAGGGGTCTAAACTTTCGAGGCATGCTTTCCCAGGTTTTCCG
TCCCATGACAACGGCGTTGAATCTCTTGCCGACTGATGGAGAGGGAAGTCCAGAGTCGCC
CGTCTTTGCAAATTTCCTGGGAAGCCACCCGTTCAGGCGACTGGCTTCTTCGGGCGTCGT
TTTTGTCACACGAGAAAAGTGTTTGAAATCTGTGGTCAAGTGGGGCCACGGGAGGCCGTT
GTTGATGCCGATGCCCCTCTTGGGGGTCATCGCGACGACCAGACACACCGGTTTATGCAT
TGATCCTTTGTAACATTTAGGTGTGTATTTATATATATAAGCAATATACAAATGAAAATA
CTATTATAAAACGGAAACTCAAAAAATGTTGAAACAAATAGTCAATTTTTAAAATGAATG
TGTAAATTGTTTAAGTTTTATGACAAAAAAAGGAAAAGTGTATATAAAATAAAAATTGGA
AAAAATTTATATATTACATGCCATATAATACCATTTATTTACTATTTATATCTTAATATA
AAAAATTGTAGCAAAAATATATAATCGTATTTTATATATTTAATATTATATAACCCCTTA
AAAAAAAAAATTTAATTGAAAAAATTTTAATTTATGCACTTTTTTATTGTATTATATATA
TTCCGTTATTTTAATTACACATAAACTTAATAGAAAAATTAATTCGCTGCTAAATAAAAA
TTATAGGGCTATATATGATGGGATCATATTCTATCTTTCTCTTTTTCAAATYCTTTATAT
TTTAGAATACAAATCGCTTTAAACAATAAAAAAGAGGTATATCCTACCCGAATTGACACA
CACAATAAATAATAAATATATAAATAATAAAAATATAAATATATAAATTATAAATATATA
AATTATATATATAAATATATTAACATGTGTGTAATTTGTTAAAATTGATATTAAACAGCT
TTGCATGGGGAAAGAGTGGCTTTTATAAGCGAATCATATAATACAGTGTTAATTTTAATA
TATAAAATATAATTTCCTTAAATTGTTATTTAACATTTTTAATTGCCTACCCARGGTAAT
ATAATATCCAAATGACAATCAAAGATAAATGAAATACCGCTCCATTTTTCCTATATATCC
ATTTTCATTTTGTGTAAATGTTCATAAAATTTTATCATATACTCATAAAAAACAGGGGAA
AAATGAAAAAATGAACGTCCACCCCGTGTGAATATGCTCATTTTGTATACTATACATTTT
TTTTTATATAATTTATATTTTCCAAATAAAAAAAATGTTTGTACATGTATATATCAATAT
TGTGTTATGGCATGATGAAAAACAATTATTTTATTCGTTTTTAAATTTGTAGAATTTCCC
TAAATTATTAATGATTTCATATTATAATATATGTATCTATATATTTTTAATGTGTAATTC
AAAATGGCGAAAAAGAAACAAACCATACTTTATAAGCAACTCTCTTTGAGCACTTACCAT
AAAAAATTATACTACTAAATTCAAATTAAATAAAAAAATATATATTATAAAATTGGCTTT
AATTATTTATAAGCACTTGCTTTGTTTTACTCAAAAAGTATTGGCATAAGTATAAACATA
AAACCAATTTTTCACAAAATATATTATATGTCCATACAACTATATCCGAACYTTCAAAAC
ACATCCAAATTTCCATTTACCACATGCAAGCAAAAGGATATTAAATAAAAAAAAATTATT
TATATTTTCATGATCACTTAAATAATATTTAAAAAAAAAAGAGACCATATATAAAATGTA
TAATTTAAATATAGCTAACTTATTAAAAAAAATGAATATTGAGCATATATCCATAATTTA
AAACACTTCTATTTTATGTTATTTGATATATAAGTCGATAAATTATTGCATGTATGCATA
TAGACAGATTAATATACACCAAAATAATAAAAATTATATATAGTTCATATATTATTCTGA
ATAATAAATTAGCTTTCCTCTTAATTCACTTGGTATGTATATATATATATATATATATAT
ATATATATATATGGATAAATAAATATAAGGTCTATAACAGGAAAAAATGTGTATTAACTT
TTTTTTATAATTATTTTATATTTTTTCTCCATTTATTTAAATATGTTAAAATTATTTCCC
ATTTTATAAACAAAAATTAATCCATAAAATAGCAAAATAACAAAATGACAAAATAACAAA
AACAAAAAAACGCATTATATGAGTTCATTTTACACAATCCAAAGAAAAAGAAAATATGCT
TAAAATATTCATAACACACTTTTAAGCTATAAAAATATATATATTTATTTGTGTCTATAT
TACCAACTCAATTTAATAGATGTGTTATGTGATTAATTCATACACAAACATACAAAAATA
AACACCATTTTTGAAAAGATTAATTTGAAATCATATATGTTATTATTATATATTTTTATA
TCAATGAAATTTAAAAAAATAAAAGGGAAATCAATGTATTAAAAATAATTATATGTTATT
TTATTTCCACAATATTTATTTATTATTTATTGTTGACCTGCAGGCATGCAAGCTTATCGA
TACCGTCGACCTCGAGGGGGGGCCCGGTACCCAGCTTTTGTTCCCTTTAGTGAGGGTTAA
TTGCGCGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCA
CAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAG
TGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGT
CGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGC
GCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGG
TATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAA
AGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGG
CGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGA
GGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCG
TGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGG
GAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTC
GCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCG
GTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCA
CTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGT
GGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAG
TTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCG
GTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATC
CTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTT
TGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTT
TTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCA
GTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCG
TCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATAC
CGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGG
CCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCC
GGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTA
CAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAAC
GATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTC
CTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCAC
TGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACT
CAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAA
TACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTT
CTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCA
CTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAA
AAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATAC
TCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCG
GATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCC
GAAAAGTGCCAC
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 modificationTo generate transgenic parasites expressing SMAC that is C-terminally tagged with mCherry construct pL1419 was generated. First the mCherry gene of pL0017-mCherry (Graewe et al., 2009) was cloned into pL0017eGFPcam (BamHI/XbaI). Next, the mCherry expression cassette of pL0017mCherrycam was subcloned in pBluescript (BamHI/EcoRI) and then cloned (HindIII/EcoRI) into pL0001 to obtain the mCherry-tagging vector. Finally, the smac targeting region (primers 3774-3775; SpeI/BglII) was cloned into the mCherry-tagging plasmid cut with SpeI/BamHI to obtain pL1419.
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 1gggACTAGTGTTACATTTATATATTGAACCC
Additional information primer 13774: smac mCherry tagging, F (SpeI)
Sequence Primer 2ggAGATCTTCCACCTCCACCGGATCCTATGGAAGTGAAATAAGCGAGTAC
Additional information primer 23775: smac mCherry tagging, F (BglII, BamHI)
Sequence Primer 3
Additional information primer 3
Sequence Primer 4
Additional information primer 4
Sequence Primer 5
Additional information primer 5
Sequence Primer 6
Additional information primer 6