RMgmDB - Rodent Malaria genetically modified Parasites

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Summary

RMgm-1436

Malaria parasite	P. berghei
Genotype
Tagged	Gene model (rodent): PBANKA_0836800; Gene model (P.falciparum): Not available; Gene product: erythrocyte membrane associated protein 1; fam-a protein (EMAP1) Name tag: a synthetic 3D7-DBL1X-6ε gene (var2csa; PF3D7_1200600)
Phenotype	Asexual bloodstage;

Last modified: 6 August 2019, 12:32

*RMgm-1436

Successful modification	The 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) : 27045035
MR4 number
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Parent parasite used to introduce the genetic modification
Rodent Malaria Parasite	P. berghei
Parent strain/line	P. berghei ANKA
Name parent line/clone	P. berghei ANKA 1037m1f1mocl1 (RMgm-32)
Other information parent line	P. 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).
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The mutant parasite was generated by
Name PI/Researcher	de Moraes LV; Janse CJ; Franke-Fayard B; Penha-Gonçalves C
Name Group/Department	Instituto Gulbenkian de Ciência
Name Institute	Instituto Gulbenkian de Ciência
City	Oeiras
Country	Portugal
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Name of the mutant parasite
RMgm number	RMgm-1436
Principal name	1653cl3
Alternative name	emap1::var2csa; Pb-VAR
Standardized name
Is the mutant parasite cloned after genetic modification	Yes
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Phenotype
Asexual blood stage	emap1::var2csa expressed in blood stages and exported into the infected red blood cell and located at the surface membrane of infected erythrocytes (IEs). Evidence is provided that DBL1-6 protein is expressed in Pb-VAR but only a proportion of IEs containing schizonts (less than 10%) exposed the protein on the outer membrane as revealed by staining live IEs using anti-DBL1-6 antibodies.
Gametocyte/Gamete	Not tested
Fertilization and ookinete	Not tested
Oocyst	Not tested
Sporozoite	Not tested
Liver stage	Not tested
Additional remarks phenotype	Mutant/mutation The mutant expresses EMAP1 (PBANKA_0836800) that is C-terminally tagged with a synthetic 3D7-DBL1X-6ε gene (var2csa; PF3D7_1200600). The mutant was generated as previously reported for C-terminally mcherry-tagged EMAP1 (RMgm-690). Protein (function) EMAP1 is a member of the variant exported multigene family fam-a of rodent malaria parasites. EMAP1 is exported into the host red blood cell and is located at the surface membrane of infected erythrocytes (see RMgm-690). Phenotype emap1::var2csa expressed in blood stages and exported into the infected red blood cell and located at the surface membrane of infected erythrocytes (IEs) (see also Additional information). Evidence is provided that DBL1-6 protein is expressed in Pb-VAR but only a proportion of IEs containing schizonts (less than 10%) exposed the protein on the outer membrane as revealed by staining live IEs using anti-DBL1-6 antibodies. Additional information The mutant is used to analyse pregnancy-associated malaria in a mouse model (P. berghei ANKA in BALB/c mice). The extracellular region of var2CSA on the P falciparum IE membrane comprises six DBL domains and a CIDRpam module arranged in the following configuration DBL1X-DBL2X-CIDRpam-DBL3X-DBL4ε -DBL5ε -DBL6ε followed by a single transmembrane helix and a cytoplasmic acidic terminal sequence. In soluble form (no specific orientation) this protein binds specifically and with high affinity to CSA. Furthermore it has to be noted that the var2CSA protein folded upon itself (20) indicates that the DBL1 and DBL6 are close to each other and therefore close to the IE membrane (26) which reinforces the relevance of the construct used. Due to the anchoring using the EMAP1 protein, the extracellular region of var2CSA on the surface on P. berghei erythrocytes was expressed in a mirror orientation DBL6ε-DBL5ε- DBL4ε- DBL3X- CIDRpam- DBL2X- DBL1X. Evidence is provided that DBL1-6 protein is expressed in Pb-VAR but only a proportion of IEs containing schizonts (less than 10%) exposed the protein on the outer membrane as revealed by staining live IEs using anti-DBL1-6 antibodies. Our data also indicate that surface DBL1-6 maintains recDBL1-6 conformation and enhances binding of IEs to placental tissue in vitro. Other mutants

Tagged: Mutant parasite with a tagged gene

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Details of the target gene

Gene Model of Rodent Parasite PBANKA_0836800

Gene Model P. falciparum ortholog Not available

Gene product erythrocyte membrane associated protein 1; fam-a protein

Gene product: Alternative name EMAP1

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Details of the genetic modification

Name of the tag a synthetic 3D7-DBL1X-6ε gene (var2csa; PF3D7_1200600)

Details of tagging C-terminal

Additional remarks: tagging A transgenic 1037cl1 that expresses C-terminally var2csa-tagged EMAP1 (gene model PBANKA_0836900; emap1::var2csa; line 1653cl3) was generated as previously reported for C-terminally mcherry-tagged EMAP1 (RMgm-690). The synthetic 3D7-DBL1X-6ε gene (var2csa gene; PF3D7_1200600) (Srivastava A et al. 2010, PNAS 107, 4884) was first subcloned into PCR2.1 Topo (Invitrogen, NL) and the C-terminally region of emap1 from pL1534 (BamHI/SpeI) was fused N-terminally to the 3D7-DBL1X-6ε gene (NdeI/SpeI). The PCR-amplified 3’utr of the calmodulin (cam) gene (PBANKA_1010600) was then cloned as a XbaI fragment downstream the emap1:3D7-DBL1X-6ε fusion using primer set: 5397: 5’ ATCTAGATAATTATTAATATATATGAATATATATACATCGTTG and 5398: 5’ ATCTAGAGGTACCGACCATATAAGAATTAAC.) Finally, after destruction of the NdeI, the KpnI fragment containing the emap1:: 3D7-DBL1X-6ε – 3’utr cam was cloned into pL0007 to create pL1593. The final DNA construct was linearized with NdeI before transfection.

Commercial source of tag-antibodies

Type of plasmid/construct (Linear) plasmid single cross-over

PlasmoGEM (Sanger) construct/vector used No

Modified PlasmoGEM construct/vector used No

Plasmid/construct map

Plasmid/construct sequence

AATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTT

AATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACC

GATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGCGCCTGATGCGGTATTTT

CTCCTTACGCATCTGTGCGGTATTTCACACCGCATATATGGTGCACTCTCAGTACAATCT

GCTCTGATGCCGCATAGTTAAGCCAGCCCCGACACCCGCCAACACCCGCTGACGCGCCCT

GACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCT

GCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGACGAAAGGGCCTCGTGA

TACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCA

CTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATA

TGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGA

GTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTC

CTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTG

CACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCC

CCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTAT

CCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACT

TGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAAT

TATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGA

TCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCC

TTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGA

TGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAG

CTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGC

GCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGT

CTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCT

ACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTG

CCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTG

ATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCA

TGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGA

TCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAA

AACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGA

AGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGT

TAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGT

TACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGAT

AGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCT

TGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCATTGAGAAAGCGCCA

CGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAG

AGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTC

GCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGA

AAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACA

TGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAG

CTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGG

AAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCT

GGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTT

AGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTG

GAATTGTGAGCGGTAAACAATTTCACACAGGAAACAGCTTGACCATGATTACGCCAAGCT

TGATAATTCCTGCAGCCCAGCTTAATTCTTTTCGAGCTCTTTATGCTTAAGTTTACAATT

TAATATTCATACTTTAAGTATTTTTTGTAGTATCCTAGATATTGTGCTTTAAATGCTCAC

CCCTCAAAGCACCAGTAATATTTTCATCCACTGAAATACCATTAAATTTTCAAAAAAATA

CTATGCATATAATGTTATACATATAAACATAAAACGCCATGTAAATCAAAAAATATATAA

AAATATGTATAAAAATAAATATGCACTAAATATAAGCTAATTATGCATAAAAATTAAAGT

GCCCTTTATTAACTAGTCGTAATTATTTATATTTCTATGTTATAAAAAAATCCTCATATA

ATAATATAATTAATATATGTAATGTTTTTTTTATTTTATAATTTTAATATAAAATAATAT

GTAAATTAATTCAAAAAATAAATATAATTGTTGTGAAACAAAAAACGTAATTTTTTCATT

TGCCTTCAAAATTTAAATTTATTTTAATATTTCCTAAAATATATATACTTTGTGTATAAA

TATATAAAAATATATATTTGCTTATAAATAAATAAAAAATTTTATAAAACATAGGGGGAT

CCATGGTTGGTTCGCTAAACTGCATCGTCGCTGTGTCCCAGAACATGGGCATCGGCAAGA

ACGGGGACCTGCCCTGGCCACCGCTCAGGAACGAATTTAGATATTTCCAGAGAATGACCA

CAACCTCTTCAGTAGAAGGTAAACAGAATCTGGTGATTATGGGTAAGAAGACCTGGTTCT

CCATTCCTGAGAAGAATCGACCTTTAAAGGGTAGAATTAATTTAGTTCTCAGCAGAGAAC

TCAAGGAACCTCCACAAGGAGCTCATTTTCTTTCCAGAAGTCTAGATGATGCCTTAAAAC

TTACTGAACAACCAGAATTAGCAAATAAAGTAGACATGGTCTGGATAGTTGGTGGCAGTT

CTGTTTATAAGGAAGCCATGAATCACCCAGGCCATCTTAAACTATTTGTGACAAGGATCA

TGCAAGACTTTGAAAGTGACACGTTTTTTCCAGAAATTGATTTGGAGAAATATAAACTTC

TGCCAGAATACCCAGGTGTTCTCTCTGATGTCCAGGAGGAGAAAGGCATTAAGTACAAAT

TTGAAGTATATGAGAAGAATGATTAAGGATCCCGTTTTTCTTACTTATATATTTATACCA

ATTGATTGTATTTATAACTGTAAAAATGTGTATGTTGTGTGCATATTTTTTTTTGTGCAT

GCACATGCATGTAAATAGCTAAAATTATGAACATTTTATTTTTTGTTCAGAAAAAAAAAA

CTTTACACACATAAAATGGCTAGTATGAATAGCCATATTTTATATAAATTAAATCCTATG

AATTTATGACCATATTAAAAATTTAGATATTTATGGAACATAATATGTTTGAAACAATAA

GACAAAATTATTATTATTATTATTATTTTTACTGTTATAATTATGTTGTCTCTTCAATGA

TTCATAAATAGTTGGACTTGATTTTTAAAATGTTTATAATATGATTAGCATAGTTAAATA

AAAAAAGTTGAAAAATTAAAAAAAAACATATAAACACAAATGATGTTTTTTCCTTCAATT

TCGGGTACCGAGCTCGGATCCACTAGTCACAAGTGTGCACTGATCCTGAAGAAATCAAAA

AAGTAACGGAAATTATGGACGAAGCTGTATATCGCTTAAAATACCATTCTAATTTTAATA

ATGGTTATAATCATTATTATGTTGGCAATGAAGAGTTATCTTTATGTTTTACGGAAGACA

ACGGAATAAAATTTGAAAGATTCCATCTTGTGATCCCCAATCCAGATAATGTGCGAAATA

AGAAACATATTTTTTTATGTTATAAAATTAAGTTCAAATAAAAAAATATAATTATTATAT

ATATCCATATATTATTTTTTGTTGCCATTAGTATGAAGAAATAGTAAAAATGTTTTACAC

ATTTAGAGGCATTCCCTATATGGATAACTTCGATTTTACGGGTATATGAAAAATAATCAA

TTAAGCATTTTAATATATTATTATTATTATTATTATTATTATTATTACTCTCATGTTATA

TTCATTGTTTTATTTTTTAATTTTATATGCTACCATTTTTCATCCGTCATATGTCATTTT

TTGTAATATATTAACTTACATGATGAAAAATTATAATTTTTTTTAGAAAAATTGGTTCAC

GAATATTATTCAAATTTATCATTGATGTATAATAGTTATGAAAGCCGAACCACACCATTC

TGTGGATATAGTTTTGTTTTATCTGCAGATATTCAAGTAAGGAAAACTTCTTTTCATTTA

CCCCTCTATATATCATATTTTTCATATTTACACAATTTATTCAAATTCAAATACATTTGT

ATTAATTTTGTAGGTATCAGAAGACATAACAATGTTTGTTCAAACATCAGTAGATATAGA

TGACCACAACGAGTTCGATACCGAAAAATATAAAAACACTATTGTAGAAAGTGCAAATTC

ATTAAATATTGATGTTTATGCGAACGAATATGACCAAATGGAAAACAAAACAAAATTGTT

TGTTAATTTATCTGGAATTATCGTTAAAAAAAAAAGCGACAGTGTTCGTGTTACATACGT

CAACTCTGTAAGCAATATAAAAATTATAATAACATAATAATTTTATTTTATCATTATCAA

AGATATATACCTTTTCTATAATTTGCCATATTTTAATATTTCTATTCATCTACAAACAAA

TTGACCTATTTTATATTTCTATTCATTCTTTTCTTTTTCCGTAGATTCATAATGGTGACT

CCTTTCCAAAAGATTTTTGTGACAAACAATTTAGAGTAAAAAATATGCTATATTTGATCG

ATTTATTAAAAAATAGTTTTAAAGGATCTATGGATTCCGGCACAAATGATCCTTGTGACC

GCATCCCCCCTCCCTATGGGGACAATGATCAGTGGAAATGTGCCATTATCCTGTCCAAAG

TGAGCGAAAAGCCTGAAAATGTGTTCGTGCCCCCACGGAGGCAGCGCATGTGTATCAATA

ATCTGGAAAAACTGAACGTGGATAAAATCCGGGATAAACACGCCTTCCTGGCCGACGTGC

TGCTGACAGCCAGAAACGAGGGCGAGAGGATTGTGCAGAACCACCCCGACACAAACTCTG

CCAATGTGTGCAACGCCCTGGAGCGGTCCTTCGCCGACATTGCCGATATCATCCGGGGCA

CCGATCTGTGGAAGGGGACCAACAGCAACCTGGAGCAGAATCTGAAACAGATGTTTGCCA

AGATCCGGGAGAACGACAAGGTGCTGCAGGATAAGTATCCCAAGGACCAGAACTACAGGA

AGCTGAGAGAGGACTGGTGGAACGCCAACAGGCAGAAAGTGTGGGAAGTGATTACCTGCG

GCGCCAGAAGCAACGACCTGCTGATCAAGAGAGGCTGGAGGACCTCTGGGAAATCCAACG

GCGACAACAAGCTGGAGCTGTGCCGGAAGTGCGGCCACTACGAGGAGAAAGTGCCAACAA

AACTGGATTACGTGCCCCAGTTTCTGAGGTGGCTGACCGAGTGGATCGAGGACTTCTACA

GAGAGAAACAGAACCTGATTGATGACATGGAGAGGCACCGGGAGGAGTGCACAAGCGAGG

ACCACAAGTCCAAAGAGGGCACATCCTATTGCAGCACCTGTAAAGATAAGTGCAAAAAAT

ACTGCGAATGCGTGAAGAAGTGGAAGTCTGAGTGGGAAAACCAGAAAAATAAGTATACCG

AACTGTATCAGCAGAACAAGAATGAGGCCTCCCAGAAGAACACCTCTAGGTACGACGACT

ACGTGAAGGACTTCTTCAAGAAACTGGAGGCCAATTATGCCTCCCTGGAGAACTATATTA

AGGGAGATCCTTACTTCGCCGAGTACGCCACCAAGCTGTCCTTTATCCTGAACTCTGCCG

ACGCCAACAATCCTAGCGAGAAAATCCAGAAGAACAATGACGAGGTGTGCAACTGCAATG

AAAGCGGGATCGCCTCCGTGGAGCAGGAACAGATTTCCGATCCTAGCTCTAATAAAGCCT

GTATTACCCACAGCTCTATCAAGGCCAACAAGAAGAAGGTGTGTAAGCACGTGAAGCTGG

GCGTGCGCGAGAATGACAAAGACCTGAGAGTGTGCGTGATCGAGCACACATCTCTGTCCG

GAGTGGAAAATTGCTGCTGTCAGGATTTCCTGCGCATTCTGCAGGAAAACTGCGCCGATA

ACAAGGCCGGCTCTTCCAGCAACGGAGCCTGCAATAATAAAAACCAGGAAGCCTGCGAGA

AGAATCTGGAGAAAGTGCTGGCCAGCCTGACAAACTGTTACAAGTGCGATAAGTGTAAGT

CCGAGCAGTCTAAAAAGAATAACAAGAATTGGATCTGGAAAAAGAGCTCCGGCAAGGAGG

GGGGGCTGCAGAAGGAGTACGCCAACACCATTGGGCTGCCACCAAGAACCCAGTCCCTGT

GCCTGGTGGTGTGTCTGGATGAGAAAGGAAAGAAAACCCAGGAGCTGAAAAACATCAGAA

CCAATTCCGAGCTGCTGAAGGAGTGGATCATCGCCGCCTTCCACGAAGGGAAGAATCTGA

AACCCTCCCACGAGAAGAAAAATGACGATAATGGCAAAAAACTGTGCAAGGCCCTGGAGT

ATTCTTTCGCCGATTATGGCGATCTGATTAAGGGGACCTCCATCTGGGACAACGAGTACA

CCAAGGACCTGGAGCTGAACCTGCAGAAGATCTTCGGGAAGCTGTTCCGCAAATATATCA

AGAAAAATAACGCCGCCGAACAGGACACATCCTACTCCTCTCTGGACGAGCTGAGGGAGT

CCTGGTGGAATACAAACAAAAAGTATATTTGGCTGGCCATGAAGCACGGAGCCGGCATGA

ACTCTACCACCTGCTGTGGCGACGGCTCTGTGACCGGAAGCGGCAGCTCCTGCGACGATA

TCCCAACCATCGACCTGATTCCCCAGTATCTGAGGTTCCTGCAGGAATGGGTGGAACACT

TCTGTAAACAGCGCCAGGAAAAGGTGAAGCCCGTGATTGAGAACTGTAAGTCCTGCAAGG

AGTCCGGGGGCACATGCAACGGCGAGTGTAAAACCGAATGCAAGAACAAGTGCGAGGTGT

ATAAGAAGTTCATCGAAGACTGCAAAGGCGGGGATGGGACCGCCGGCTCTTCCTGGGTGA

AAAGGTGGGATCAGATCTATAAGAGGTACAGCAAGTATATTGAAGACGCCAAAAGGAATA

GAAAAGCCGGGACAAAAAATTGCGGGCCATCTTCTACCACCAATGCCGCCGAGAATAAGT

GTGTGCAGTCCGATATTGACTCCTTCTTTAAGCACCTGATCGATATCGGCCTGACAACCC

CAAGCTCTTACCTGTCCATCGTGCTGGATGATAATATTTGCGGGGCCGACAAGGCCCCAT

GGACAACCTATACCACATATACAACCACCGAGAAATGTAACAAGGAGACCGACAAGTCCA

AGCTGCAGCAGTGCAATACCGCCGTGGTGGTGAACGTGCCTTCCCCACTGGGAAACACCC

CCCACGGGTATAAGTATGCCTGCCAGTGCAAAATCCCTACCAATGAAGAAACCTGTGATG

ACAGGAAAGAATACATGAACCAGTGGAGCTGCGGAAGCGCCAGAACAATGAAGAGAGGAT

ATAAAAACGACAACTATGAGCTGTGTAAATACAATGGGGTGGATGTGAAACCAACAACCG

TGAGAAGCAATTCCGCCAAACTGGATGACAAGGATGTGACCTTCTTCAATCTGTTCGAGC

AGTGGAACAAGGAGATTCAGTACCAGATTGAGCAGTACATGACCAACACAAAGATCAGCT

GCAACAACGAGAAGAATGTGCTGTCTCGGGTGAGCGATGAAGCCGCCCAGCCTAAGTTCT

CCGATAATGAGAGAGATAGGAACAGCATTACCCACGAAGACAAGAATTGCAAGGAAAAAT

GCAAGTGCTATTCTCTGTGGATTGAGAAGATCAACGATCAGTGGGATAAACAGAAGGATA

ACTACAACAAATTCCAGCGGAAGCAGATCTATGATGCCAACAAAGGAAGCCAGAACAAGA

AAGTGGTGAGCCTGTCCAATTTCCTGTTTTTCAGCTGTTGGGAAGAGTACATTCAGAAGT

ATTTTAACGGAGACTGGAGCAAGATTAAGAATATCGGGTCTGATACCTTTGAGTTTCTGA

TCAAGAAATGCGGAAACGACAGCGGGGATGGCGAAACCATCTTCAGCGAGAAACTGAACA

ACGCCGAGAAGAAATGTAAAGAAAATGAGTCCACCAACAATAAGATGAAAAGCTCTGAGA

CCAGCTGTGACTGTTCTGAGCCCATTTATATCCGCGGGTGTCAGCCTAAAATCTACGACG

GGAAAATCTTTCCAGGCAAAGGGGGAGAGAAACAGTGGATCTGCAAGGACACAATCATCC

ACGGCGACACCAACGGCGCCTGTATCCCTCCCAGAACCCAGAACCTGTGTGTGGGGGAAC

TGTGGGACAAGCGGTACGGAGGCAGGTCTAACATCAAAAACGACACCAAAGAATCTCTGA

AGCAGAAGATCAAAAATGCCATTCAGAAAGAAACCGAGCTGCTGTACGAGTATCACGATA

AGGGCACCGCCATCATCTCTAGGAACCCTATGAAAGGCCAGAAAGAGAAGGAGGAGAAAA

ACAACGATTCTAACGGCCTGCCTAAGGGCTTCTGCCACGCCGTGCAGCGCTCCTTCATTG

ACTACAAAAATATGATCCTGGGAACATCTGTGAATATCTACGAGTACATTGGGAAACTGC

AGGAGGATATTAAGAAGATCATCGAAAAAGGGACCACCAAGCAGAACGGGAAGACAGTGG

GGAGCGGGGCCGAGAATGTGAATGCCTGGTGGAAGGGCATCGAAGGGGAGATGTGGGACG

CCGTGAGGTGTGCCATTACAAAGATTAATAAAAAACAGAAGAAAAATGGAACCTTTTCCA

TCGACGAGTGCGGCATCTTCCCTCCTACCGGCAATGACGAGGATCAGAGCGTGAGCTGGT

TCAAGGAGTGGTCTGAGCAGTTTTGCATCGAGCGCCTGCAGTACGAGAAAAACATTAGAG

ACGCCTGCACAAACAACGGGCAGGGCGATAAGATCCAGGGGGACTGCAAGCGGAAGTGCG

AGGAATACAAAAAATACATCAGCGAGAAGAAACAGGAGTGGGACAAACAGAAGACAAAGT

ACGAAAACAAATATGTGGGCAAAAGCGCCTCCGACCTGCTGAAGGAAAACTACCCCGAAT

GTATTAGCGCCAATTTTGATTTTATCTTCAATGACAACATCGAGTATAAGACCTATTATC

CTTATGGCGATTACTCTTCCATCTGTAGCTGTGAACAGGTGAAATATTATGAATACAACA

ACGCCGAAAAGAAAAATAACAAGCTTCTGTGCCACGAGAAAGGAAATGACAGAACCTGGT

CCAAAAAATACATTAAGAAGCTGGAAAACGGAAGAACCCTGGAAGGCGTGTACGTGCCCC

CTAGGAGGCAGCAGCTGTGCCTGTACGAGCTGTTCCCCATCATCATCAAGAACAAAAACG

ACATTACAAATGCCAAAAAAGAACTGCTGGAGACACTGCAGATCGTGGCCGAGCGCGAAG

CCTACTACCTGTGGAAGCAGTATCACGCCCACAATGACGCCACCTATCTGGCCCACAAGA

AGGCCTGTTGCGCCATCAGGGGATCTTTTTACGACCTGGAAGACATTATCAAGGGAAACG

ATCTGGTGCACGACGAGTATACAAAGTATATTGATTCCAAGCTGAACGAGATTTTCGACA

GCAGCAATAAAAACGATATTGAGACCAAGCGGGCCCGGACAGACTGGTGGGAGAATGAGG

CCATCGCCGTGCCTAATATTGCCGGGGCCAACAAGGCCGACCCAAAGACAATCCGCCAGC

TGGTGTGGGACGCCATGCAGTCCGGAGTGCGCAAGGCCATCGATGAGGAAAAGGAGAAGA

AAAAGCCCAATGAAAATTTCCCCCCCTGTATGGGAGTGCAGCACATCGGCATCGCCAAGC

CCCAGTTCATCCGCTGGCTGGAAGAGTGGACCAATGAGTTCTGCGAGAAGTACACCAAGT

ACTTCGAGGATATGAAAAGCAACTGCAACCTGAGAAAAGGCGCCGACGATTGCGACGATA

ACTCCAATATCGAGTGTAAGAAGGCCTGTGCCAATTATGCCAACTGGCTGAACCCAAAGA

GAATCGAATGGAATGGAATGAGCAATTACTACAATAAAATTTACAGAAAAAGCAACAAAG

AGTCTGAGGATGGCAAGGACTACAGCATGATCATGGAGCCAACAGTGATCGATTATCTGA

ACAAACGCTGCAATGGCGAAATCAATGGAAACTACATCTGCTGTAGCTGCAAGAACATCG

GCGAAAACAGCGCCTCCGGCACAGTGAACAAGAAGCTGCAGAAAAAGGAGACCCAGTGTG

AAGACAACAAAGGCCCCCTGGATCTGATGAACAAGGTGCTGAACAAAATGGATCCCAAGT

ATTCTGAGCACAAGATGAAATGCACCGAGGTGTACCTGGAGCACGTGGAGGAGCAGCTGA

AGGAGATCGACAATGCCATCAAGGATTATAAGCTGTACCCTCTGGACCGCTGTTTCGACG

ATAAATCCAAGATGAAGGTGTGCGACCTGATCGGAGACGCCATCGGCTGTAAGCACAAGA

CAAAGCTGGACGAACTGGATGAGTGGAATGACGTGGATATGAGGGACCCATATAACAAAT

ACAAAGGCGTGCTGATTCCCCCCCGCCGCAGGCAGCTGTGTTTTTCCAGAATTGTGAGAG

GCCCAGCCAACCTGAGGAACCTGAAGGAGTTCAAGGAGGAAATCCTGAAGGGGGCCCAGT

CCGAAGGAAAGTTTCTGGGAAATTATTACAATGAGGACAAGGACAAGGAAAAGGCCCTGG

AAGCCATGAAAAACTCCTTCTACGACTACGAGTATATTATTAAGGGGTCCGACATGCTGA

CAAATATCCAGTTTAAAGATATCAAGAGGAAGCTGGACCGCCTGCTGGAAAAGGAGACCA

ATAATGCCGAGAAGGTGGACGACTGGTGGGAGACAAACAAGAAAAGCATTTGGAATGCCA

TGCTGTGTGGGTACAAGAAAAGCGGAAACAAAATTATCGATCCCAGCTGGTGCACCATCC

CCACCACCGAGACCCCACCCCAGTTTCTGCGCTGGATCAAGGAGTGGGGGACCAACGTGT

GCATTCAGAAGGAGGAACACAAAGAGTATGTGAAGTCTAAGTGTTCCAACGTGGCCAACC

TGGGCGCCCAGGAGTCCGAATCTAAGAACTGTACCAGCGAAATTAAGAAGTATCAGGAGT

GGAGCAGAAAGAGATCTATTCAGTGGGAGGCCATTTCCGAAGGATATAAGAAGTATAAAG

GGATGGACGAGTTTAAGAATACCTTTAAGAATATTAAGGAGCCAGATGCCAATGAGCCCA

ACGCCAATGAGTATCTGAAAAAGCACTGTTCTAAGTGCCCCTGCGGCTTCAATGACATGC

AGGAGATCACCAAGTATACCAATATCGGGAATGAGGCCTTTAAACAGATCAAGGAACAGG

TGGACATCCCAGCCGAGCTGGAGGATGTGATCTACAGGCTGAAGCACCACGAATACGATA

AAGGCAACGATTACATCTGTAATAAATACAAGAACATTAATGTGAATATGAAGAAAAATA

ACGATGACACCTGGACAGATCTGGTGAAAAATAGCTCCGATATTAACAAAGGAGTGCTGC

TGCCTCCAAGACGGAAAAACCTGTTCCTGAAGATCGATGAAAGCGACATCTGTAAGTATA

AACGGGACCCCAAGCTGTTTAAAGATTTTATCTACTCCAGCGCCATCTCCGAGGTGGAAC

GCCTGAAGAAGGTGTACGGCGAGGCCAAAACAAAGGTGGTGCACGCCATGAAGTACTCTT

TTGCCGACATTGGGTCCATCATCAAGGGCGATGACATGATGGAAAACAATTCCTCCGATA

AAATCGGCAAGATCCTGGGGGATGGAGTGGGCCAGAATGAGAAGAGAAAAAAGTGGTGGG

ACATGAACAAGTATCACATTTGGGAGTCCATGCTGTGCGGCTATAAACACGCCTACGGAA

ACATCGCCGAGAATGACCGCAAGATGCTGGACATCCCAAATAATGATGACGAGCACCAGT

TCCTGCGGTGGTTCCAGGAGTGGACCGAGAATTTCTGCACCAAAAGAAACGAGCTGTACG

AGAACATGGTGACCGCCTGCAACTCTGCCAAATGTAACACCTCCAACGGATCTGTGGATA

AAAAGGAGTGCACAGAGGCCTGCAAGAACTATGCCAACTTCATCCTGATCAAGAAAAAGG

AGTATCAGTCTCTGAACTCCCAGTATGACATGAATTATAAAGAGACCAAGGCCGAGAAGA

AAGAGAGCCCAGAGTACTTTAAGGACAAGTGCAACGGCGAATGTAGCTGCCTGAGCGAGT

ATTTTAAGGACGAGACCAGGTGGAAGAACCCTTACGAGACCCTGGACGACACCGAGGTGA

AAAATAACTGCATGTGCAAGCCCCCTCCACCCGCCTCTAGATAATTATTAATATATATGA

ATATATATACATCGTTGTATGCGTAATATACGTGTAACTGTTTTTTTTAATTTTTATATT

AACTTTTCCTTATACATTTTAATGCCATTAACTAATAATATTTTACTTGAAAAAATTCTA

AATTTTTATAACTCTAATATAAAATCTGTGAATAATAAAAAATTAGCATAAAGTACGAAT

TAAACATCGAATATTTGTTTTAAAGGGGCACACATTATGTGCATATAATTTTATTCTTTG

AATTTAATTTTTTGCTTATAATTAGATTAAATTAATATAATTTTTTTATTGAAAATAAAA

ACTTATTTTAAAAAATTAACAAATGGAAGTTAATGAAAAATACGAAAAAAGTAAAGGGTT

AATTCTTATATGGTCGGTACCCTCGAGGCTAGCGATATCTGATCACCCGGGGCGGCCGCG

Restriction sites to linearize plasmid NdeI

Selectable marker used to select the mutant parasite hdhfr

Promoter of the selectable marker eef1a

Selection (positive) procedure pyrimethamine

Selection (negative) procedure No

Additional remarks genetic modification A transgenic 1037cl1 that expresses C-terminally var2csa-tagged EMAP1 (gene model PBANKA_0836900; emap1::var2csa; line 1653cl3) was generated as previously reported for C-terminally mcherry-tagged EMAP1 (RMgm-690). The synthetic 3D7-DBL1X-6ε gene (var2csa gene; PF3D7_1200600) (Srivastava A et al. 2010, PNAS 107, 4884) was first subcloned into PCR2.1 Topo (Invitrogen, NL) and the C-terminally region of emap1 from pL1534 (BamHI/SpeI) was fused N-terminally to the 3D7-DBL1X-6ε gene (NdeI/SpeI). The PCR-amplified 3’utr of the calmodulin (cam) gene (PBANKA_1010600) was then cloned as a XbaI fragment downstream the emap1:3D7-DBL1X-6ε fusion using primer set: 5397: 5’ ATCTAGATAATTATTAATATATATGAATATATATACATCGTTG and 5398: 5’ ATCTAGAGGTACCGACCATATAAGAATTAAC.) Finally, after destruction of the NdeI, the KpnI fragment containing the emap1:: 3D7-DBL1X-6ε – 3’utr cam was cloned into pL0007 to create pL1593. The final DNA construct was linearized with NdeI before transfection.

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 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
Sequence Primer 5
Additional information primer 5
Sequence Primer 6
Additional information primer 6

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