Summary

RMgm-628
Malaria parasiteP. berghei
Genotype
TaggedGene model (rodent): PBANKA_0932000; Gene model (P.falciparum): PF3D7_1116000; Gene product: rhoptry neck protein 4 (RON4)
Name tag: mCherry
Phenotype Asexual bloodstage;
Last modified: 20 October 2011, 16:38
  *RMgm-628
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene tagging
Reference (PubMed-PMID number) Not published (yet)
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/ResearcherC.J. Janse; O. Klop; B.M. Franke-Fayard
Name Group/DepartmentLeiden Malaria Research Group
Name InstituteLeiden University Medical Center
CityLeiden
CountryThe Netherlands
Name of the mutant parasite
RMgm numberRMgm-628
Principal name1964
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationNo
Phenotype
Asexual blood stageSchizonts express mCherry-tagged RON4 as shown by fluorescence microscopy. Merozoites of mature schizonts show a 'rhoptry-like' fluorescence pattern (see also '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 an mCherry-tagged version of RON4 (rhoptry neck protein 4)

Protein (function)
RON4 forms a complex with different other proteins (e.g. AMA1, RON2, RON5) during invasion of the merozoite at the tight junction, an aperture in the host cell through which the parasite pulls itself before settling into a newly formed parasitophorous vacuole.

Phenotype
Both the genotype and phenotype of this mutant has not been analysed in detail. The mutant parasites have not been cloned after transfection and drug-selection.
Schizonts express mCherry-tagged RON4 as shown by fluorescence microscopy. Merozoites of mature schizonts show a 'rhoptry-like' fluorescence pattern.
The mutant can be obtained from the
Leiden Malaria Research Group.

Additional information

Figure: Southern analysis of PFG-separated schizonts of transfected parasites after drug-selection


 

Other mutants
RMgm-214: Unsuccessful attempts to disrupt the ron2 gene (PBANKA_131570)
RMgm-213: A mutant expressing a cmyc-tagged RON2 protein (PBANKA_131570)


  Tagged: Mutant parasite with a tagged gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0932000
Gene Model P. falciparum ortholog PF3D7_1116000
Gene productrhoptry neck protein 4
Gene product: Alternative nameRON4
Details of the genetic modification
Name of the tagmCherry
Details of taggingC-terminal
Additional remarks: tagging
Commercial source of tag-antibodies
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
CCGCGGTGGCGGCCGCTCTAGAACTAGTGCATCCATATTAATGCAACAATGGAAATATTA
TCAAAATGCTTTAGGGTTTGAAGATAATAATGATCATGCATTTTTAGGTTTAATCGATGA
ATTATTAACAATAGACAAAAGATATAGTAAAAATGAAGAACATAGTAAATCTTTAGAACA
AATGAAAAAATCAAAAGTATTTAATTATTGTACTAAACTTATGAGAACAAGTGGAAACAT
TTCAACAATACCATTTAATCATGAAAACAATAAAACTCCAAGTTCATCCATTATTGGTTC
ATTAGGAAATTTAGTTAAGGCACATATAAGTGGATATTATGCAGCTACTGCTCAAAGAAT
TAACTCTTATTTTAATTATTCTAACAATAAAGATCAAAAGAAAGCACTATACAAAGTCAT
ATCAGTTTGTACTCTCTTAAATTTAACAGATATTTTATTTAATTGCACAGATCCACAATC
AACTAATTCAGCAAATTTGAACATTTTGAAGCTAAATGTATTAAATACTAAAGGAAAAGA
AATTCTATATCGCCTTTATAATTTAAGCTACTTATTAGAGAGCGAAAACGAAGCAATTAA
AGAAGTGTGTAATCCTAAAAATGCCTCAGTTGGTAATGCTAAAATGCGATGAATTATTTC
TTTGTATATTTTTTTGTGTGGATATGTGCTTTTTTTGTAGGTGTTATACGGGATTTCAAA
TATTGCAAAATATGCATATATTTCATATGCAATATTTTCGAATTTCATTTTATCATTTGA
TATGAACTTATATACATATTTGCGCTTGTTTGATTTTCATAAGAATATATGTATTTATAA
TTTCACTTTTATTTAATTATTTTTTACAGATAAATCGTTACAAAAATTGTTAGAATTACT
TTCATCAAACTCACACAACGTATTAGCACATGAAATGGAAAAGAGAGCTCTTGATACTGA
TTATATTAACGAGGAAATAAACAATATAAGTGATTTTGATCATAATGAAAAAGACAATGG
AGAAGATGATGTTGAGAAAGCCATTTTTGATGATTTAGGATCCGGTGGAGGTGGAAGATC
CATGGTGAGCAAGGGCGAGGAGGATAACATGGCCATCATCAAGGAGTTCATGCGCTTCAA
GGTGCACATGGAGGGCTCCGTGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGG
CCGCCCCTACGAGGGCACCCAGACCGCCAAGCTGAAGGTGACCAAGGGTGGCCCCCTGCC
CTTCGCCTGGGACATCCTGTCCCCTCAGTTCATGTACGGCTCCAAGGCCTACGTGAAGCA
CCCCGCCGACATCCCCGACTACTTGAAGCTGTCCTTCCCCGAGGGCTTCAAGTGGGAGCG
CGTGATGAACTTCGAGGACGGCGGCGTGGTGACCGTGACCCAGGACTCCTCCCTGCAGGA
CGGCGAGTTCATCTACAAGGTGAAGCTGCGCGGCACCAACTTCCCCTCCGACGGCCCCGT
AATGCAGAAGAAGACCATGGGCTGGGAGGCCTCCTCCGAGCGGATGTACCCCGAGGACGG
CGCCCTGAAGGGCGAGATCAAGCAGAGGCTGAAGCTGAAGGACGGCGGCCACTACGACGC
TGAGGTCAAGACCACCTACAAGGCCAAGAAGCCCGTGCAGCTGCCCGGCGCCTACAACGT
CAACATCAAGTTGGACATCACCTCCCACAACGAGGACTACACCATCGTGGAACAGTACGA
ACGCGCCGAGGGCCGCCACTCCACCGGCGGCATGGACGAGCTGTACAAGTAATCTAGAAT
TATTAATATATATGAATATATATACATCGTTGTATGCGTAATATACGTGTAACTGTTTTT
TTTAATTTTTATATTAACTTTTCCTTATACATTTTAATGCCATTAACTAATAATATTTTA
CTTGAAAAAATTCTAAATTTTTATAACTCTAATATAAAATCTGTGAATAATAAAAAATTA
GCATAAAGTACGAATTAAACATCGAATATTTGTTTTAAAGGGGCACACATTATGTGCATA
TAATTTTATTCTTTGAATTTAATTTTTTGCTTATAATTAGATTAAATTAATATAATTTTT
TTATTGAAAATAAAAACTTATTTTAAAAAATTAACAAATGGAAGTTAATGAAAAATACGA
AAAAAGTAAAGGGTTAATTCTTATATGGTCGGTACCGAGCTCGAATTCGATATCGAAATT
GAAGGAAAAAACATCATTTGTGTTTATATGTTTTTTTTTAATTTTTCAACTTTTTTTATT
TAACTATGCTAATCATATTATAAACATTTTAAAAATCAAGTCCAACTATTTATGAATCAT
TGAAGAGACAACATAATTATAACAGTAAAAATAATAATAATAATAATAATTTTGTCTTAT
TGTTTCAAACATATTATGTTCCATAAATATCTAAATTTTTAATATGGTCATAAATTCATA
GGATTTAATTTATATAAAATATGGCTATTCATACTAGCCATTTTATGTGTGTAAAGTTTT
TTTTTTCTGAACAAAAAATAAAATGTTCATAATTTTAGCTATTTACATGCATGTGCATGC
ACAAAAAAAAATATGCACACAACATACACATTTTTACAGTTATAAATACAATCAATTGGT
ATAAATATATAAGTAAGAAAAACGGGATCAAAGCTAGCTTCTGTATTTCCGCTAGACAGC
CATCTCCATCTGGATTCGTCCGTGCGGGACGTAGCCCACGACCTCAAAATCCTCGGCGGT
GAAATCGTCGATTTCCTTGATGCGTTCCTTGTTGAGGATGTTCACAATGGGGAACGGTCT
CGGTTCTCTCCGCAGCTGCTCTTTTAAAGCCTCGACATGGTTCGTGTAGACATGCGTGTT
CCCCATGAAGTGAATGAACTCCTTAGGTTTTAGGTTGCAGACGTGTGCAACCATGAGCGT
CAAAAGCGAATAGGAAGCGATGTTGAAGGGGACGCCGAGGCCGACATCGCACGACCGCTG
ATACATGATGCACGACAGCTCCTTCTGGTCGTTCACGTAGAACTGGCACAACAAGTGACA
AGGCGGCAGCGCCATTTCGTCCAGCGCTGCAGGATTCCAGGCAGTCATGAGCATGCGACG
ATCTGTTGGATTCGTTCTCAGCATCTGGATCACATTCTTCAGCTGGTCGACGCCCTGCCC
TGTGTAGTCTGTGTGCATGTCTTTGTATGCCGCGCCGAAGTGTCTCCACTGGAAGCCGTA
GCCCGGGCCGATGTCTCCGACCTCTCGGTGGGGGAGATTGCGCGAATCGAGGAACTCGCG
TGTCACATTCTTGTCCCAGATCTTCACGCCCTTCTCAGAAAGATGGTTTGCGTTCGTGTC
GCCGCGAATGAACCACAGCAACTCTTCGAGGACCCCTTTCCAGAACACACGCTTTGTGGT
GAGAAGTGGAAAGGCCTGATCCAGCGAGTAGCGCATAGTGCAGCCGAATTTGGAGATGAC
ACCAACGCCCGTTCGGTCATCCATTGTCCTTCCATTGTTAATAATGTCGGCAATGAGATC
AAGGTACTGGAACTCTTCATGGCCTCTAAAGTGAACATGCGGAACGGCCCGAATCAGTTC
CTTTTGCTCGCGTTTTTTCCGGTCTTCTTCGTCCATCCACGCCAACACCGGGGCAATGGC
TGCGGCCGAAGAAGGAGCCTGCAACCCGTGCACGGGAGTTGTCTCCCTCGTGGACGTCAA
GGAGCTCATTGCGTTGCTCGGTTCCGCAGTGGCTGCGTCGTCAGTCTTCCTTCTCTTCTC
GAGAACCACAAAGTCGTAGGGAACCCCGTTGTCTGAGAAGGTCTTGGAAATGAAGATGGG
TCGATACGTCGCTTCATTGTCCTTCTCTCTTCCGAGCTCCGGACAAAAGGGAACGAACAC
AGACTCGGCAGGAGCTGCAGCCTGCGCAGCAGTTGATTTGTTTGAAAGAATGTCATCTCC
GGGGAACGCAGGGAAGAAAACGTCGCACGGAAACTCGCGGGCTACACGCGTGATGTACAG
GTGAGAGGCAACGCCCAGAGACAGCGCTGCCTCGTACAGTCCCGCTCCTCCCACGACAAA
AATCTGGTCGACAGAATCCTTGTACTCTTCCTCCAGAAGGCTGAGAGCTGCTGGGAGTGA
AGCACAGACTCGGACGCGCTGCTGGCCTTCAGCTTGAGGCTTCTCCGCCGCAATGTCTTC
TTCTTTGAGGGAAGAGGAAACGACGATGTTCAATCTGTCCACGAGGGGTCTAAACTTTCG
AGGCATGCTTTCCCAGGTTTTCCGTCCCATGACAACGGCGTTGAATCTCTTGCCGACTGA
TGGAGAGGGAAGTCCAGAGTCGCCCGTCTTTGCAAATTTCCTGGGAAGCCACCCGTTCAG
GCGACTGGCTTCTTCGGGCGTCGTTTTTGTCACACGAGAAAAGTGTTTGAAATCTGTGGT
CAAGTGGGGCCACGGGAGGCCGTTGTTGATGCCGATGCCCCTCTTGGGGGTCATCGCGAC
GACCAGACACACCGGTTTATGCATTGATCCTTTGTAACATTTAGGTGTGTATTTATATAT
ATAAGCAATATACAAATGAAAATACTATTATAAAACGGAAACTCAAAAAATGTTGAAACA
AATAGTCAATTTTTAAAATGAATGTGTAAATTGTTTAAGTTTTATGACAAAAAAAGGAAA
AGTGTATATAAAATAAAAATTGGAAAAAATTTATATATTACATGCCATATAATACCATTT
ATTTACTATTTATATCTTAATATAAAAAATTGTAGCAAAAATATATAATCGTATTTTATA
TATTTAATATTATATAACCCCTTAAAAAAAAAAATTTAATTGAAAAAATTTTAATTTATG
CACTTTTTTATTGTATTATATATATTCCGTTATTTTAATTACACATAAACTTAATAGAAA
AATTAATTCGCTGCTAAATAAAAATTATAGGGCTATATATGATGGGATCATATTCTATCT
TTCTCTTTTTCAAATYCTTTATATTTTAGAATACAAATCGCTTTAAACAATAAAAAAGAG
GTATATCCTACCCGAATTGACACACACAATAAATAATAAATATATAAATAATAAAAATAT
AAATATATAAATTATAAATATATAAATTATATATATAAATATATTAACATGTGTGTAATT
TGTTAAAATTGATATTAAACAGCTTTGCATGGGGAAAGAGTGGCTTTTATAAGCGAATCA
TATAATACAGTGTTAATTTTAATATATAAAATATAATTTCCTTAAATTGTTATTTAACAT
TTTTAATTGCCTACCCARGGTAATATAATATCCAAATGACAATCAAAGATAAATGAAATA
CCGCTCCATTTTTCCTATATATCCATTTTCATTTTGTGTAAATGTTCATAAAATTTTATC
ATATACTCATAAAAAACAGGGGAAAAATGAAAAAATGAACGTCCACCCCGTGTGAATATG
CTCATTTTGTATACTATACATTTTTTTTTATATAATTTATATTTTCCAAATAAAAAAAAT
GTTTGTACATGTATATATCAATATTGTGTTATGGCATGATGAAAAACAATTATTTTATTC
GTTTTTAAATTTGTAGAATTTCCCTAAATTATTAATGATTTCATATTATAATATATGTAT
CTATATATTTTTAATGTGTAATTCAAAATGGCGAAAAAGAAACAAACCATACTTTATAAG
CAACTCTCTTTGAGCACTTACCATAAAAAATTATACTACTAAATTCAAATTAAATAAAAA
AATATATATTATAAAATTGGCTTTAATTATTTATAAGCACTTGCTTTGTTTTACTCAAAA
AGTATTGGCATAAGTATAAACATAAAACCAATTTTTCACAAAATATATTATATGTCCATA
CAACTATATCCGAACYTTCAAAACACATCCAAATTTCCATTTACCACATGCAAGCAAAAG
GATATTAAATAAAAAAAAATTATTTATATTTTCATGATCACTTAAATAATATTTAAAAAA
AAAAGAGACCATATATAAAATGTATAATTTAAATATAGCTAACTTATTAAAAAAAATGAA
TATTGAGCATATATCCATAATTTAAAACACTTCTATTTTATGTTATTTGATATATAAGTC
GATAAATTATTGCATGTATGCATATAGACAGATTAATATACACCAAAATAATAAAAATTA
TATATAGTTCATATATTATTCTGAATAATAAATTAGCTTTCCTCTTAATTCACTTGGTAT
GTATATATATATATATATATATATATATATATATATGGATAAATAAATATAAGGTCTATA
ACAGGAAAAAATGTGTATTAACTTTTTTTTATAATTATTTTATATTTTTTCTCCATTTAT
TTAAATATGTTAAAATTATTTCCCATTTTATAAACAAAAATTAATCCATAAAATAGCAAA
ATAACAAAATGACAAAATAACAAAAACAAAAAAACGCATTATATGAGTTCATTTTACACA
ATCCAAAGAAAAAGAAAATATGCTTAAAATATTCATAACACACTTTTAAGCTATAAAAAT
ATATATATTTATTTGTGTCTATATTACCAACTCAATTTAATAGATGTGTTATGTGATTAA
TTCATACACAAACATACAAAAATAAACACCATTTTTGAAAAGATTAATTTGAAATCATAT
ATGTTATTATTATATATTTTTATATCAATGAAATTTAAAAAAATAAAAGGGAAATCAATG
TATTAAAAATAATTATATGTTATTTTATTTCCACAATATTTATTTATTATTTATTGTTGA
CCTGCAGGCATGCAAGCTTATCGATACCGTCGACCTCGAGGGGGGGCCCGGTACCCAGCT
TTTGTTCCCTTTAGTGAGGGTTAATTGCGCGCTTGGCGTAATCATGGTCATAGCTGTTTC
CTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGT
GTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGC
CCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGG
GGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCT
CGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCA
CAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGA
ACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATC
ACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGG
CGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGAT
ACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGT
ATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTC
AGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACG
ACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCG
GTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTG
GTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCG
GCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCA
GAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGA
ACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGA
TCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGT
CTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTT
CATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCAT
CTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAG
CAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCT
CCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTT
TGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGG
CTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCA
AAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGT
TATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGAT
GCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGAC
CGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAA
AAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGT
TGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTT
TCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAA
GGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTT
ATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAA
TAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCAC
Restriction sites to linearize plasmid NdeI
Selectable marker used to select the mutant parasitetgdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modification
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 1gggactagtGCATCCATATTAATGCAACAATGG
Additional information primer 1L6686 (SpeI); PBANKA_093200 tag F
Sequence Primer 2cccggatccTAAATCATCAAAAATGGCTTTCTC
Additional information primer 2L6687 (BamHI); PBANKA_093200 tag R
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