Summary

RMgm-263
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_1463000; Gene model (P.falciparum): PF3D7_1250100; Gene product: osmiophilic body protein G377 (PbG377)
Phenotype Gametocyte/Gamete;
Last modified: 26 October 2014, 12:34
  *RMgm-263
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene disruption
Reference (PubMed-PMID number) Reference 1 (PMID number) : 25262869
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/ResearcherA. Oliviera; B. Franke-Fayard, C.J. Janse; M. Ponzi
Name Group/DepartmentDipartimento di Malattie Infettive, Parassitarie ed Immunomediate
Name InstituteIstituto Superiore di Sanità
CityRome
CountryItaly
Name of the mutant parasite
RMgm numberRMgm-263
Principal nameΔPbg377
Alternative name600cl1, 622cl1
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageNot different from wild type
Gametocyte/GameteProduction of gametocytes was slightly reduced in clone ΔPbg377-b (622cl1) compared to WT, while the other clone (600cl1) examined was similar to WT.

In female gametocytes typical osmiophilic bodies (OB) were not detected by Transmission Electron Microscopy (TEM) but only tiny osmiophilic vesicles, which were reduced in size compared to OB of WT females. The altered morphology of OB in mutant parasites was confirmed by determining minimum and maximum size values of more than a hundred OB sections present in TEM micrographs of WT and ΔPbg377 gametocytes. These observations indicate that Pbg377 may have a structural role in defining the shape of female OB. Absence of this protein does not result in a near complete lack of OB, as described for P. falciparum mutant lacking Pfg377, but instead results in a dramatic change in OB morphology.

15 min post activation of gametocytes a reduction of approximately 60% in egress of females from the host erythrocyte was observed in mutant parasites compared to the WT, while males emerged normally, as expected. At 20 min, egress of mutant female gametocytes/gametes was comparable to the WT. These observations indicate that in the absence of normal OB, female gametocytes are able to egress from the host erythrocyte, although less efficiently than the WT.
Fertilization and ookineteNot different from wild type
OocystNot different from wild type
SporozoiteNot different from wild type
Liver stageNot different from wild type
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of PbG377.

Protein (function)
The orthologous gene In P. falciparum, Pfg377, is associated with osmiophilic bodies (OB) and plays a role in the emergence of female gametocytes from the host erythrocyte after induction of gamete formation (de Koning-Ward, T.F. et al. Mol. Microbiol. (2008) 67:278-90).

Phenotype
Production of gametocytes was slightly reduced in clone ΔPbg377-b (622cl1) compared to WT, while the other clone (600cl1) examined was similar to WT.

In female gametocytes typical osmiophilic bodies (OB) were not detected by Transmission Electron Microscopy (TEM) but only tiny osmiophilic vesicles, which were reduced in size compared to OB of WT females. The altered morphology of OB in mutant parasites was confirmed by determining minimum and maximum size values of more than a hundred OB sections present in TEM micrographs of WT and ΔPbg377 gametocytes. These observations indicate that Pbg377 may have a structural role in defining the shape of female OB. Absence of this protein does not result in a near complete lack of OB, as described for P. falciparum mutant lacking Pfg377, but instead results in a dramatic change in OB morphology.

15 min post activation of gametocytes a reduction of approximately 60% in egress of females from the host erythrocyte was observed in mutant parasites compared to the WT, while males emerged normally, as expected. At 20 min, egress of mutant female gametocytes/gametes was comparable to the WT. These observations indicate that in the absence of normal OB, female gametocytes are able to egress from the host erythrocyte, although less efficiently than the WT.

Additional information
Expression in female gametocytes has been demonstrated using anti-PbG377 antibodies. No expression was detected in males.

Evidence is presented for co-localisation with MDV1/PEG3, a protein localised in osmiophilic bodies (OB). Immune electron microscopy (IEM), revealed that both Pbg377 and MDV1/PEG3 reside in the characteristic oval-shaped OB of female gametocytes.

A P. falciparum has been generated lacking expression of Pfg377.  Female gametocytes of this mutant are significantly less efficient in their emergence from the erythrocytes upon induction of gamete formation (de Koning-Ward, T.F. et al. Mol. Microbio. (2008) 67:278-90).

Other mutants


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1463000
Gene Model P. falciparum ortholog PF3D7_1250100
Gene productosmiophilic body protein G377
Gene product: Alternative namePbG377
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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GTGCTTATAAATAATTAAAGCCAATTTTATAATATATATTTTTTTATTTAATTTGAATTT
AGTAGTATAATTTTTTATGGTAAGTGCTCAAAGAGAGTTGCTTATAAAGTATGGTTTGTT
TCTTTTTCGCCATTTTGAATTACACATTAAAAATATATAGATACATATATTATAATATGA
AATCATTAATAATTTAGGGAAATTCTACAAATTTAAAAACGAATAAAATAATTGTTTTTC
ATCATGCCATAACACAATATTGATATATACATGTACAAACATTTTTTTTATTTGGAAAAT
ATAAATTATATAAAAAAAAATGTATAGTATACAAAATGAGCATATTCACACGGGGTGGAC
GTTCATTTTTTCATTTTTCCCCTGTTTTTTATGAGTATATGATAAAATTTTATGAACATT
TACACAAAATGAAAATGGATATATAGGAAAAATGGAGCGGTATTTCATTTATCTTTGATT
GTCATTTGGATATTATATTACCYTGGGTAGGCAATTAAAAATGTTAAATAACAATTTAAG
GAAATTATATTTTATATATTAAAATTAACACTGTATTATATGATTCGCTTATAAAAGCCA
CTCTTTCCCCATGCAAAGCTGTTTAATATCAATTTTAACAAATTACACACATGTTAATAT
ATTTATATATATAATTTATATATTTATAATTTATATATTTATATTTTTATTATTTATATA
TTTATTATTTATTGTGTGTGTCAATTCGGGTAGGATATACCTCTTTTTTATTGTTTAAAG
CGATTTGTATTCTAAAATATAAAGRATTTGAAAAAGAGAAAGATAGAATATGATCCCATC
ATATATAGCCCTATAATTTTTATTTAGCAGCGAATTAATTTTTCTATTAAGTTTATGTGT
AATTAAAATAACGGAATATATATAATACAATAAAAAAGTGCATAAATTAAAATTTTTTCA
ATTAAATTTTTTTTTTTAAGGGGTTATATAATATTAAATATATAAAATACGATTATATAT
TTTTGCTACAATTTTTTATATTAAGATATAAATAGTAAATAAATGGTATTATATGGCATG
TAATATATAAATTTTTTCCAATTTTTATTTTATATACACTTTTCCTTTTTTTGTCATAAA
ACTTAAACAATTTACACATTCATTTTAAAAATTGACTATTTGTTTCAACATTTTTTGAGT
TTCCGTTTTATAATAGTATTTTCATTTGTATATTGCTTATATATATAAATACACACCTAA
ATGTTACAAAGGATCAATGCATAAACCGGTGTGTCTGGTCGTCGCGATGACCCCCAAGAG
GGGCATCGGCATCAACAACGGCCTCCCGTGGCCCCACTTGACCACAGATTTCAAACACTT
TTCTCGTGTGACAAAAACGACGCCCGAAGAAGCCAGTCGCCTGAACGGGTGGCTTCCCAG
GAAATTTGCAAAGACGGGCGACTCTGGACTTCCCTCTCCATCAGTCGGCAAGAGATTCAA
CGCCGTTGTCATGGGACGGAAAACCTGGGAAAGCATGCCTCGAAAGTTTAGACCCCTCGT
GGACAGATTGAACATCGTCGTTTCCTCTTCCCTCAAAGAAGAAGACATTGCGGCGGAGAA
GCCTCAAGCTGAAGGCCAGCAGCGCGTCCGAGTCTGTGCTTCACTCCCAGCAGCTCTCAG
CCTTCTGGAGGAAGAGTACAAGGATTCTGTCGACCAGATTTTTGTCGTGGGAGGAGCGGG
ACTGTACGAGGCAGCGCTGTCTCTGGGCGTTGCCTCTCACCTGTACATCACGCGTGTAGC
CCGCGAGTTTCCGTGCGACGTTTTCTTCCCTGCGTTCCCCGGAGATGACATTCTTTCAAA
CAAATCAACTGCTGCGCAGGCTGCAGCTCCTGCCGAGTCTGTGTTCGTTCCCTTTTGTCC
GGAGCTCGGAAGAGAGAAGGACAATGAAGCGACGTATCGACCCATCTTCATTTCCAAGAC
CTTCTCAGACAACGGGGTTCCCTACGACTTTGTGGTTCTCGAGAAGAGAAGGAAGACTGA
CGACGCAGCCACTGCGGAACCGAGCAACGCAATGAGCTCCTTGACGTCCACGAGGGAGAC
AACTCCCGTGCACGGGTTGCAGGCTCCTTCTTCGGCCGCAGCCATTGCCCCGGTGTTGGC
GTGGATGGACGAAGAAGACCGGAAAAAACGCGAGCAAAAGGAACTGATTCGGGCCGTTCC
GCATGTTCACTTTAGAGGCCATGAAGAGTTCCAGTACCTTGATCTCATTGCCGACATTAT
TAACAATGGAAGGACAATGGATGACCGAACGGGCGTTGGTGTCATCTCCAAATTCGGCTG
CACTATGCGCTACTCGCTGGATCAGGCCTTTCCACTTCTCACCACAAAGCGTGTGTTCTG
GAAAGGGGTCCTCGAAGAGTTGCTGTGGTTCATTCGCGGCGACACGAACGCAAACCATCT
TTCTGAGAAGGGCGTGAAGATCTGGGACAAGAATGTGACACGCGAGTTCCTCGATTCGCG
CAATCTCCCCCACCGAGAGGTCGGAGACATCGGCCCGGGCTACGGCTTCCAGTGGAGACA
CTTCGGCGCGGCATACAAAGACATGCACACAGACTACACAGGGCAGGGCGTCGACCAGCT
GAAGAATGTGATCCAGATGCTGAGAACGAATCCAACAGATCGTCGCATGCTCATGACTGC
CTGGAATCCTGCAGCGCTGGACGAAATGGCGCTGCCGCCTTGTCACTTGTTGTGCCAGTT
CTACGTGAACGACCAGAAGGAGCTGTCGTGCATCATGTATCAGCGGTCGTGCGATGTCGG
CCTCGGCGTCCCCTTCAACATCGCTTCCTATTCGCTTTTGACGCTCATGGTTGCACACGT
CTGCAACCTAAAACCTAAGGAGTTCATTCACTTCATGGGGAACACGCATGTCTACACGAA
CCATGTCGAGGCTTTAAAAGAGCAGCTGCGGAGAGAACCGAGACCGTTCCCCATTGTGAA
CATCCTCAACAAGGAACGCATCAAGGAAATCGACGATTTCACCGCCGAGGATTTTGAGGT
CGTGGGCTACGTCCCGCACGGACGAATCCAGATGGAGATGGCTGTCTAGCGGAAATACAG
AAGCTAGCTTTGATCCCGTTTTTCTTACTTATATATTTATACCAATTGATTGTATTTATA
ACTGTAAAAATGTGTATGTTGTGTGCATATTTTTTTTTGTGCATGCACATGCATGTAAAT
AGCTAAAATTATGAACATTTTATTTTTTGTTCAGAAAAAAAAAACTTTACACACATAAAA
TGGCTAGTATGAATAGCCATATTTTATATAAATTAAATCCTATGAATTTATGACCATATT
AAAAATTTAGATATTTATGGAACATAATATGTTTGAAACAATAAGACAAAATTATTATTA
TTATTATTATTTTTACTGTTATAATTATGTTGTCTCTTCAATGATTCATAAATAGTTGGA
CTTGATTTTTAAAATGTTTATAATATGATTAGCATAGTTAAATAAAAAAAGTTGAAAAAT
TAAAAAAAAACATATAAACACAAATGATGTTTTTTCCTTCAATTTCGATATCGAATTCGC
ACCCCCAGAATCAAACATAGTAAAAGAAACACAAGATGCAAAAGAAACATTACCAATATT
TATAAGCTTAAAAGATGCAAATAGACATAATCGTAGAGTTCTATCTGACGATCTTTATGA
TTCTGATATTGATGAAATTGATAGTCATGAAATGTCAGAAGATAATGACACGCCAGATGA
AAACAATAAATATTTAAAAGAATTAAAAATAACAGTTGAAAATGAATTAATAGAGGAACG
AAAAAAATATGTACTTGAAAGAAATCAAGAAAAACAAAAAAAAGAACAATTTATTAAAAA
AGAAATAGAAGAAATTGATAAAAAACATGAATTAGAAACACAAAAATTAGAAAAAGAAGA
ACCACAAAAAGAAATATTACAAGATGAACAAATTGACAGGGAATATGAAAATGGCTTAGA
AGATGAAACAATAATTGAAAGGGACCGACTAATAGCTTTAGGAAAAGAAAATATTAGATC
AACAGTAGATAAAAGTAAAAGATATGATAAAATGTATTATGAATTAGCACTTAAAACATT
AACAGACGACCCAGATGAAGTATATGGTAGAATTGTTAAGGCAGGATATGGATTTGCATC
AAAATCAGAACAACAATTTCAACAAATTGTTTACACATTAAACGATACTACAGCACCGGA
TCCACTAGTTCTAGAGCGGCCGCCACCGCGGTGGAGCTCCAATTCGCCCTATAGTGAGTC
GTATTACAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTAC
CCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGC
CCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGGACGCGCCCTG
TAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGC
CAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGG
CTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACG
GCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTG
ATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTT
CCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTT
GCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTT
TAACAAAATATTAACGCTTACAATTTAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACC
CCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCC
TGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTC
GCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTG
GTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGAT
CTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGC
ACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAA
CTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAA
AAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGT
GATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCT
TTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAAT
GAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTG
CGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGG
ATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTT
ATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGG
CCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATG
GATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTG
TCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAA
AGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTT
TCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTT
TTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGT
TTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAG
ATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTA
GCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGAT
AAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCG
GGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTG
AGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGAC
AGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGA
AACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTT
TTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTA
CGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGAT
TCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACG
ACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCT
CTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAA
GCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCT
TTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCAC
ACAGGAAACAGCTATGACCATGATTACGCCAAGCTCGAAATTAACCCTCACTAAAGGGAA
CAAAAGCTGGTACCCAACAAGCAATATGTGCAGACAAATTTACACTTATGATAATGTATA
AGACGCCTTTTTAATCGTTTTTTTGTATGTTTCTTTAATACATGGATCAAATATACATAC
ATGTTACATATTAATGCACGCGTTATATATATATATATATATATATATATATATATATAT
ATATCAATACATTTATATATTCATTTAGTTATATAATTACTTTTGCACACACTTATACTC
ATTCCTGCATTTGCTAAGTCTACATAACTCATAAATATAGTAGTTGCTTACCCGATAAAA
AAGTAATTTTTTCATCACCAATAAAATTTTTTTTTCATTACATTGTGTTTGCCGCCATCA
TAAGGTCTTTATTTCGTGCTTTATTATTTTGTTAAATTTAACATCTACATTTAAGCATGA
TAATTATTTTAAGTTTATTCATAAACTCATGAATATTTCAAATAAAACAATATATGAACG
AAAATACATTGTTTGAAACTTCGAAGCCAATCCCAAAAAGAATGTTGGCAAAATGAAAAT
ACTGTTGTATACTTTTCTTTTTTGTATTTGCTCTGGTTTTGCAAAATGCCAGAAAGCATA
TAATTTTGATAATATAATAAAATATCTTAAAGAAATAAAAGTAATACCTAGTTATATACC
AGATAAACTAGAAGCTTGCATGCCTGCAGGTCAACAATAAATAATAAATAAATATTGTGG
AAATAAAATAACATATAATTATTTTTAATACATTGATTTCCCTTTTATTTTTTTAAATTT
CATTGATATAAAAATATATAATAATAACATATATGATTTCAAATTAATCTTTTCAAAAAT
GGTGTTTATTTTTGTATGTTTGTGTATGAATTAATCACATAACACATCTATTAAATTGAG
TTGGTAATATAGACACAAATAAATATATATATTTTTATAGCTTAAAAGTGTGTTATGAAT
ATTTTAAGCATATTTTCTTTTTCTTTGGATTGTGTAAAATGAACTCATATAATGCGTTTT
TTTGTTTTTGTTATTTTGTCATTTTGTTATTTTGCTATTTTATGGATTAATTTTTGTTTA
TAAAATGGGAAATAATTTTAACATATTTAAATAAATGGAGAAAAAATATAAAATAATTAT
AAAAAAAAGTTAATACACATTTTTTCCTGTTATAGACCTTATATTTATTTATCCATATAT
ATATATATATATATATATATATATATATACATACCAAGTGAATTAAGAGGAAAGCTAATT
TATTATTCAGAATAATATATGAACTATATATAATTTTTATTATTTTGGTGTATATTAATC
TGTCTATATGCATACATGCAATAATTTATCGACTTATATATCAAATAACATAAAATAGAA
GTGTTTTAAATTATGGATATATGCTCAATATTCATTTTTTTTAATAAGTTAGCTATATTT
AAATTATACATTTTATATATGGTCTCTTTTTTTTTTAAATATTATTTAAGTGATCATGAA
AATATAAATAATTTTTTTTTATTTAATATCCTTTTGCTTGCATGTGGTAAATGGAAATTT
GGATGTGTTTTGAARGTTCGGATATAGTTGTATGGACATATAATATATTTTGTGAAAAAT
TGGTTTTATGTTTATACTTATGCCAATACTTTTTGAGTAAAACAAAGCAA
Restriction sites to linearize plasmid
Partial or complete disruption of the geneComplete
Additional remarks partial/complete disruption The ORF of PbG377 reads 1839 bp (PlasmoDB). The selectable marker was inserted between bp positions 138 and 1630, replacing everything in between.
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 1CGGGGTACCcaacaagcaatatgtgcagac
Additional information primer 1L2307 (Asp718); 5'
Sequence Primer 2CCCAAGCTTctagtttatctggtatataactagg
Additional information primer 2L2308 (HindIII); 5'
Sequence Primer 3CCGGAATTCgcacccccagaatcaaacatag
Additional information primer 3L2309 (EcoRI); 3'
Sequence Primer 4CGCGGATCCggtgctgtagtatcgtttaatg
Additional information primer 4L2310 (BamHI); 3'
Sequence Primer 5
Additional information primer 5
Sequence Primer 6
Additional information primer 6