Summary

RMgm-100
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_1217700; Gene model (P.falciparum): PF3D7_0320800; Gene product: ATP-dependent RNA helicase DDX6 (DOZI, protein development of zygote inhibited)
Phenotype Gametocyte/Gamete; Fertilization and ookinete;
Last modified: 5 February 2010, 22:18
  *RMgm-100
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) : 16888139
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/ResearcherG.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-100
Principal name560cl1; 611cl1
Alternative namepbdozi–
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageNot different from wild type
Gametocyte/GameteNormal production of gametocytes and gametes. A complete block of development of fertilized female gametes (zygotes) into mature ookinetes. Northern analysis of mRNA of gametocytes showed a nearly complete loss of transcripts (mRNA) of genes that are translationally repressed (for example genes encoding the ookinete surface proteins P25, P28). Microarray analysis of mRNA of gametocytes showed a significant reduction (> 2x) of transcripts of 370 genes; including seven of nine genes previously shown to be translationally repressed.
Fertilization and ookineteNormal production of gametocytes and gametes (see also phenotype 'Gametocyte/gamete'). A complete block of development of fertilized female gametes (zygotes) into mature ookinetes. Normal development into ookinetes requires meiotic DNA replication in the zygote 2-3 hours after fertilization of female gametes. In the mutant, all zygotes aborted development before meiosis.
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of DOZI (protein development of zygote inhibited; ATP-dependent RNA helicase, putative).

Protein (function)
DOZI is an RNA helicase that is highly up-regulated in female gametocytes and shows high sequence homology to the DDX6 family of RNA helicases.  It contains domains involved in RNA-binding and RNA-unwinding activity.  In other eukaryotes the DDX6 family of DEAD-box RNA helicases is tightly linked both to storage of mRNAs encoding proteins associated with progression through meiosis into translationally silent mRNPs and with the transport of mRNA to degradation centers in the cell (P-bodies). These helicases are found in organisms as diverse as yeast (e.g., Dhh1p) and humans (e.g., RCK/p54).

Phenotype
The phenotype analyses indicate an essential role of DOZI in the development of fertilized female gametes (zygotes).
The phenotype analyses indicate that DOZI has a central role in the silencing and maintenance of steady-state levels of a population of gametocyte-specific transcripts. The loss of DOZI severely affected the capacity of the parasite to store and stabilize a discrete subset of mRNAs in the female gametocyte, resulting in a failure to synthesize specific proteins and to complete normal zygote development.  Translational repression of transcripts encoding these proteins in Plasmodium may function to specifically regulate gene expression during meiosis in the zygote.

Additional information
DOZI is also produced in male gametocytes, although in much lower abundance than in females. Male and female gametes of pbdozi have been crossed with gametes of mutant parasite lines that are defective in male or in female gamete production. These cross-fertilization assays revealed that male pbdozi gametes were able to fertilize wild-type DOZI-expressing female gametes, resulting in the development of ookinetes. By contrast, development of zygotes from pbdozi females fertilized by wild-type males was incomplete and similar to development of zygotes from the pbdozi line. These crosses demonstrate that the block in zygote/ookinete development is essentially due to a lack of DOZI of female gametocyte origin.

Other mutants
RMgm-133: A mutant expressing a (c-terminal) GFP tagged DOZI
RMgm-361: A mutant which lacks expression of DOZI  and expresses GFP under control of a male and RFP under control of a female gametocyte specific promoter.


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1217700
Gene Model P. falciparum ortholog PF3D7_0320800
Gene productATP-dependent RNA helicase DDX6
Gene product: Alternative nameDOZI, protein development of zygote inhibited
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct usedPlasmid 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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GTACCGAGCTCGGATCCACTAGTAACGGCCGCCAGTGTGCTGGAATTCGGCTTAAAAAGA
ATTCAATATACAGAAAATATATCCTGTTTAGGCTTTATATTGTTATTTAGTCTCTGATAT
CTTTATTTATTATATATATTTTTATTTATTCTATTTTGTTCCTTTTTTTTTATTTGTATG
TAGTTTATTATTATACTATTATTATTTTTTTTTTTGCTTTCATTCTTTAATTCATATCGA
GTTATTTTTCTTTCTTATAACATTTATATTCCTTTCCATATTCTATTCATATATTTCTTT
TATTACGCTGTTAATTTTATAATATTCTTTTTTACGGCATAATTCTTTCATCTCAATACG
TTGCATATATATAATCCATATATGTATATAACATATCCTCTCCATGTTTTTCATATTTTT
TTCTACTATTCCCTTTTTCAACAAATGATATAAACAGCTATTCTCTCTCTCTCTATATAT
ATATATATTACTATTCACGCGTTTCATTTTAAAATTCTTATATAAATATAAAATTAAACT
GTAATATATATAACACTTGTAAGCTTGCATGCCTGCAGGTCAACAATAAATAATAAATAA
ATATTGTGGAAATAAAATAACATATAATTATTTTTAATACATTGATTTCCCTTTTATTTT
TTTAAATTTCATTGATATAAAAATATATAATAATAACATATATGATTTCAAATTAATCTT
TTCAAAAATGGTGTTTATTTTTGTATGTTTGTGTATGAATTAATCACATAACACATCTAT
TAAATTGAGTTGGTAATATAGACACAAATAAATATATATATTTTTATAGCTTAAAAGTGT
GTTATGAATATTTTAAGCATATTTTCTTTTTCTTTGGATTGTGTAAAATGAACTCATATA
ATGCGTTTTTTTGTTTTTGTTATTTTGTCATTTTGTTATTTTGCTATTTTATGGATTAAT
TTTTGTTTATAAAATGGGAAATAATTTTAACATATTTAAATAAATGGAGAAAAAATATAA
AATAATTATAAAAAAAAGTTAATACACATTTTTTCCTGTTATAGACCTTATATTTATTTA
TCCATATATATATATATATATATATATATATATATATACATACCAAGTGAATTAAGAGGA
AAGCTAATTTATTATTCAGAATAATATATGAACTATATATAATTTTTATTATTTTGGTGT
ATATTAATCTGTCTATATGCATACATGCAATAATTTATCGACTTATATATCAAATAACAT
AAAATAGAAGTGTTTTAAATTATGGATATATGCTCAATATTCATTTTTTTTAATAAGTTA
GCTATATTTAAATTATACATTTTATATATGGTCTCTTTTTTTTTTAAATATTATTTAAGT
GATCATGAAAATATAAATAATTTTTTTTTATTTAATATCCTTTTGCTTGCATGTGGTAAA
TGGAAATTTGGATGTGTTTTGAARGTTCGGATATAGTTGTATGGACATATAATATATTTT
GTGAAAAATTGGTTTTATGTTTATACTTATGCCAATACTTTTTGAGTAAAACAAAGCAAG
TGCTTATAAATAATTAAAGCCAATTTTATAATATATATTTTTTTATTTAATTTGAATTTA
GTAGTATAATTTTTTATGGTAAGTGCTCAAAGAGAGTTGCTTATAAAGTATGGTTTGTTT
CTTTTTCGCCATTTTGAATTACACATTAAAAATATATAGATACATATATTATAATATGAA
ATCATTAATAATTTAGGGAAATTCTACAAATTTAAAAACGAATAAAATAATTGTTTTTCA
TCATGCCATAACACAATATTGATATATACATGTACAAACATTTTTTTTATTTGGAAAATA
TAAATTATATAAAAAAAAATGTATAGTATACAAAATGAGCATATTCACACGGGGTGGACG
TTCATTTTTTCATTTTTCCCCTGTTTTTTATGAGTATATGATAAAATTTTATGAACATTT
ACACAAAATGAAAATGGATATATAGGAAAAATGGAGCGGTATTTCATTTATCTTTGATTG
TCATTTGGATATTATATTACCYTGGGTAGGCAATTAAAAATGTTAAATAACAATTTAAGG
AAATTATATTTTATATATTAAAATTAACACTGTATTATATGATTCGCTTATAAAAGCCAC
TCTTTCCCCATGCAAAGCTGTTTAATATCAATTTTAACAAATTACACACATGTTAATATA
TTTATATATATAATTTATATATTTATAATTTATATATTTATATTTTTATTATTTATATAT
TTATTATTTATTGTGTGTGTCAATTCGGGTAGGATATACCTCTTTTTTATTGTTTAAAGC
GATTTGTATTCTAAAATATAAAGRATTTGAAAAAGAGAAAGATAGAATATGATCCCATCA
TATATAGCCCTATAATTTTTATTTAGCAGCGAATTAATTTTTCTATTAAGTTTATGTGTA
ATTAAAATAACGGAATATATATAATACAATAAAAAAGTGCATAAATTAAAATTTTTTCAA
TTAAATTTTTTTTTTTAAGGGGTTATATAATATTAAATATATAAAATACGATTATATATT
TTTGCTACAATTTTTTATATTAAGATATAAATAGTAAATAAATGGTATTATATGGCATGT
AATATATAAATTTTTTCCAATTTTTATTTTATATACACTTTTCCTTTTTTTGTCATAAAA
CTTAAACAATTTACACATTCATTTTAAAAATTGACTATTTGTTTCAACATTTTTTGAGTT
TCCGTTTTATAATAGTATTTTCATTTGTATATTGCTTATATATATAAATACACACCTAAA
TGTTACAAAGGATCAATGCATAAACCGGTGTGTCTGGTCGTCGCGATGACCCCCAAGAGG
GGCATCGGCATCAACAACGGCCTCCCGTGGCCCCACTTGACCACAGATTTCAAACACTTT
TCTCGTGTGACAAAAACGACGCCCGAAGAAGCCAGTCGCCTGAACGGGTGGCTTCCCAGG
AAATTTGCAAAGACGGGCGACTCTGGACTTCCCTCTCCATCAGTCGGCAAGAGATTCAAC
GCCGTTGTCATGGGACGGAAAACCTGGGAAAGCATGCCTCGAAAGTTTAGACCCCTCGTG
GACAGATTGAACATCGTCGTTTCCTCTTCCCTCAAAGAAGAAGACATTGCGGCGGAGAAG
CCTCAAGCTGAAGGCCAGCAGCGCGTCCGAGTCTGTGCTTCACTCCCAGCAGCTCTCAGC
CTTCTGGAGGAAGAGTACAAGGATTCTGTCGACCAGATTTTTGTCGTGGGAGGAGCGGGA
CTGTACGAGGCAGCGCTGTCTCTGGGCGTTGCCTCTCACCTGTACATCACGCGTGTAGCC
CGCGAGTTTCCGTGCGACGTTTTCTTCCCTGCGTTCCCCGGAGATGACATTCTTTCAAAC
AAATCAACTGCTGCGCAGGCTGCAGCTCCTGCCGAGTCTGTGTTCGTTCCCTTTTGTCCG
GAGCTCGGAAGAGAGAAGGACAATGAAGCGACGTATCGACCCATCTTCATTTCCAAGACC
TTCTCAGACAACGGGGTTCCCTACGACTTTGTGGTTCTCGAGAAGAGAAGGAAGACTGAC
GACGCAGCCACTGCGGAACCGAGCAACGCAATGAGCTCCTTGACGTCCACGAGGGAGACA
ACTCCCGTGCACGGGTTGCAGGCTCCTTCTTCGGCCGCAGCCATTGCCCCGGTGTTGGCG
TGGATGGACGAAGAAGACCGGAAAAAACGCGAGCAAAAGGAACTGATTCGGGCCGTTCCG
CATGTTCACTTTAGAGGCCATGAAGAGTTCCAGTACCTTGATCTCATTGCCGACATTATT
AACAATGGAAGGACAATGGATGACCGAACGGGCGTTGGTGTCATCTCCAAATTCGGCTGC
ACTATGCGCTACTCGCTGGATCAGGCCTTTCCACTTCTCACCACAAAGCGTGTGTTCTGG
AAAGGGGTCCTCGAAGAGTTGCTGTGGTTCATTCGCGGCGACACGAACGCAAACCATCTT
TCTGAGAAGGGCGTGAAGATCTGGGACAAGAATGTGACACGCGAGTTCCTCGATTCGCGC
AATCTCCCCCACCGAGAGGTCGGAGACATCGGCCCGGGCTACGGCTTCCAGTGGAGACAC
TTCGGCGCGGCATACAAAGACATGCACACAGACTACACAGGGCAGGGCGTCGACCAGCTG
AAGAATGTGATCCAGATGCTGAGAACGAATCCAACAGATCGTCGCATGCTCATGACTGCC
TGGAATCCTGCAGCGCTGGACGAAATGGCGCTGCCGCCTTGTCACTTGTTGTGCCAGTTC
TACGTGAACGACCAGAAGGAGCTGTCGTGCATCATGTATCAGCGGTCGTGCGATGTCGGC
CTCGGCGTCCCCTTCAACATCGCTTCCTATTCGCTTTTGACGCTCATGGTTGCACACGTC
TGCAACCTAAAACCTAAGGAGTTCATTCACTTCATGGGGAACACGCATGTCTACACGAAC
CATGTCGAGGCTTTAAAAGAGCAGCTGCGGAGAGAACCGAGACCGTTCCCCATTGTGAAC
ATCCTCAACAAGGAACGCATCAAGGAAATCGACGATTTCACCGCCGAGGATTTTGAGGTC
GTGGGCTACGTCCCGCACGGACGAATCCAGATGGAGATGGCTGTCTAGCGGAAATACAGA
AGCTAGCTTTGATCCCGTTTTTCTTACTTATATATTTATACCAATTGATTGTATTTATAA
CTGTAAAAATGTGTATGTTGTGTGCATATTTTTTTTTGTGCATGCACATGCATGTAAATA
GCTAAAATTATGAACATTTTATTTTTTGTTCAGAAAAAAAAAACTTTACACACATAAAAT
GGCTAGTATGAATAGCCATATTTTATATAAATTAAATCCTATGAATTTATGACCATATTA
AAAATTTAGATATTTATGGAACATAATATGTTTGAAACAATAAGACAAAATTATTATTAT
TATTATTATTTTTACTGTTATAATTATGTTGTCTCTTCAATGATTCATAAATAGTTGGAC
TTGATTTTTAAAATGTTTATAATATGATTAGCATAGTTAAATAAAAAAAGTTGAAAAATT
AAAAAAAAACATATAAACACAAATGATGTTTTTTCCTTCAATTTCGATGGGACCACATCA
TAATGATGAACACATATATATACATATACACATACACATTGCGGCTTCAATGCAAAATCT
ATTTGTGCACAGTATTACTAACTTACCCCACATATACAATACTTTTTTTATTTATCATCA
ATTTTTTTCTTTTTTCCCATTATACTTAATTTTTTTAAGCTATTGACATTATATTGGAGT
ATAAATTAATTAAAGCATAATATATTAATACACAAAACATATATACATACATATATATGC
ATATTTGTACATTTTTTTTTGTTAAATTACTATATATAATTTCACCCAATTAAACATGCG
AACACAAATATAATTGATTAACTAGGAATTCTGTTTATATATATTTATACACAATATACC
TATAGATATATAGTGCAACCTATATATCCAGAATGACTCTAAATTCGACGTTATGGAACT
CTTTAGTTTTTTCAAGGAATTTATTAATTAAAAAATTAATGAAAATAAAACGTACAAATA
GGAGAATACATTTTTTTTTGTTCTTTTTTGCTCGTAGTCACACACAAAAAATGTAATAAA
AATGCAATTTTTCGTGTTTATTGGCTTTCACCATTTTTATAATTTTATAATTTATAATGC
ATTGTTATGATATAAAATTTATTAATCATTATTCTTCTGCATTTCACGATTTTTCAAATA
TCGATTTCTTAATATCACAAATAAAAATCAAATTATGTAAAAGGTGTGATATCCACACTT
TTCAACCTATACAAAATGTAATAACATATATTTATATTTACATTAATATTTTTAACTCTT
TTGTGAAAAAATTTAAAAGAAAAAAAAAAATTTATAGCGATTTACATGTTTTTAAAATGG
TTAAAAAGGTAAAAAAATTAATTGTTTCTTATTGATACTACATTTGCATGCTTATATATC
AAACAATGTATTGGATCACGTTTTGTTCGCATTAGGATCCACTAGTTCTAGAGCGGCCGC
CACCGCGGTGGAGCTCCAATTCGCCCTATAGTGAGTCGTATTACGCGCGCTCACTGGCCG
TCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAG
CACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCC
AACAGTTGCGCAGCCTGAATGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGG
CGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTC
CTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAA
ATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAAC
TTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTT
TGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCA
ACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGT
TAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGCTTA
CAATTTAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTA
AATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATA
TTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGC
GGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGA
AGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCT
TGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATG
TGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTA
TTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCAT
GACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTT
ACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGA
TCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGA
GCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGA
ACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGC
AGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGC
CGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCG
TATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGAT
CGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATA
TATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCT
TTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGA
CCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTG
CTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACC
AACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCT
AGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGC
TCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTT
GGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTG
CACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCT
ATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAG
GGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAG
TCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGG
GCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTG
GCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTAC
CGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGT
GAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGAT
TCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGC
AATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGC
TCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCA
TGATTACGCCAAGCTCGAAATTAACCCTCACTAAAGGGAACAAAAGCTG
Restriction sites to linearize plasmid
Partial or complete disruption of the geneComplete
Additional remarks partial/complete disruption
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 1AAAGAATTCAATATACAGAAAATATATCCTG
Additional information primer 12238EcoRI; 5'-region
Sequence Primer 2AAAAAGCTTACAAGTGTTATATATATTACAG
Additional information primer 22239HindIII; 5'-region
Sequence Primer 3AAACCCGGGACCACATCATAATGATGAACAC
Additional information primer 32240SmaI; 3'-region
Sequence Primer 4AAAGGATCCTAATGCGAACAAAACGTGATCC
Additional information primer 42241BamHI; 3'-region
Sequence Primer 5
Additional information primer 5
Sequence Primer 6
Additional information primer 6