Summary

RMgm-178
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_0702700; Gene model (P.falciparum): PF3D7_0828000; Gene product: rhomboid protease ROM3 (ROM3)
Phenotype Oocyst; Sporozoite;
Last modified: 16 March 2013, 12:01
  *RMgm-178
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) : 23490234
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. Lin, G.R. Mair, C.J. Janse, S.M. Khan
Name Group/DepartmentLeiden Malaria Research Group
Name InstituteLeiden University Medical Center (LUMC)
CityLeiden
CountryThe Netherlands
Name of the mutant parasite
RMgm numberRMgm-178
Principal name430cl1; 687cl1; 687cl2
Alternative name∆rom3
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageNot different from wild type
Gametocyte/GameteNot different from wild type
Fertilization and ookineteNot different from wild type
OocystNormal numbers of oocysts are produced. Mature (11-13 day) mutant oocysts have the same size as wild type oocysts. No sporozoites are formed within the oocysts. Oocysts appear vacuolated.
SporozoiteNormal numbers of oocysts are produced. Mature (11-13 day) mutant oocysts have the same size as wild type oocysts. No sporozoites are formed within the oocysts. Oocysts appear vacuolated.
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of rhomboid protease ROM3.

Protein (function)
Rhomboid proteins are intra-membrane proteases that play a role in multiple processes. They belong to a family of serine proteases that cleave cell-surface proteins within their transmembrane domains. The Plasmodium  genome encodes a total of 8 rhomboid proteases (ROM1, 3, 4, 6, 7, 8, 9 and 10). . 

Phenotype
The phenotype analyses indicate that ROM3 plays an essential role in oocyst development and in the formation of sporozoites. Sporozoite formation is completely blocked in the absence of ROM3.

Additional information
Transcripts of rom3 were not detected in asexual blood stage parasites. Relatively high transcription levels were observed in the gametocyte and ookinete stage. See also RMgm-763 for a P. berghei mutant expressing  a (C-terminal) GFP-tagged version of rhomboid protease ROM3. ROM3-GFP expression was observed in female gametocytes and ookinetes while no GFP-signals were detected in developing oocysts.

Other mutants
RMgm-176: A P. berghei mutant lacking expression of rhomboid protease ROM1 (partial disruption)
RMgm-177: A P. berghei mutant lacking expression of rhomboid protease ROM1 (partial disruption)
RMgm-761: A P. berghei mutant lacking expression of rhomboid protease ROM1 (complete disruption)
RMgm-762: A P. berghei mutant lacking expression of rhomboid protease ROM9
RMgm-179: A P. berghei mutant lacking expression of rhomboid protease ROM10

RMgm-187: Unsuccessful attempts to disrupt P. berghei rhomboid protease rom4
RMgm-758: Unsuccessful attempts to disrupt P. berghei rhomboid protease rom6
RMgm-759: Unsuccessful attempts to disrupt P. berghei rhomboid protease rom7
RMgm-760: Unsuccessful attempts to disrupt P. berghei rhomboid protease rom8

RMgm-763: A P. berghei mutant expressing  a (C-terminal) GFP-tagged version of rhomboid protease ROM3
RMgm-764: A P. berghei mutant expressing  a (C-terminal) mCherry-tagged version of rhomboid protease ROM4

RMgm-659: A P. y. yoelii 17XNL mutant lacking expression of rhomboid protease ROM1
RMgm-660: A P. y. yoelii 17XNL mutant expressing  a (N-terminal) HA-tagged version of rhomboid protease ROM1


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0702700
Gene Model P. falciparum ortholog PF3D7_0828000
Gene productrhomboid protease ROM3
Gene product: Alternative nameROM3
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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CGATGAATTCCAACAATGAATGTAATAAAGGTAATGTTTGAATATATCCCATAATTATTT
TAATAGACTTTGAACTATAGTTTGCGCTTTTTTTAGCAAGCTAATAAAAATAAAATAGTG
TAAAATAATAATATATATAGTTATTATCAAAAGACCACAATTTTACACACATAAAAAAAT
ATATACTCAACTAAAGTTTATATAATAAATAGCTGTTATAAATTATAAAATATTTTTTAT
GCATTATTTTTAAACGTAATAACAAAAATCCAACTATATTTTGTACAAACGTTTGTATTT
TTCATTTTTTTTTATGTTTATATAAATGCAAGTAAACAAATATATATATAAATAATGTTA
AATAAATATGCAGAAAATGATGAAAATTCAAATTTGTTACCTGGGACTACTGAGCTGATT
GAAAAAAATGCACAAAATAAATTTTACGATGTTTTATTTCCTCAAATATCACTAAATAGG
TAATATATGGAAACCTATAATATTATATTAAACAAGTGAAACAAAATAGACATAAGCTTG
CATGCCTGCAGGTCAACAATAAATAATAAATAAATATTGTGGAAATAAAATAACATATAA
TTATTTTTAATACATTGATTTCCCTTTTATTTTTTTAAATTTCATTGATATAAAAATATA
TAATAATAACATATATGATTTCAAATTAATCTTTTCAAAAATGGTGTTTATTTTTGTATG
TTTGTGTATGAATTAATCACATAACACATCTATTAAATTGAGTTGGTAATATAGACACAA
ATAAATATATATATTTTTATAGCTTAAAAGTGTGTTATGAATATTTTAAGCATATTTTCT
TTTTCTTTGGATTGTGTAAAATGAACTCATATAATGCGTTTTTTTGTTTTTGTTATTTTG
TCATTTTGTTATTTTGCTATTTTATGGATTAATTTTTGTTTATAAAATGGGAAATAATTT
TAACATATTTAAATAAATGGAGAAAAAATATAAAATAATTATAAAAAAAAGTTAATACAC
ATTTTTTCCTGTTATAGACCTTATATTTATTTATCCATATATATATATATATATATATAT
ATATATATATACATACCAAGTGAATTAAGAGGAAAGCTAATTTATTATTCAGAATAATAT
ATGAACTATATATAATTTTTATTATTTTGGTGTATATTAATCTGTCTATATGCATACATG
CAATAATTTATCGACTTATATATCAAATAACATAAAATAGAAGTGTTTTAAATTATGGAT
ATATGCTCAATATTCATTTTTTTTAATAAGTTAGCTATATTTAAATTATACATTTTATAT
ATGGTCTCTTTTTTTTTTAAATATTATTTAAGTGATCATGAAAATATAAATAATTTTTTT
TTATTTAATATCCTTTTGCTTGCATGTGGTAAATGGAAATTTGGATGTGTTTTGAARGTT
CGGATATAGTTGTATGGACATATAATATATTTTGTGAAAAATTGGTTTTATGTTTATACT
TATGCCAATACTTTTTGAGTAAAACAAAGCAAGTGCTTATAAATAATTAAAGCCAATTTT
ATAATATATATTTTTTTATTTAATTTGAATTTAGTAGTATAATTTTTTATGGTAAGTGCT
CAAAGAGAGTTGCTTATAAAGTATGGTTTGTTTCTTTTTCGCCATTTTGAATTACACATT
AAAAATATATAGATACATATATTATAATATGAAATCATTAATAATTTAGGGAAATTCTAC
AAATTTAAAAACGAATAAAATAATTGTTTTTCATCATGCCATAACACAATATTGATATAT
ACATGTACAAACATTTTTTTTATTTGGAAAATATAAATTATATAAAAAAAAATGTATAGT
ATACAAAATGAGCATATTCACACGGGGTGGACGTTCATTTTTTCATTTTTCCCCTGTTTT
TTATGAGTATATGATAAAATTTTATGAACATTTACACAAAATGAAAATGGATATATAGGA
AAAATGGAGCGGTATTTCATTTATCTTTGATTGTCATTTGGATATTATATTACCYTGGGT
AGGCAATTAAAAATGTTAAATAACAATTTAAGGAAATTATATTTTATATATTAAAATTAA
CACTGTATTATATGATTCGCTTATAAAAGCCACTCTTTCCCCATGCAAAGCTGTTTAATA
TCAATTTTAACAAATTACACACATGTTAATATATTTATATATATAATTTATATATTTATA
ATTTATATATTTATATTTTTATTATTTATATATTTATTATTTATTGTGTGTGTCAATTCG
GGTAGGATATACCTCTTTTTTATTGTTTAAAGCGATTTGTATTCTAAAATATAAAGRATT
TGAAAAAGAGAAAGATAGAATATGATCCCATCATATATAGCCCTATAATTTTTATTTAGC
AGCGAATTAATTTTTCTATTAAGTTTATGTGTAATTAAAATAACGGAATATATATAATAC
AATAAAAAAGTGCATAAATTAAAATTTTTTCAATTAAATTTTTTTTTTTAAGGGGTTATA
TAATATTAAATATATAAAATACGATTATATATTTTTGCTACAATTTTTTATATTAAGATA
TAAATAGTAAATAAATGGTATTATATGGCATGTAATATATAAATTTTTTCCAATTTTTAT
TTTATATACACTTTTCCTTTTTTTGTCATAAAACTTAAACAATTTACACATTCATTTTAA
AAATTGACTATTTGTTTCAACATTTTTTGAGTTTCCGTTTTATAATAGTATTTTCATTTG
TATATTGCTTATATATATAAATACACACCTAAATGTTACAAAGGATCAATGCATAAACCG
GTGTGTCTGGTCGTCGCGATGACCCCCAAGAGGGGCATCGGCATCAACAACGGCCTCCCG
TGGCCCCACTTGACCACAGATTTCAAACACTTTTCTCGTGTGACAAAAACGACGCCCGAA
GAAGCCAGTCGCCTGAACGGGTGGCTTCCCAGGAAATTTGCAAAGACGGGCGACTCTGGA
CTTCCCTCTCCATCAGTCGGCAAGAGATTCAACGCCGTTGTCATGGGACGGAAAACCTGG
GAAAGCATGCCTCGAAAGTTTAGACCCCTCGTGGACAGATTGAACATCGTCGTTTCCTCT
TCCCTCAAAGAAGAAGACATTGCGGCGGAGAAGCCTCAAGCTGAAGGCCAGCAGCGCGTC
CGAGTCTGTGCTTCACTCCCAGCAGCTCTCAGCCTTCTGGAGGAAGAGTACAAGGATTCT
GTCGACCAGATTTTTGTCGTGGGAGGAGCGGGACTGTACGAGGCAGCGCTGTCTCTGGGC
GTTGCCTCTCACCTGTACATCACGCGTGTAGCCCGCGAGTTTCCGTGCGACGTTTTCTTC
CCTGCGTTCCCCGGAGATGACATTCTTTCAAACAAATCAACTGCTGCGCAGGCTGCAGCT
CCTGCCGAGTCTGTGTTCGTTCCCTTTTGTCCGGAGCTCGGAAGAGAGAAGGACAATGAA
GCGACGTATCGACCCATCTTCATTTCCAAGACCTTCTCAGACAACGGGGTTCCCTACGAC
TTTGTGGTTCTCGAGAAGAGAAGGAAGACTGACGACGCAGCCACTGCGGAACCGAGCAAC
GCAATGAGCTCCTTGACGTCCACGAGGGAGACAACTCCCGTGCACGGGTTGCAGGCTCCT
TCTTCGGCCGCAGCCATTGCCCCGGTGTTGGCGTGGATGGACGAAGAAGACCGGAAAAAA
CGCGAGCAAAAGGAACTGATTCGGGCCGTTCCGCATGTTCACTTTAGAGGCCATGAAGAG
TTCCAGTACCTTGATCTCATTGCCGACATTATTAACAATGGAAGGACAATGGATGACCGA
ACGGGCGTTGGTGTCATCTCCAAATTCGGCTGCACTATGCGCTACTCGCTGGATCAGGCC
TTTCCACTTCTCACCACAAAGCGTGTGTTCTGGAAAGGGGTCCTCGAAGAGTTGCTGTGG
TTCATTCGCGGCGACACGAACGCAAACCATCTTTCTGAGAAGGGCGTGAAGATCTGGGAC
AAGAATGTGACACGCGAGTTCCTCGATTCGCGCAATCTCCCCCACCGAGAGGTCGGAGAC
ATCGGCCCGGGCTACGGCTTCCAGTGGAGACACTTCGGCGCGGCATACAAAGACATGCAC
ACAGACTACACAGGGCAGGGCGTCGACCAGCTGAAGAATGTGATCCAGATGCTGAGAACG
AATCCAACAGATCGTCGCATGCTCATGACTGCCTGGAATCCTGCAGCGCTGGACGAAATG
GCGCTGCCGCCTTGTCACTTGTTGTGCCAGTTCTACGTGAACGACCAGAAGGAGCTGTCG
TGCATCATGTATCAGCGGTCGTGCGATGTCGGCCTCGGCGTCCCCTTCAACATCGCTTCC
TATTCGCTTTTGACGCTCATGGTTGCACACGTCTGCAACCTAAAACCTAAGGAGTTCATT
CACTTCATGGGGAACACGCATGTCTACACGAACCATGTCGAGGCTTTAAAAGAGCAGCTG
CGGAGAGAACCGAGACCGTTCCCCATTGTGAACATCCTCAACAAGGAACGCATCAAGGAA
ATCGACGATTTCACCGCCGAGGATTTTGAGGTCGTGGGCTACGTCCCGCACGGACGAATC
CAGATGGAGATGGCTGTCTAGCGGAAATACAGAAGCTAGCTTTGATCCCGTTTTTCTTAC
TTATATATTTATACCAATTGATTGTATTTATAACTGTAAAAATGTGTATGTTGTGTGCAT
ATTTTTTTTTGTGCATGCACATGCATGTAAATAGCTAAAATTATGAACATTTTATTTTTT
GTTCAGAAAAAAAAAACTTTACACACATAAAATGGCTAGTATGAATAGCCATATTTTATA
TAAATTAAATCCTATGAATTTATGACCATATTAAAAATTTAGATATTTATGGAACATAAT
ATGTTTGAAACAATAAGACAAAATTATTATTATTATTATTATTTTTACTGTTATAATTAT
GTTGTCTCTTCAATGATTCATAAATAGTTGGACTTGATTTTTAAAATGTTTATAATATGA
TTAGCATAGTTAAATAAAAAAAGTTGAAAAATTAAAAAAAAACATATAAACACAAATGAT
GTTTTTTCCTTCAATTTCGATATCAATTAAGTATATTAGGATAACATATTTTTATATATT
TTAAATCGAAACGACTTCGTTGCAATTTATTTATATTCCTTTTTTTTTAGTTTTCAGGAA
TGTTTACATTATGGATTTGCCTCGATTTGTGCTTTAATTATTTTTTATTTGCCACTTCGT
CTATTCATTTTGAATATGCCATGTGAATTTGCTTAGCTTATTTTTAAAAAAATTAATTTT
AACAATTGTTTAATAATAATGAAGTGTTTTGACAACAAAAAATAAATAAAATTACATCAA
AAAAATATATAAAATTACAACAAAAAAATATATAAAATTACAACAAAAAAATATATAAAA
TTACAAAAAAATAAATATATAAAATTCAACAAAACAAATTATTAAATTGACGTTTTATTT
TCGTTGTTCTTTAGTATATGCTTATTTAAATTAGATGAATTTTTAGTGATATAATCAAAA
GTACTTATATTTATTTCTTCATATAACGAATGAATAAAATTTGTCATCTGGGCTTTTGTT
GTTTCAAATTGTATGGTATAAGTTTTGGAAATGTCTTTTAAGAAATATTCATATATATAT
TCGGATTTTGAGATTTTATACAAACATAAGATATACATATATGATGGTCTTATAAATTCA
CCTGCCTGTATAAAATCAAGTATATGCATTTTTTCTTTTATTTTTTTTCTTATTTTCCTT
TTATTATTTATTGTTATGATATTATTTTCCCATTTGTACCATCTTTTAAAAATTACATCA
TTACTAAGTTTAAAATCGATATTGTTAATATTATTAGTATATTTCGAAGAATGTGATATA
ACAGTAGCTGATAATTGTAATAGTTTTTTAACAAATGGTGATGATATAATGTGTGCAATA
ACTTGCTTCGTGGATCCACTAGTTCTAGAGCGGCCGCCACCGCGGTGGAGCTCCAATTCG
CCCTATAGTGAGTCGTATTACGCGCGCTCACTGGCCGTCGTTTTACAACGTCGTGACTGG
GAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGG
CGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGC
GAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGC
GTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTT
CTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTC
CGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGT
AGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTT
AATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTT
GATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAA
AAATTTAACGCGAATTTTAACAAAATATTAACGCTTACAATTTAGGTGGCACTTTTCGGG
GAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGC
TCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTA
TTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTG
CTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGG
GTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAAC
GTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTG
ACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGT
ACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTG
CTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGAC
CGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTT
GGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAG
CAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGC
AACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCC
TTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTA
TCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGG
GGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGA
TTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAAC
TTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAA
TCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGAT
CTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGC
TACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTG
GCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACC
ACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGG
CTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGG
ATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAA
CGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCG
AAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGA
GGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCT
GACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCA
GCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTC
CTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCG
CTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCC
CAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACA
GGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTC
ATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGA
GCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCTCGAAATTA
ACCCTCACTAAAGGGAACAAAAGCTGGTACCGGGCCCCCCCTCGAGGTCGACGGTAT
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 1AAATCGATGAATTCCAACAATGAATGTAATAAAGG
Additional information primer 1L1754 (ClaI,EcoRI); Pb 5' rho-7 sense
Sequence Primer 2TTAAGCTTATGTCTATTTTGTTTCACTTG
Additional information primer 2L1755 (HindIII); Pb 5' rho-7 antisense
Sequence Primer 3AAGATATCAATTAAGTATATTAGGATAAC
Additional information primer 3L1756 (EcoRV); Pb 3' rho-7 sense
Sequence Primer 4TTGGATCCACGAAGCAAGTTATTGCACAC
Additional information primer 4L1757 (BamHI); Pb 3' rho-7 antisense
Sequence Primer 5
Additional information primer 5
Sequence Primer 6
Additional information primer 6