IGEM:Harvard/2010/Team Allergy: Difference between revisions
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|''CCTTGAATTCGCGGCCGCATCTAGA'' GATGACACCACCACCGGCTA | |''CCTTGAATTCGCGGCCGCATCTAGA'' GATGACACCACCACCGGCTA | ||
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|Fra a 1 | |Fra a 1 Exon2 (for hair pin) | ||
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|''CCTTGAATTCGCGGCCGCATCTAGA'' acgtcaagcacaagatccac | |''CCTTGAATTCGCGGCCGCATCTAGA'' acgtcaagcacaagatccac |
Revision as of 08:56, 21 June 2010
Description
Allergies to fruits and vegetables are (increasingly) common, affecting millions of people around the world with symptoms ranging from mild itchiness to life-threatening anaphylaxis. Allergy is caused by an inappropriate immune response to harmless proteins present in the environment. Several common food allergens are structurally similar to pollens that cause seasonal allergies and are present in a wide range of fruits and vegetables. Many of these proteins have been knocked down in plants using RNA interference, leading to plants with reduced allergenicity. As part of our iGarden project we are designing modular BioBrick intron-containing self-complementary hairpin forming RNA (ihpRNA) constructs for the targeted knockdown of proteins with homology to allergens in arabidopsis and strawberry, as well as designing ihpRNA constructs against allergens in a range of other plants common in home gardens, including lettuce, carrots, celery, tomato, and several herbs. Our goal is to use genetic engineering to make food safer, and to specially tailor gardens to the needs of each person with a different set of allergies.
Bibliography
Plant shRNA
- Wesley SV, Helliwell CA, Smith NA, Wang MB, Rouse DT, Liu Q, Gooding PS, Singh SP, Abbott D, Stoutjesdijk PA, Robinson SP, Gleave AP, Green AG, and Waterhouse PM. Construct design for efficient, effective and high-throughput gene silencing in plants. Plant J. 2001 Sep;27(6):581-90. DOI:10.1046/j.1365-313x.2001.01105.x |
- Schwab R, Ossowski S, Riester M, Warthmann N, and Weigel D. Highly specific gene silencing by artificial microRNAs in Arabidopsis. Plant Cell. 2006 May;18(5):1121-33. DOI:10.1105/tpc.105.039834 |
Allergen silencing
- Dodo HW, Konan KN, Chen FC, Egnin M, and Viquez OM. Alleviating peanut allergy using genetic engineering: the silencing of the immunodominant allergen Ara h 2 leads to its significant reduction and a decrease in peanut allergenicity. Plant Biotechnol J. 2008 Feb;6(2):135-45. DOI:10.1111/j.1467-7652.2007.00292.x |
- Singh MB and Bhalla PL. Genetic engineering for removing food allergens from plants. Trends Plant Sci. 2008 Jun;13(6):257-60. DOI:10.1016/j.tplants.2008.04.004 |
- Le LQ, Mahler V, Lorenz Y, Scheurer S, Biemelt S, Vieths S, and Sonnewald U. Reduced allergenicity of tomato fruits harvested from Lyc e 1-silenced transgenic tomato plants. J Allergy Clin Immunol. 2006 Nov;118(5):1176-83. DOI:10.1016/j.jaci.2006.06.031 |
- Gallo M and Sayre R. Removing allergens and reducing toxins from food crops. Curr Opin Biotechnol. 2009 Apr;20(2):191-6. DOI:10.1016/j.copbio.2009.03.005 |
- Lorenz Y, Enrique E, Lequynh L, Fötisch K, Retzek M, Biemelt S, Sonnewald U, Vieths S, and Scheurer S. Skin prick tests reveal stable and heritable reduction of allergenic potency of gene-silenced tomato fruits. J Allergy Clin Immunol. 2006 Sep;118(3):711-8. DOI:10.1016/j.jaci.2006.05.014 |
- Herman EM, Helm RM, Jung R, and Kinney AJ. Genetic modification removes an immunodominant allergen from soybean. Plant Physiol. 2003 May;132(1):36-43. DOI:10.1104/pp.103.021865 |
- Gilissen LJ, Bolhaar ST, Matos CI, Rouwendal GJ, Boone MJ, Krens FA, Zuidmeer L, Van Leeuwen A, Akkerdaas J, Hoffmann-Sommergruber K, Knulst AC, Bosch D, Van de Weg WE, and Van Ree R. Silencing the major apple allergen Mal d 1 by using the RNA interference approach. J Allergy Clin Immunol. 2005 Feb;115(2):364-9. DOI:10.1016/j.jaci.2004.10.014 |
- Bhalla PL and Singh MB. Knocking out expression of plant allergen genes. Methods. 2004 Mar;32(3):340-5. DOI:10.1016/j.ymeth.2003.08.011 |
Allergen proteins
- Chardin H, Mayer C, Sénéchal H, Wal JM, Poncet P, Desvaux FX, and Peltre G. Lipid transfer protein 1 is a possible allergen in Arabidopsis thaliana. Int Arch Allergy Immunol. 2003 Jun;131(2):85-90. DOI:10.1159/000070923 |
- San Miguel-Moncín M, Krail M, Scheurer S, Enrique E, Alonso R, Conti A, Cisteró-Bahíma A, and Vieths S. Lettuce anaphylaxis: identification of a lipid transfer protein as the major allergen. Allergy. 2003 Jun;58(6):511-7. DOI:10.1034/j.1398-9995.2003.00097.x |
- Salcedo G, Sanchez-Monge R, Diaz-Perales A, Garcia-Casado G, and Barber D. Plant non-specific lipid transfer proteins as food and pollen allergens. Clin Exp Allergy. 2004 Sep;34(9):1336-41. DOI:10.1111/j.1365-2222.2004.02018.x |
- Gajhede M, Osmark P, Poulsen FM, Ipsen H, Larsen JN, Joost van Neerven RJ, Schou C, Løwenstein H, and Spangfort MD. X-ray and NMR structure of Bet v 1, the origin of birch pollen allergy. Nat Struct Biol. 1996 Dec;3(12):1040-5. DOI:10.1038/nsb1296-1040 |
- Zuidmeer L, Salentijn E, Rivas MF, Mancebo EG, Asero R, Matos CI, Pelgrom KT, Gilissen LJ, and van Ree R. The role of profilin and lipid transfer protein in strawberry allergy in the Mediterranean area. Clin Exp Allergy. 2006 May;36(5):666-75. DOI:10.1111/j.1365-2222.2006.02453.x |
- Karlsson AL, Alm R, Ekstrand B, Fjelkner-Modig S, Schiött A, Bengtsson U, Björk L, Hjernø K, Roepstorff P, and Emanuelsson CS. Bet v 1 homologues in strawberry identified as IgE-binding proteins and presumptive allergens. Allergy. 2004 Dec;59(12):1277-84. DOI:10.1111/j.1398-9995.2004.00585.x |
- Musidlowska-Persson A, Alm R, and Emanuelsson C. Cloning and sequencing of the Bet v 1-homologous allergen Fra a 1 in strawberry (Fragaria ananassa) shows the presence of an intron and little variability in amino acid sequence. Mol Immunol. 2007 Feb;44(6):1245-52. DOI:10.1016/j.molimm.2006.06.004 |
- Breiteneder H and Ebner C. Molecular and biochemical classification of plant-derived food allergens. J Allergy Clin Immunol. 2000 Jul;106(1 Pt 1):27-36. DOI:10.1067/mai.2000.106929 |
- Jensen-Jarolim E, Schmid B, Bernier F, Berna A, Kinaciyan T, Focke M, Ebner C, Scheiner O, and Boltz-Nitulescu G. Allergologic exploration of germins and germin-like proteins, a new class of plant allergens. Allergy. 2002 Sep;57(9):805-10. DOI:10.1034/j.1398-9995.2002.23686.x |
Primers and Sequences
name | GenBank Accession | forward sense primer | reverse sense primer | forward antisense primer | reverse antisense primer |
---|---|---|---|---|---|
pdk intron | AJ311872 | CCTTGAATTCGCGGCCGCATCTAGA CCAATTGGTAAGGAAATAAT | AAGGCTGCAGCGGCCGCTACTAGT TTCGAACCCAATTTCCCAAC | ||
GFP | AF218816.1 | CCTTGAATTCGCGGCCGCATCTAGA atggtgagcaagggcgagga | AAGGCTGCAGCGGCCGCTACTAGT gaagatggtgcgctcctgga | AAGGCTGCAGCGGCCGCTACTAGT atggtgagcaagggcgagga | CCTTGAATTCGCGGCCGCATCTAGA gaagatggtgcgctcctgga |
A. thaliana LTP1 | ATHSEQB | CCTTGAATTCGCGGCCGCATCTAGA ATGGCTGGAGTGATGAAGTT | AAGGCTGCAGCGGCCGCTACTAGT CAGCTGCACGGCCAGCGTTG | AAGGCTGCAGCGGCCGCTACTAGT ATGGCTGGAGTGATGAAGTT | CCTTGAATTCGCGGCCGCATCTAGA CAGCTGCACGGCCAGCGTTG |
Fra a 1A (Bet v 1) | DQ385511 | CCTTGAATTCGCGGCCGCATCTAGA ATGGGTGTTTACACTTATGA | AAGGCTGCAGCGGCCGCTACTAGT GTAGTCGATCTTCTCGATGT | AAGGCTGCAGCGGCCGCTACTAGT ATGGGTGTTTACACTTATGA | CCTTGAATTCGCGGCCGCATCTAGA GTAGTCGATCTTCTCGATGT |
L. sativa LTP 1.1 | EF101532.1 | CCTTGAATTCGCGGCCGCATCTAGA ATGGCAAGGATGGCAACGAT | AAGGCTGCAGCGGCCGCTACTAGT TGACACCACACTTGCCGGGG | AAGGCTGCAGCGGCCGCTACTAGT ATGGCAAGGATGGCAACGAT | CCTTGAATTCGCGGCCGCATCTAGA TGACACCACACTTGCCGGGG |
L. sativa LTP 1.2 | EF101531.1 | CCTTGAATTCGCGGCCGCATCTAGA GTGACATGTGGTCAGGTGGT | AAGGCTGCAGCGGCCGCTACTAGT CTGGATCCTATTGCAGTCGG | AAGGCTGCAGCGGCCGCTACTAGT GTGACATGTGGTCAGGTGGT | CCTTGAATTCGCGGCCGCATCTAGA CTGGATCCTATTGCAGTCGG |
S. lycopersicum Lyc E 1.01 (profilin) | AJ417553 | CCTTGAATTCGCGGCCGCATCTAGA ATGTCGTGGCAAACATATGT | AAGGCTGCAGCGGCCGCTACTAGT AGCCTGATTGGTCTTCTTAA | AAGGCTGCAGCGGCCGCTACTAGT ATGTCGTGGCAAACATATGT | CCTTGAATTCGCGGCCGCATCTAGA AGCCTGATTGGTCTTCTTAA |
D. Carota Dau C 1 | Z84376.1 | CCTTGAATTCGCGGCCGCATCTAGA ATGGGTGCCCAGAGCCATTCAC | AAGGCTGCAGCGGCCGCTACTAGT GGTTTCAATGGATTCGATGA | AAGGCTGCAGCGGCCGCTACTAGT ATGGGTGCCCAGAGCCATTCAC | CCTTGAATTCGCGGCCGCATCTAGA GGTTTCAATGGATTCGATGA |
Api g 4 (celery profilin) | AF129423.1 | CCTTGAATTCGCGGCCGCATCTAGA ATGTCGTGGCAGGCGTACGT | AAGGCTGCAGCGGCCGCTACTAGT GGTTCATCATAGACTCCGAA | AAGGCTGCAGCGGCCGCTACTAGT ATGTCGTGGCAGGCGTACGT | CCTTGAATTCGCGGCCGCATCTAGA GGTTCATCATAGACTCCGAA |
Api g 1.0201 (celery) | Z75662.2 | CCTTGAATTCGCGGCCGCATCTAGA ATGGGTGTCCAAAAGACCGT | AAGGCTGCAGCGGCCGCTACTAGT CACCACTGACTCGAGTACGT | AAGGCTGCAGCGGCCGCTACTAGT ATGGGTGTCCAAAAGACCGT | CCTTGAATTCGCGGCCGCATCTAGA CACCACTGACTCGAGTACGT |
Api g 1 (celery) | Z48967.1 | CCTTGAATTCGCGGCCGCATCTAGA ATGGGAGTGCAGACACATGT | AAGGCTGCAGCGGCCGCTACTAGT GTTTTCAATGGATTCGATAA | AAGGCTGCAGCGGCCGCTACTAGT ATGGGAGTGCAGACACATGT | CCTTGAATTCGCGGCCGCATCTAGA GTTTTCAATGGATTCGATAA |
A. Thaliana (BLAST) Bet v 1 (#1) | :12937059-12937689 | CCTTGAATTCGCGGCCGCATCTAGA ATGATAGAAGAGGAGATTGA | AAGGCTGCAGCGGCCGCTACTAGT TACCATATTAAAAAATACGT | AAGGCTGCAGCGGCCGCTACTAGT ATGATAGAAGAGGAGATTGA | CCTTGAATTCGCGGCCGCATCTAGA TACCATATTAAAAAATACGT |
A. Thaliana (BLAST) Bet v 1 (#2) | :12937059-12937689 | CCTTGAATTCGCGGCCGCATCTAGA AAATTCATTTATAAACGCAT | AAGGCTGCAGCGGCCGCTACTAGT TTAATCCTTAGACAAGAGGT | AAGGCTGCAGCGGCCGCTACTAGT AAATTCATTTATAAACGCAT | CCTTGAATTCGCGGCCGCATCTAGA TTAATCCTTAGACAAGAGGT |
A. thaliana GER3 | NM_122070 | CCTTGAATTCGCGGCCGCATCTAGA ATGAAGATGATAATCCAAAT | AAGGCTGCAGCGGCCGCTACTAGT GATGACACCACCACCGGCTA | AAGGCTGCAGCGGCCGCTACTAGT ATGAAGATGATAATCCAAAT | CCTTGAATTCGCGGCCGCATCTAGA GATGACACCACCACCGGCTA |
Fra a 1 Exon2 (for hair pin) | CCTTGAATTCGCGGCCGCATCTAGA acgtcaagcacaagatccac | AAGGCTGCAGCGGCCGCTACTAGT gtggatcttgtgcttgacgt | AAGGCTGCAGCGGCCGCTACTAGT acgtcaagcacaagatccac | CCTTGAATTCGCGGCCGCATCTAGA gtggatcttgtgcttgacgt |
Protocols
Isolating Genes From Plants
Strawberry Transformation
- Schaart JG, Krens FA, Pelgrom KT, Mendes O, and Rouwendal GJ. Effective production of marker-free transgenic strawberry plants using inducible site-specific recombination and a bifunctional selectable marker gene. Plant Biotechnol J. 2004 May;2(3):233-40. DOI:10.1111/j.1467-7652.2004.00067.x |
- Oosumi T, Gruszewski HA, Blischak LA, Baxter AJ, Wadl PA, Shuman JL, Veilleux RE, and Shulaev V. High-efficiency transformation of the diploid strawberry (Fragaria vesca) for functional genomics. Planta. 2006 May;223(6):1219-30. DOI:10.1007/s00425-005-0170-3 |
- Husaini AM. Pre- and post-agroinfection strategies for efficient leaf disk transformation and regeneration of transgenic strawberry plants. Plant Cell Rep. 2010 Jan;29(1):97-110. DOI:10.1007/s00299-009-0801-4 |
- Graham J, McNicol RJ, and Kumar A. Agrobacterium-mediated transformation of soft fruit Rubus, Ribes, and Fragaria. Methods Mol Biol. 1995;44:129-33. DOI:10.1385/0-89603-302-3:129 |
Plasmids
Introns
ihpRNA production requires the selection of an intron. Here's one (between || in the range 295..396): D64156.1
1 tgtagtaatg tgtatatata ggtggtcgtg ttacgcgagg aggaacacag caaactcatc 61 aaatctgggg ttgacaagaa tctggtactg tctacaagca atagttatta ctccccaaag 121 gtgtggttgg tgggggatgg aatagagaac gaagagcaga tgaaagcaaa agaaggaacc 181 ctctttgttc ccttttctca ctttccgccc aacaaactcc gcaaggactg tttctaccag 241 tccactccag ctatgcgtgt tcccaagtct gcccaaaaca tcgactcctg tgag|gtacat 301 ctttgaattc ttatagatat atctgtaact tttatattat ataagctgat agatgtgttc 361 atctataatg aatgaatggt tgttatatat atatag|aact ggctggggag gagggtgatg 421 agtgcatgga aaataggagg tatagtgcat gcacttgagg gttgggagga gcatgactgc 481 ggcaacactt gcaacgtcct ccgtctccac gccatatggg aagctgctct tcgccatgat 541 ttccaacctc tcccaccatc tcctctatga gcttttttca tattcataca tctatgtccc 601 ct
- PAL 3 intron 2303-2992
1 ttttatctat ctttttattt tcttgtgtgg attgcttgtg attttgtttg ttggcctaat 61 aatcagcttg gattttttag ctttcttgga aataaaagaa gatcacgata attaattact 121 ttatggtccc aacctacaaa ttgttggact actattggaa ctaacaaata aatacacgaa 181 attgattggg tgagttataa tattaccttt ttagtttttg atatagtcaa attttatagt 241 ataacagatt tgttaaaatt aaattgtatc gaaacttctt atagtaatgt actaatgtga 301 gaaaatctgt gtactgttat tatagtatac gcgttttaat acttttatta ttattcctac 361 ttaaagaaaa taccaaatat ttttccgttt tttgggtgtg tttagttctt ttttttttaa 421 ctgaaggatt ttggtgaatt ttctgttgcc attaaaaata tttgtgttta gttttttgat 481 attttaattc tttacttttt tttaagataa tcaatgwwgg accaacatcc caaatgtgaa 541 ccaaactccg acccgaaanc cgctgctttc aatactttca atcgaaatag cgtgtgacgt 601 gtgttaatat ctgcatgcat tgattatcgg acaatgacaa atattctatt tatcctaaat 661 tttattctcc gtgaagtaca catttggaca aaccaaacgt agatttgaga aaaaaatgcg 721 gaaaacccaa agtctatccg tggcgaagaa taagaggcaa aaacggggaa tataacccaa 781 agtctaaacc caataattat tttattgata atcaaaatct atcacgagaa agacgacgtt 841 ttgacatctc agtcgttata aatttcaata aattatgttt cccttgaact gattcacatg 901 tcattttcat tccacatata tacgccaatc gtgtaatctt ttagattatc gatcgaacat 961 ctaaattttg tcaacatgtt aatgaatatc taaggaacgc tatcgsstat cgcttttttc 1021 aacataagtc taatttgaga gatgttaatc caccgcatgt tgagtggtgt aagagagaaa 1081 gagccgccac taggctactt gtctcccaac aacaatatat atcctgaatt atgaattatg 1141 caattatact tttctggtca ttcaattatt attacttttg ttttgttcaa aatgctttag 1201 tgtactactt tcaaatttca acatccttct ttatttattt caactttttt ctcaacatta 1261 tcgtttcttt tttsctgtcg ataacaacaa cttgttgagt gtcttgcaaa gattagcctt 1321 ttactcgatt ccccgtttga ttagatttta aggctttccg ttaaccttta acagttaaca 1381 aagtgtggag tgcctagcaa agacaattgt gctttgtkky ggcggcaacg tcagcaacgr 1441 ttttaggact tctacttctg atgagagata cagakcgttg ccwygttgaa aaagaaatta 1501 aaaggaaaac ataacacatt atacgagaaa gaagtttaaa aaaaaaaaaa actggttgaa 1561 actaatcgaa tatttgaagc attttttata tctactagaa agtaagtttt ttaatggcta 1621 aaaacgaata atcctattca ttattttaar tcaaacacta ataagatgtt cttaaacatt 1681 tcaactagtt aaccccattg ttttccctct atatatatac cccaccaagg cacaaaccaa 1741 cccactcact cacattattt aaaacacatg ttttactact tgtctacata tatctatata 1801 atcatcgtag aaatcgatac ttgaggccag cccaaaatca cactcgagtc gaatatatat 1861 atatatatat atatcttaat ccggctcgtt atataaaagc tccagacttg tcactagggt 1921 tgccgtcaga ttttgtgatc atggagtttc gtcaaccaaa cgcaacagca ttgagcgacc 1981 cacttaactg gaatgtagcg gcggaggctt taaaagggag ccacctggag gaggtgaaga 2041 agatggtgaa ggattatagg aaaggaacgg tgcagctagg cggagagacg ctgaccatcg 2101 gtcaggttgc ggcagtagcg agtggaggag gaccgacagt ggagctttct gaggaggctc 2161 ggggcggtgt gaaggcgagt agtgactggg tgatggagag catgaaccgt gatacggaca 2221 catatgggat caccactgga tttggttcat cttctcgtag gaggactgac caaggtgctg 2281 ctcttcaaaa agagcttatt aggtaatt|ca tattcatagt tgaatcagac gaaaaataaa 2341 tagaatcata gaaaactagg tttagaaacc cgtacataaa ctagtttttt taaaaaggaa 2401 tssatattta tatccagcgc ctatagataa agaacaagac attttataat ctgtctcgac 2461 gtttttacac caacaaatga gaataattag tcgtactata aaagttcaaa tttctttaaa 2521 tatgattaaa cgaagactaa aacgctctaa gttttactac atttttaaat aaatacgact 2581 ttttgccgaa agaaaacata gtacttaaga taagagcatt ttgtgttcta atgaaaatta 2641 aacgtttctg cattctctat tcttttgact gtcttatact tttattgaaa taagcttttt 2701 ttaacttttg taaatcagct ttaacgaaaa aagaatgtag tgactagtgg agcacggagg 2761 caaagttgct agctttgtac accttttttt tatttttgtc ttttgggttg taacttagat 2821 aagaagaggt ctcagtccgt aaaaagttta aacggtatcg attaaaattg tttaagctaa 2881 aagaggtagt agtcgaagtt attaaatgca ttatattata tagttgacaa aagtaattaa 2941 aaaataaatg taatcggttt aaaactgacg gatcccaaat tatatcaacc ag|gtatttga 3001 acgccgggat attcgctacc ggcaacgaag atgacgacag gtcaaacacg cttccccggc 3061 cggctactag agcagcgatg ctcatccgtg taaacaccct cctccaaggc tactctggta 3121 tacgctttga gatcctcgaa gccatcacaa cactcctcaa ctgcaaaatt acaccgctcc 3181 ttcctctccg aggcaccatt accgcctccg gggatctcgt tccgttatcc tatatcgctg 3241 gattcctcat cgggcgcccc aactcccgat ccgtgggccc ctctggcgag atcctcactg 3301 ccttggaggc cttcaagctc gctggagtat cgtctttttt cgaactcagg cctaaagaag 3361 ggcttgcgct cgtgaatggg actgcggtgg ggtctgcttt agcctctacg gtactgtacg 3421 atgccaacat tttggtggtt ttctccgaag ttgcttccgc catgtttgca gaggttatgc 3481 aggggaaacc agagtttacc gatcatctta cgcataaact caagcaccat cctggtcaga 3541 tcgaagccgc cgctatcatg gagcatattc tagacggaag ctcttatgta aaagaagctc 3601 tacatctcca caagattgat ccgcttcaga aacctaaaca agatcggtat gttcttgggt 3661 atcaagcaac tagtttcttg taactcaagt aacccttttg atttgctttc tgattaaatt 3721 gacttatagt ttcgtgttta aattaacagt tacgctctgc gaacatctcc gcaatggctt 3781 ggaccgcaga ttgaggtgat aagagcagcg actaagatga tcgaacgtga gataaactca 3841 gtaaacgata accctttgat cgatgtttca agaaacaaag ctatccatgg tgggaacttc 3901 caggggacac caattggtgt cgccatggat aacactcgtc tagcacttgc ttctatcggg 3961 aagctaatgt tcgctcagtt cactgaactc gtaaatgatt tctacaacaa cgggttaccc 4021 tctaatctat ctggtggtag aaaccctagt cttgattacg ggttaaaagg cgcagaagtc 4081 gccatggctt cttattgctc agagcttcag ttcctagcaa atcctgtgac gaaccatgtc 4141 gaaagcgctt ctcaacacaa tcaagatgtt aactctcttg ggctgatctc gagccgaacg 4201 acagcagaag ctgtggttat cctcaagctc atgtcaacga cttacttggt agctttgtgc 4261 caagcctttg atctgagaca ccttgaagaa attctcaaga aagcggttaa tgaggttgtg 4321 agccacactg ccaaaagcgt tctagcaatc gaaccattcc gcaaacacga cgatattctt 4381 ggagttgtca accgcgaata cgtcttctcc tatgttgatg acccaagcag cctcactaac 4441 cctttaatgc agaagctgag acatgtcctc ttcgacaaag ctttagctga accggaaggc 4501 gagaccgaca cggtttttcg gaaaatcgga gcgttcgagg ccgagctgaa atttctcctc 4561 cctaaagaag tggaacgagt caggacagag tacgagaacg gtacatttaa tgtggctaac 4621 cgaatcaaga agtgtcgatc gtatccgctg taccggtttg tgcggaatga actcgagacg 4681 aggttgctaa ccggagagga tgttcggtca ccgggagagg attttgacaa agtcttcagg 4741 gctatatctc aaggaaaact catagawcct ctgtttgaat gtctgaaaga gtggaacggt 4801 gctccgattt ctatctgcta aaacgtttcg tcagatctcc atatatcgag aataataaac 4861 acaccattcg aaaatatttg gttgtgtagt aatcgtcttg ccttcgcctt ttcaataatg 4921 tcctg