An innovative multigenic plasmid for high levels of expression of the GFP and LacZ reporter genes Catalog # pvitro2-gfplacz

Transcription

1 pvitro2-hygro-gfp/lacz An innovative multigenic plasmid for high levels of expression of the GFP and LacZ reporter genes Catalog # pvitro2-gfplacz For research use only Version # 04K05-SV PRODUCT INFORMATION Contents: - 20 µg of pvitro2-hygro-gfp/lacz provided as lyophilized DNA - 4 pouches of E. coli FastMedia Hygro Storage and Stability: - Product is shipped at room temperature. - Lyophilized DNA is stable for 12 months when stored at - 20 C - Resuspended DNA is stable for 12 months at -20 C. Avoid repeated freeze-thaw cycles. - Store E. coli FastMedia Hygro pouches at room temperature. FastMedia is stable 18 months when stored properly. Quality control: - Plasmid construct has been confirmed by restriction analysis and sequencing. - Plasmid DNA was purified by ion exchange chromatography and lyophilized. GENERAL PRODUCT USE pvitro is a new family of vectors with improved features. pvitro1 and pvitro2 allow the co-expression of two or more genes from two diff e r e n t transcription units. pvitro plasmids can be stably transfected in mammalian cells and yield high levels of expression. pvitro2-hygro-gfp/lacz contains the reporter genes GFP and LacZ and can be used as a control vector. pvitro2-hygro-gfp/lacz also can be used for cloning of open reading frames (ORF). Both reporter genes are flanked by unique sites (BspH I/Avr II for GFP and Nco I/Nhe I for LacZ) that allow for convenient cloning of ORF s which can be selected from InvivoGen s extensive porf and pblast lists. PLASMID FEATURES hferh and hferl composite promoters: Ferritin is a 24 subunit protein composed of two subunit types, termed H (heavy) and L (light), which perform complementary functions in the protein. Ferritin is ubiquitously expressed. Its synthesis is highly regulated by the iron status of the cell. The iron regulation is achieved at the translational level through the interaction between the iron-responsive element (IRE), located in the 5 untranslated region (5 UTR) of the ferritin mrnas, and the iron regulatory protein 1. To eliminate the iron regulation of the ferritin promoters, the 5 UTR of FerH and FerL have been replaced by the 5 UTR of the mouse and chimpanzee elongation factor 1 (EF1) genes, respectively. SV40 enhancer which is comprised of a 72-base-pair repeat allows the enhancement of gene expression in a large host range. The enhancement varies from 2-fold in non-permissive cells to 20-fold in permissive cells. Furthermore, the SV40 enhancer is able to direct nuclear localization of plasmids 2. CMV enhancer: The major immediate early enhancer of the human cytomegalovirus (HCMV), located between nucleotides -118 and -524, is composed of unique and repeated sequence motifs. The HCMV enhancer can substitute for the 72-bp repeats of SV40 and is severalfold more active than the SV40 enhancer 3. pmb1 ori: A minimal E. coli origin of replication to limit vector size, but with the same activity as the longer Ori. GFP gene: This red-shifted variant of the jellyfish GFP gene encodes a green fluorescent protein that absorbs blue light (major peak at 480 nm) and emits green light (major peak at 505 nm). FMDV IRES: The internal ribosome entry site of the Foot and Mouth Disease Virus enables the translation of two open reading frames from one mrna with high levels of expression 4. EM7 is a bacterial promoter that enables the constitutive expression of the antibiotic resistance gene in E. coli. hph gene: hph confers resistance to Hygromycin B both in E. coli and mammalin cells. In bacteria, hph is expressed from the constitutive E. coli EM7 p r o- m o t e r. In mammalian cells, h p h is transcribed from the human FerH c o m- posite promoter as a polycistronic mrna and translated via the FMDV I R E S. EF1 pan is a strong polyadenylation signal. InvivoGen uses a s e q u e n c e starting after the stop codon of the EF1 cdna and finishing after a bent structure rich in GT. LacZ gene: T h e E. coli lacz gene codes for the enzyme β-galactosidase which catalyzes the hydrolysis of the substrate X-Gal to produce a blue color that is easily visualized under a microscope. SV40 pan: the Simian Virus 40 late polyadenylation signal enables efficient cleavage and polyadenylation reactions resulting in high levels of steady-state mrna. The efficiency of this signal was first described by Carswell et al. 5 METHODS Plasmid resuspension: Quickly spin the tube containing the lyophilized plasmid to pellet the DNA. To obtain a plasmid solution at 1 µg/µl, resuspend the DNA in 20 µl of sterile H 2 O. Store resuspended plasmid at -20 C. Selection of bacteria with E. coli FastMedia Hygro: E. coli F a s t M e d i a Hygro is a n e w, fast and convenient way to prepare liquid and solid media for bacterial culture by using only a microwave. E. coli FastMedia Hygro is a TB (liquid) or LB (solid) based medium with hygromycin B, and contains stabilizers. E. coli FastMedia Hygro can be ordered separately (catalog code # fas-hg-l, fas-hg-s). Method: 1- Pour the contents of a pouch into a clean borosilicate glass bottle or flask. 2- Add 200 ml of distilled water to the flask 3- Heat in a microwave on MEDIUM power setting (about 400Watts), until bubbles start appearing (approximately 3 minutes). Do not heat a closed container. Do not autoclave FastMedia. 4- Swirl gently to mix the preparation. Be careful, the bottle and media are hot, use heatproof pads or gloves and care when handling. 5- Reheat the media for 30 seconds and gently swirl again. Repeat as necessary to completely dissolve the powder into solution. But be careful to avoid overboiling and volume loss. 6- Let agar medium cool to 45 C before pouring plates. Let liquid media cool to 37 C before seeding bacteria. Note: Do not reheat solidified FastMedia as the antibiotic will be permanently destroyed by the procedure. References: 1. Eisenstein RS. and Munro HN Translational regulation of ferritin synthesis by iron. Enzyme 44(1-4): Dean DA. et al Sequence requirements for plasmid nuclear import. Exp. Cell. Res. 253: Boshart M. et al A very strong enhancer is located upstream of an immediate early gene of human cytomegalovirus. Cell 141(2): Ramesh N et al High-titer bicistronic retroviral vectors employing foot-and-mouth disease virus internal ribosome entry site. Nucleic Acids Res. 24(14): Carswell S., and Alwine JC Efficiency of utilization of the simian virus 40 late polyadenylation site: effects of upstream sequences. Mol. Cell Biol. 10: TECHNICAL SUPPORT Toll free (US): Outside US: (+1) info@invivogen.com Website: Sorrento Valley Blvd. Suite A San Diego, CA USA

2 HindIII (679) BglII (1039) HindIII (8278) EM7 hph EF1 pan CMV enh hferl prom chef1 5'UTR XhoI (1438) NcoI (1673) AvrII (7874) FMDV IRES GFP pvitro2-gfp/lacz (10034 bp) LacZ BspHI (7154) mef1 5'UTR hferh prom SV40 enh HindIII (6153) pmb1 ori SV40 pan NotI (6000) PacI (5718) PacI (4978) NheI (4738) 250

3 CCTGCAGGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAA CGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCC TATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATC GCTATTACCATGATGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATG GGAGTTTGTTTTGACTAGTCAGGGCCCCAACCCCCCCAAGCCCCCATTTCACAACACGCTGGCGCTACAGGCGCGTGACTTCCCCTTGCTTTGGGGCGGG GGGCTGAGACTCCTATGTGCTCCGGATTGGTCAGGCACGGCCTTCGGCCCCGCCTCCTGCCACCGCAGATTGGCCGCTAGGCCTCCCCGAGCGCCCTGCC HindIII (679) TCCGAGGGCCGGCGCACCATAAAAGAAGCCGCCCTAGCCACGTCCCCTCGCAGTTCGGCGGTCCCGCGGGTCTGTCTCAAGCTTGCCGCCAGAACACAGg taagtgccgtgtgtggttcccgcgggcctggcctctttacgggttatggcccttgcgtgccttgaattacttccatgcccctggctgcagtacgtgattc ttgatcccgagcttcgggttggaagtgggtgggagagttcgaggccttgcgcttaaggagccccttcgcctcgtgcttgagttgaggcctggcttgggcg ctggggccgccgcgtgctaatctggtggcaccttcgcgcctgtctcgctgctttcgctaagtctctagccatttaaaatttttgataaccagctgcgacg BglII (1039) ctttttttctggcgagatagtcttgtaaatgcgggccaagatctgcacactggtatttcggtttttggggccgcgggcggcgacggggcccgtgcgtccc agcgcacatgttcggcgaggcggggcctgcgagcgcggccaccgagaatcggacgggggtagtctcaaactggccggcctgctctggtgcctggcctcgc gccgccgtgtatcgccccgccctgggcggcaaggctggcccggtcggcaccagttgcgtgagcggaaagatggccgcttcccggccctgctgcagggagc tcaaaatggaggacgcggcgcccgggagagcgggcgggtgagtcacccacacaaaggaaaagggcctttccttcctcatccgtcgcttcatgtgactcca XhoI (1438) cggagtaccgggcgccgtccaggcacctcgattagttctcgagcttttggagtacgtcgtctttaggttggggggaggggttttatgcgatggagtttcc ccacactgagtgggtggagactgaagagttaggccagcttggcacttgatgtaattctccttggaatttgccctttttgagtttggatcttgcctcattc NcoI (1673) 1601 tcaagcctcagacagtggttcaaagtttttttcttccatttcaggtgtcgtgaaaactacccctaaaagccaccatggaccctgttgtgctgcaaaggag 1 MetAspProValValLeuGlnArgAr 1701 AGACTGGGAGAACCCTGGAGTGACCCAGCTCAACAGACTGGCTGCCCACCCTCCCTTTGCCTCTTGGAGGAACTCTGAGGAAGCCAGGACAGACAGGCCC 9 gasptrpgluasnproglyvalthrglnleuasnargleualaalahisproprophealasertrpargasnsergluglualaargthraspargpro 1801 AGCCAGCAGCTCAGGTCTCTCAATGGAGAGTGGAGGTTTGCCTGGTTCCCTGCCCCTGAAGCTGTGCCTGAGTCTTGGCTGGAGTGTGACCTCCCAGAGG 43 SerGlnGlnLeuArgSerLeuAsnGlyGluTrpArgPheAlaTrpPheProAlaProGluAlaValProGluSerTrpLeuGluCysAspLeuProGluA 1901 CTGACACTGTTGTGGTGCCCAGCAACTGGCAGATGCATGGCTATGATGCCCCCATCTACACCAATGTCACCTACCCCATCACTGTGAACCCCCCTTTTGT 76 laaspthrvalvalvalproserasntrpglnmethisglytyraspalaproi letyrthrasnvalthrtyrproi lethrvalasnpropropheva 2001 GCCCACTGAGAACCCCACTGGCTGCTACAGCCTGACCTTCAATGTTGATGAGAGCTGGCTGCAAGAAGGCCAGACCAGGATCATCTTTGATGGAGTCAAC 109 lprothrgluasnprothrglycystyrserleuthrpheasnvalaspglusertrpleuglngluglyglnthrargi lei lepheaspglyvalasn 2101 TCTGCCTTCCACCTCTGGTGCAATGGCAGGTGGGTTGGCTATGGCCAAGACAGCAGGCTGCCCTCTGAGTTTGACCTCTCTGCCTTCCTCAGAGCTGGAG 143 SerAlaPheHisLeuTrpCysAsnGlyArgTrpValGlyTyrGlyGlnAspSerArgLeuProSerGluPheAspLeuSerAlaPheLeuArgAlaGlyG 2201 AGAACAGGCTGGCTGTCATGGTGCTCAGGTGGTCTGATGGCAGCTACCTGGAAGACCAAGACATGTGGAGGATGTCTGGCATCTTCAGGGATGTGAGCCT 176 luasnargleualavalmetvalleuargtrpseraspglysertyrleugluaspglnaspmettrpargmetserglyi lepheargaspvalserle 2301 GCTGCACAAGCCCACCACCCAGATTTCTGACTTCCATGTTGCCACCAGGTTCAATGATGACTTCAGCAGAGCTGTGCTGGAGGCTGAGGTGCAGATGTGT 209 uleuhislysprothrthrglni leseraspphehisvalalathrargpheasnaspasppheserargalavalleuglualagluvalglnmetcys 2401 GGAGAACTCAGAGACTACCTGAGAGTCACAGTGAGCCTCTGGCAAGGTGAGACCCAGGTGGCCTCTGGCACAGCCCCCTTTGGAGGAGAGATCATTGATG 243 GlyGluLeuArgAspTyrLeuArgValThrValSerLeuTrpGlnGlyGluThrGlnValAlaSerGlyThrAlaProPheGlyGlyGluI lei leaspg 2501 AGAGAGGAGGCTATGCTGACAGAGTCACCCTGAGGCTCAATGTGGAGAACCCCAAGCTGTGGTCTGCTGAGATCCCCAACCTCTACAGGGCTGTTGTGGA 276 luargglyglytyralaaspargvalthrleuargleuasnvalgluasnprolysleutrpseralaglui leproasnleutyrargalavalvalgl 2601 GCTGCACACTGCTGATGGCACCCTGATTGAAGCTGAAGCCTGTGATGTTGGATTCAGAGAAGTCAGGATTGAGAATGGCCTGCTGCTGCTCAATGGCAAG 309 uleuhisthralaaspglythrleui leglualaglualacysaspvalglyphearggluvalargi legluasnglyleuleuleuleuasnglylys 2701 CCTCTGCTCATCAGGGGAGTCAACAGGCATGAGCACCACCCTCTGCATGGACAAGTGATGGATGAACAGACAATGGTGCAAGATATCCTGCTAATGAAGC 343 ProLeuLeuI leargglyvalasnarghisgluhishisproleuhisglyglnvalmetaspgluglnthrmetvalglnaspi leleuleumetlysg 2801 AGAACAACTTCAATGCTGTCAGGTGCTCTCACTACCCCAACCACCCTCTCTGGTACACCCTGTGTGACAGGTATGGCCTGTATGTTGTTGATGAAGCCAA 376 lnasnasnpheasnalavalargcysserhistyrproasnhisproleutrptyrthrleucysaspargtyrglyleutyrvalvalaspglualaas 2901 CATTGAGACACATGGCATGGTGCCCATGAACAGGCTCACAGATGACCCCAGGTGGCTGCCTGCCATGTCTGAGAGAGTGACCAGGATGGTGCAGAGAGAC 409 ni legluthrhisglymetvalprometasnargleuthraspaspproargtrpleuproalametsergluargvalthrargmetvalglnargasp 3001 AGGAACCACCCCTCTGTGATCATCTGGTCTCTGGGCAATGAGTCTGGACATGGAGCCAACCATGATGCTCTCTACAGGTGGATCAAGTCTGTTGACCCCA 443 ArgAsnHisProSerVal I lei letrpserleuglyasngluserglyhisglyalaasnhisaspalaleutyrargtrpi lelysservalasppros 3101 GCAGACCTGTGCAGTATGAAGGAGGTGGAGCAGACACCACAGCCACAGACATCATCTGCCCCATGTATGCCAGGGTTGATGAGGACCAGCCCTTCCCTGC 476 erargprovalglntyrgluglyglyglyalaaspthrthralathraspi lei lecyspromettyralaargvalaspgluaspglnpropheproal 3201 TGTGCCCAAGTGGAGCATCAAGAAGTGGCTCTCTCTGCCTGGAGAGACCAGACCTCTGATCCTGTGTGAATATGCACATGCAATGGGCAACTCTCTGGGA 509 avalprolystrpseri lelyslystrpleuserleuproglygluthrargproleui leleucysglutyralahisalametglyasnserleugly 3301 GGCTTTGCCAAGTACTGGCAAGCCTTCAGACAGTACCCCAGGCTGCAAGGAGGATTTGTGTGGGACTGGGTGGACCAATCTCTCATCAAGTATGATGAGA 543 GlyPheAlaLysTyrTrpGlnAlaPheArgGlnTyrProArgLeuGlnGlyGlyPheValTrpAspTrpValAspGlnSerLeuI lelystyraspglua 3401 ATGGCAACCCCTGGTCTGCCTATGGAGGAGACTTTGGTGACACCCCCAATGACAGGCAGTTCTGCATGAATGGCCTGGTCTTTGCAGACAGGACCCCTCA 576 snglyasnprotrpseralatyrglyglyasppheglyaspthrproasnaspargglnphecysmetasnglyleuvalphealaaspargthrprohi 3501 CCCTGCCCTCACAGAGGCCAAGCACCAGCAACAGTTCTTCCAGTTCAGGCTGTCTGGACAGACCATTGAGGTGACATCTGAGTACCTCTTCAGGCACTCT 609 sproalaleuthrglualalyshisglnglnglnphepheglnpheargleuserglyglnthri legluvalthrserglutyrleuphearghisser

4 3601 GACAATGAGCTCCTGCACTGGATGGTGGCCCTGGATGGCAAGCCTCTGGCTTCTGGTGAGGTGCCTCTGGATGTGGCCCCTCAAGGAAAGCAGCTGATTG 643 AspAsnGluLeuLeuHisTrpMetValAlaLeuAspGlyLysProLeuAlaSerGlyGluValProLeuAspValAlaProGlnGlyLysGlnLeuI leg 3701 AACTGCCTGAGCTGCCTCAGCCAGAGTCTGCTGGACAACTGTGGCTAACAGTGAGGGTGGTTCAGCCCAATGCAACAGCTTGGTCTGAGGCAGGCCACAT 676 luleuprogluleuproglnprogluseralaglyglnleutrpleuthrvalargvalvalglnproasnalathralatrpserglualaglyhisi l 3801 CTCTGCATGGCAGCAGTGGAGGCTGGCTGAGAACCTCTCTGTGACCCTGCCTGCTGCCTCTCATGCCATCCCTCACCTGACAACATCTGAAATGGACTTC 709 eseralatrpglnglntrpargleualagluasnleuservalthrleuproalaalaserhisalai leprohisleuthrthrserglumetaspphe 3901 TGCATTGAGCTGGGCAACAAGAGATGGCAGTTCAACAGGCAGTCTGGCTTCCTGTCTCAGATGTGGATTGGAGACAAGAAGCAGCTCCTCACCCCTCTCA 743 CysI legluleuglyasnlysargtrpglnpheasnargglnserglypheleuserglnmettrpi leglyasplyslysglnleuleuthrproleua 4001 GGGACCAATTCACCAGGGCTCCTCTGGACAATGACATTGGAGTGTCTGAGGCCACCAGGATTGACCCAAATGCTTGGGTGGAGAGGTGGAAGGCTGCTGG 776 rgaspglnphethrargalaproleuaspasnaspi leglyvalserglualathrargi leaspproasnalatrpvalgluargtrplysalaalagl 4101 ACACTACCAGGCTGAGGCTGCCCTGCTCCAGTGCACAGCAGACACCCTGGCTGATGCTGTTCTGATCACCACAGCCCATGCTTGGCAGCACCAAGGCAAG 809 yhistyrglnalaglualaalaleuleuglncysthralaaspthrleualaaspalavalleui lethrthralahisalatrpglnhisglnglylys 4201 ACCCTGTTCATCAGCAGAAAGACCTACAGGATTGATGGCTCTGGACAGATGGCAATCACAGTGGATGTGGAGGTTGCCTCTGACACACCTCACCCTGCAA 843 ThrLeuPheI leserarglysthrtyrargi leaspglyserglyglnmetalai lethrvalaspvalgluvalalaseraspthrprohisproalaa 4301 GGATTGGCCTGAACTGTCAACTGGCACAGGTGGCTGAGAGGGTGAACTGGCTGGGCTTAGGCCCTCAGGAGAACTACCCTGACAGGCTGACAGCTGCCTG 876 rgi leglyleuasncysglnleualaglnvalalagluargvalasntrpleuglyleuglyproglngluasntyrproaspargleuthralaalacy 4401 CTTTGACAGGTGGGACCTGCCTCTGTCTGACATGTACACCCCTTATGTGTTCCCTTCTGAGAATGGCCTGAGGTGTGGCACCAGGGAGCTGAACTATGGT 909 spheaspargtrpaspleuproleuseraspmettyrthrprotyrvalpheprosergluasnglyleuargcysglythrarggluleuasntyrgly 4501 CCTCACCAGTGGAGGGGAGACTTCCAGTTCAACATCTCCAGGTACTCTCAGCAACAGCTCATGGAAACCTCTCACAGGCACCTGCTCCATGCAGAGGAGG 943 ProHisGlnTrpArgGlyAspPheGlnPheAsnI leserargtyrserglnglnglnleumetgluthrserhisarghisleuleuhisalagluglug 4601 GAACCTGGCTGAACATTGATGGCTTCCACATGGGCATTGGAGGAGATGACTCTTGGTCTCCTTCTGTGTCTGCTGAGTTCCAGTTATCTGCTGGCAGGTA 976 lythrtrpleuasni leaspglyphehismetglyi leglyglyaspaspsertrpserproservalseralaglupheglnleuseralaglyargty NheI (4738) 4701 CCACTATCAGCTGGTGTGGTGCCAGAAGTAAACCTGAGCTAGCTGGCCAGACATGATAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAG 1009 rhistyrglnleuvaltrpcysglnlys 4801 TGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTCATT PacI (4978) TTATGTTTCAGGTTCAGGGGGAGGTGTGGGAGGTTTTTTAAAGCAAGTAAAACCTCTACAAATGTGGTATGGAAATGTTAATTAACTAGCCATGACCAAA ATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCT TGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGAT ACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTG GCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCA CACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAG GTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTC TGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGC PacI (5718) CTTTTGCTCACATGTTCTTAATTAACCTGCAGGGCCTGAAATAACCTCTGAAAGAGGAACTTGGTTAGGTACCTTCTGAGGCTGAAAGAACCAGCTGTGG AATGTGTGTCAGTTAGGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCAGGTGTGGAAAGT NotI (6 CCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCATAGTCCCACTAGTTCCGCCAGAGCGCGCGAGGGCCTCCAG CGGCCGCCCCTCCCCCACAGCAGGGGCGGGGTCCCGCGCCCACCGGAAGGAGCGGGCTCGGGGCGGGCGGCGCTGATTGGCCGGGGCGGGCCTGACGCCG HindIII (6153) ACGCGGCTATAAGAGACCACAAGCGACCCGCAGGGCCAGACGTTCTTCGCCGAAGCTTGCCGTCAGAACGCAGGTGAGGGGCGGGTGTGGCTTCCGCGGG CCGCCGAGCTGGAGGTCCTGCTCCGAGCGGGCCGGGCCCCGCTGTCGTCGGCGGGGATTAGCTGCGAGCATTCCCGCTTCGAGTTGCGGGCGGCGCGGGA GGCAGAGTGCGAGGCCTAGCGGCAACCCCGTAGCCTCGCCTCGTGTCCGGCTTGAGGCCTAGCGTGGTGTCCGCGCCGCCGCCGCGTGCTACTCCGGCCG CACTCTGGTCTTTTTTTTTTTTGTTGTTGTTGCCCTGCTGCCTTCGATTGCCGTTCAGCAATAGGGGCTAACAAAGGGAGGGTGCGGGGCTTGCTCGCCC GGAGCCCGGAGAGGTCATGGTTGGGGAGGAATGGAGGGACAGGAGTGGCGGCTGGGGCCCGCCCGCCTTCGGAGCACATGTCCGACGCCACCTGGATGGG GCGAGGCCTGGGGTTTTTCCCGAAGCAACCAGGCTGGGGTTAGCGTGCCGAGGCCATGTGGCCCCAGCACCCGGCACGATCTGGCTTGGCGGCGCCGCGT TGCCCTGCCTCCCTAACTAGGGTGAGGCCATCCCGTCCGGCACCAGTTGCGTGCGTGGAAAGATGGCCGCTCCCGGGCCCTGTTGCAAGGAGCTCAAAAT GGAGGACGCGGCAGCCCGGTGGAGCGGGCGGGTGAGTCACCCACACAAAGGAAGAGGGCCTGGTCCCTCACCGGCTGCTGCTTCCTGTGACCCCGTGGTC CTATCGGCCGCAATAGTCACCTCGGGCTTTTGAGCACGGCTAGTCGCGGCGGGGGGAGGGGATGTAATGGCGTTGGAGTTTGTTCACATTTGGTGGGTGG AGACTAGTCAGGCCAGCCTGGCGCTGGAAGTCATTTTTGGAATTTGTCCCCTTGAGTTTTGAGCGGAGCTAATTCTCGGGCTTCTTAGCGGTTCAAAGGT BspHI (7154) ATCTTTTAAACCCTTTTTTAGGTGTTGTGAAAACCACCGCTAATTCAAAGCAATCATGAGCAAGGGAGAAGAACTCTTTACTGGTGTTGTCCCAATTCTG 1 MetSerLysGlyGluGluLeuPheThrGlyValValProI leleu

5 7201 GTTGAGCTGGATGGTGATGTGAATGGCCACAAATTCTCTGTGTCTGGTGAAGGTGAAGGAGATGCAACTTATGGAAAGCTGACTCTGAAGTTCATTTGTA 16 ValGluLeuAspGlyAspValAsnGlyHisLysPheSerValSerGlyGluGlyGluGlyAspAlaThrTyrGlyLysLeuThrLeuLysPheI lecyst 7301 CAACAGGAAAGCTGCCAGTGCCTTGGCCAACTCTGGTGACCACCCTGACTTATGGTGTTCAATGTTTCAGCAGGTACCCTGACCACATGAAGCAGCATGA 49 hrthrglylysleuprovalprotrpprothrleuvalthrthrleuthrtyrglyvalglncyspheserargtyrproasphismetlysglnhisas 7401 CTTCTTTAAATCTGCAATGCCAGAAGGTTATGTTCAGGAGAGGACAATCTTCTTTAAGGATGATGGAAATTATAAGACAAGGGCAGAAGTGAAGTTTGAA 82 pphephelysseralametprogluglytyrvalglngluargthri lephephelysaspaspglyasntyrlysthrargalagluvallyspheglu 7501 GGTGATACACTGGTTAACAGAATTGAGCTGAAAGGCATTGATTTTAAGGAAGATGGAAACATTCTGGGTCACAAGCTGGAGTACAACTATAATTCTCACA 116 GlyAspThrLeuValAsnArgI legluleulysglyi leaspphelysgluaspglyasni leleuglyhislysleuglutyrasntyrasnserhisa 7601 ATGTTTACATTATGGCAGATAAGCAGAAGAATGGAATTAAGGTTAATTTCAAGATTAGACACAACATTGAGGATGGATCTGTCCAACTGGCAGACCATTA 149 snvaltyri lemetalaasplysglnlysasnglyi lelysvalasnphelysi learghisasni legluaspglyservalglnleualaasphisty 7701 CCAGCAGAACACCCCTATTGGTGATGGCCCAGTTCTCCTCCCAGATAATCACTATCTCCGCACTCAATCTGCTCTGTCCAAAGACCCTAATGAGAAAAGA 182 rglnglnasnthrproi leglyaspglyprovalleuleuproaspasnhistyrleuargthrglnseralaleuserlysaspproasnglulysarg AvrII (7874) 7801 GACCACATGGTCCTCCTGGAGTTTGTGACAGCAGCAGGAATTACTCTGGGAATGGATGAGCTGTACAAGTAAACCTAGGAGCAGGTTTCCCCAATGACAC 216 AspHisMetValLeuLeuGluPheValThrAlaAlaGlyI lethrleuglymetaspgluleutyrlys 7901 AAAACGTGCAACTTGAAACTCCGCCTGGTCTTTCCAGGTCTAGAGGGGTAACACTTTGTACTGCGTTTGGCTCCACGCTCGATCCACTGGCGAGTGTTAG TAACAGCACTGTTGCTTCGTAGCGGAGCATGACGGCCGTGGGAACTCCTCCTTGGTAACAAGGACCCACGGGGCCAAAAGCCACGCCCACACGGGCCCGT CATGTGTGCAACCCCAGCACGGCGACTTTACTGCGAAACCCACTTTAAAGTGACATTGAAACTGGTACCCACACACTGGTGACAGGCTAAGGATGCCCTT HindIII (8278) CAGGTACCCCGAGGTAACACGCGACACTCGGGATCTGAGAAGGGGACTGGGGCTTCTATAAAAGCGCTCGGTTTAAAAAGCTTCTATGCCTGAATAGGTG ACCGGAGGTCGGCACCTTTCCTTTGCAATTACTGACCCTATGAATACAACTGACTGTTTGACAATTAATCATCGGCATAGTATATCGGCATAGTATAATA CGACTCACTATAGGAGGGCCACCATGAAGAAACCTGAACTGACAGCAACTTCTGTTGAGAAGTTTCTCATTGAAAAATTTGATTCTGTTTCTGATCTCAT 1 MetLysLysProGluLeuThrAlaThrSerValGluLysPheLeuI leglulyspheaspservalseraspleume GCAGCTGTCTGAAGGTGAAGAAAGCAGAGCCTTTTCTTTTGATGTTGGAGGAAGAGGTTATGTTCTGAGGGTCAATTCTTGTGCTGATGGTTTTTACAAA tglnleusergluglyglugluserargalapheserpheaspvalglyglyargglytyrvalleuargvalasnsercysalaaspglyphetyrlys GACAGATATGTTTACAGACACTTTGCCTCTGCTGCTCTGCCAATTCCAGAAGTTCTGGACATTGGAGAATTTTCTGAATCTCTCACCTACTGCATCAGCA AspArgTyrValTyrArgHisPheAlaSerAlaAlaLeuProI leprogluvalleuaspi leglygluphesergluserleuthrtyrcysi lesera GAAGAGCACAAGGAGTCACTCTCCAGGATCTCCCTGAAACTGAGCTGCCAGCTGTTCTGCAACCTGTTGCTGAAGCAATGGATGCCATTGCAGCAGCTGA rgargalaglnglyvalthrleuglnaspleuprogluthrgluleuproalavalleuglnprovalalaglualametaspalai lealaalaalaas TCTGAGCCAAACCTCTGGATTTGGTCCTTTTGGTCCCCAAGGCATTGGTCAGTACACCACTTGGAGGGATTTCATTTGTGCCATTGCTGATCCTCATGTC pleuserglnthrserglypheglypropheglyproglnglyi leglyglntyrthrthrtrpargaspphei lecysalai lealaaspprohisval TATCACTGGCAGACTGTGATGGATGACACAGTTTCTGCTTCTGTTGCTCAGGCACTGGATGAACTCATGCTGTGGGCAGAAGATTGTCCTGAAGTCAGAC TyrHisTrpGlnThrValMetAspAspThrValSerAlaSerValAlaGlnAlaLeuAspGluLeuMetLeuTrpAlaGluAspCysProGluValArgH ACCTGGTCCATGCTGATTTTGGAAGCAACAATGTTCTGACAGACAATGGCAGAATCACTGCAGTCATTGACTGGTCTGAAGCCATGTTTGGAGATTCTCA isleuvalhisalaasppheglyserasnasnvalleuthraspasnglyargi lethralaval I leasptrpserglualametpheglyaspsergl ATATGAGGTTGCCAACATTTTTTTTTGGAGACCTTGGCTGGCTTGCATGGAACAACAAACAAGATATTTTGAAAGAAGACACCCAGAACTGGCTGGTTCC ntyrgluvalalaasni lephephetrpargprotrpleualacysmetgluglnglnthrargtyrphegluargarghisprogluleualaglyser CCCAGACTGAGAGCCTACATGCTCAGAATTGGCCTGGACCAACTGTATCAATCTCTGGTTGATGGAAACTTTGATGATGCTGCTTGGGCACAAGGAAGAT ProArgLeuArgAlaTyrMetLeuArgI leglyleuaspglnleutyrglnserleuvalaspglyasnpheaspaspalaalatrpalaglnglyargc GTGATGCCATTGTGAGGTCTGGTGCTGGAACTGTTGGAAGAACTCAAATTGCAAGAAGGTCTGCTGCTGTTTGGACTGATGGATGTGTTGAAGTTCTGGC ysaspalai levalargserglyalaglythrvalglyargthrglni lealaargargseralaalavaltrpthraspglycysvalgluvalleual TGACTCTGGAAACAGGAGACCCTCCACAAGACCCAGAGCCAAGGAATGAATATTAGCTAGATTATCCCTAATACCTGCCACCCCACTCTTAATCAGTGGT aaspserglyasnargargproserthrargproargalalysglu GGAAGAACGGTCTCAGAACTGTTTGTTTCAATTGGCCATTTAAGTTTAGTAGTAAAAGACTGGTTAATGATAACAATGCATCGTAAAACCTTCAGAAGGA AAGGAGAATGTTTTGTGGACCACTTTGGTTTTCTTTTTTGCGTGTGGCAGTTTTAAGTTATTAGTTTTTAAAATCAGTACTTTTTAATGGAAACAACTTG ACCAAAAATTTGTCACAGAATTTTGAGACCCATTAAAAAAGTTAAATGAGAAACCTGTGTGTTCCTTTGGTCAACACCGAGACATTTAGGTGAAAGACAT CTAATTCTGGTTTTACGAATCTGGAAACTTCTTGAAAATGTAATTCTTGAGTTAACACTTCTGGGTGGAGAATAGGGTTGTTTTCCCCCCACATAATTGG AAGGGGAAGGAATATCATTTAAAGCTATGGGAGGGTTGCTTTGATTACAACACTGGAGAGAAATGCAGCATGTTGCTGATTGCCTGTCACTAAAACAGGC CAAAAACTGAGTCCTTGGGTTGCATAGAAAGCTG