pvivo1-gfp/lacz An innovative multigenic plasmid for high levels of expression of the GFP and LacZ reporter genes in tumors Catalog # pvivo1-gfp-lacz

Transcription

1 pvivo1-gfp/lacz An innovative multigenic plasmid for high levels of expression of the GFP and LacZ reporter genes in tumors Catalog # pvivo1-gfp-lacz For research use only Version # 12H31-MM PROduCT information Content: - 20 µg of pvivo1-gfp/lacz provided as lyophilized DNA. - 4 pouches of E. coli Fast-Media Hygro (2 TB and 2 Agar) 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 6 months when stored at -20 C. Avoid repeated freeze-thaw cycles. - Store E. coli Fast-Media Hygro pouches at room temperature. Fast-Media pouches are 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 pvivo1 is a new generation multigenic vector with TWO transcription units allowing the combined expression of TWO genes of interest from a SINGLE vector. pvivo1-gfp/lacz contains the reporter genes GFP and LacZ and can be used as a control vector. pvivo1-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 hagrp78 and hgrp94 prom: The hamster GRP78 and human GRP94 promoters drive weak levels of expression in normal conditions and are induced in stress conditions prevailing inside tumors, such as glucose deprivation and hypoxia 1. Within the tumor micro-environment, the GRP promoters yield persistent expression whereas the activity of viral promoters declines rapidly 2. 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 3. CMV enhancer: The major immediate early enhancer of the human cytomegalovirus (HCMV), 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 4. LacZ-ΔCpG gene: The 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. In order to reduce the immunogenicity of this bacterial gene the 298 CpG motifs from the wildtype have been removed by chemically synthesizing the gene. 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 pmb1 Ori is a minimal E. coli origin of replication to limit vector size, but with the same activity as the longer Ori. EM7 is a bacterial promoter that enables the constitutive expression of the antibiotic resistance gene in E. coli. Hygro- CpG is a new allele of the hph gene conferring resistance to hygromycin B. In order to reduce the immunogenicity of this bacterial gene all CpG motifs have been removed by chemically synthesizing the gene. The Hygro- CpG gene allows the selection of E. coli clones transformed with a pvivo plasmid. Note: Stable transfection of mammalian clones cannot be performed due to the absence of a eukaryotic promoter upstream of the Hygro- CpG gene. Term: The E. coli rps O terminator allows efficient transcription termination of the Hygro- CpG gene. LGFP is a new allele of the green fluorescent protein. The gene has been chemically synthesized to remove the CpGs to ensure long-lasting expression. EF1 pan is a strong polyadenylation signal. InvivoGen uses a sequence starting after the stop codon of the EF1 cdna and finishing after a bent structure rich in GT. References: 1. Eisenstein RS. and Munro HN Enzyme 44(1-4): Gazit G. et al Cancer Res 59: Dean DA. et al Exp. Cell. Res. 253: Boshart M. et al Cell 141(2): Carswell S., and Alwine JC Mol. Cell Biol. 10: METHOdS Plasmid resuspension: Quickly spin the tube containing the lyophilized plasmid to pellet the DNA. To obtain a plasmid solution at 1µg/µl, resuspend the DNA in 20µl of sterile H2O. Store resuspended plasmid at -20 C. Plasmid amplification and cloning: Plasmid amplification and cloning can be preformed in E.coli GT116 or other commonly used laboratory E.coli strains, such as DH5a. TECHNICAL SUPPORT Toll free (US): Outside US: (+1) info@invivogen.com Website: Sorrento Valley Blvd. Suite 100 San Diego, CA USA

2 Selection of bacteria with E. coli Fast-Media Hygro: E. coli Fast-Media Hygro is a new, fast and convenient way to prepare liquid and solid media for bacterial culture by using only a microwave. E. coli Fast-Media Hygro is a TB (liquid) or LB (solid) based medium with hygromycin B, and contains stabilizers. E. coli Fast-Media Hygro can be ordered separately. 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 Fast-Media. 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 Fast-Media as the antibiotic will be permanently destroyed by the procedure. RELATEd PROduCTS Product HygroGold Fast-Media Hygro Agar Fast-Media Hygro TB ChemiComp GT116 LyoComp GT116 Catalog Code ant-hg-1 fas-hg-s fas-hg-l gt lyo TECHNICAL SUPPORT Toll free (US): Outside US: (+1) info@invivogen.com Website: Sorrento Valley Blvd. Suite 100 San Diego, CA USA

3 AvrII (7846) NcoI (963) BspHI (7126) BamHI (7052) LGFP EF1 pan CMV enh hagrp78 prom BamHI (6660) hgrp94 prom PacI (6179) SV40 enh pvivo1-gfp/lacz (8424 bp) LacZ- CpG Hygro- CpG EM7 pmb1 Ori SV40 pan PacI (5012) PacI (4272) NheI (4028) 225

4 CCTGCAGGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAA CGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCC TATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATC GCTATTACCATGATGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATG GGAGTTTGTTTTGACTAGTTACCGGCGGAAACGGTCTCGGGTTGAGAGGTCACCCGAGGGACAGGCAGCTGCTGAACCAATAGGACCGGCGCACAGGGCG GATGCTGCCCCTCATTGGCGGCCGTTGAGAGTGACCAAGAGCCAATGAGTCAGCCCGGGGGGCGTAGCAGTGACGTAAGTTGCGGAGGAGGCCGCTTCGA ATCGGCAGCGGCCAGCTTGGTGGCATGGACCAATCAGCGTCCTCCAACGAGGAGCGCCTTCGCCAATCGGAGGCCTCCACGACGGGGCTGGGGGGAGGGT ATATAAGCCGAGTCGGCGGCGGCGCGCTCCACACGGGCCGAGACCACAGCGACGGGAGCGTCTGCCTCTGCGGGGCCGAGAGGTAAGCGCCGCGGCCTGC CCTTTCCAGGCCAACTCGGAGCCCGTCTCGTGGCTCCGCCTGATCGGGGGCTCCTGTCGCCCTCAGATCGGTCGGAACGCCGTCGCGCTCCGGGACTACA NcoI (963) 901 AGCCTGTTGCTGGGCCCGGAGACTGCCGAAGGACCGCTGAGCACTGTCCTCAGCGCCGGCAccATGGACCCTGTTGTGCTGCAAAGGAGAGACTGGGAGA 1 MetAspProValValLeuGlnArgArgAspTrpGluA 1001 ACCCTGGAGTGACCCAGCTCAACAGACTGGCTGCCCACCCTCCCTTTGCCTCTTGGAGGAACTCTGAGGAAGCCAGGACAGACAGGCCCAGCCAGCAGCT 13 snproglyvalthrglnleuasnargleualaalahisproprophealasertrpargasnsergluglualaargthraspargproserglnglnle 1101 CAGGTCTCTCAATGGAGAGTGGAGGTTTGCCTGGTTCCCTGCCCCTGAAGCTGTGCCTGAGTCTTGGCTGGAGTGTGACCTCCCAGAGGCTGACACTGTT 46 uargserleuasnglyglutrpargphealatrppheproalaproglualavalproglusertrpleuglucysaspleuproglualaaspthrval 1201 GTGGTGCCCAGCAACTGGCAGATGCATGGCTATGATGCCCCCATCTACACCAATGTCACCTACCCCATCACTGTGAACCCCCCTTTTGTGCCCACTGAGA 80 ValValProSerAsnTrpGlnMetHisGlyTyrAspAlaProI letyrthrasnvalthrtyrproi lethrvalasnproprophevalprothrglua 1301 ACCCCACTGGCTGCTACAGCCTGACCTTCAATGTTGATGAGAGCTGGCTGCAAGAAGGCCAGACCAGGATCATCTTTGATGGAGTCAACTCTGCCTTCCA 113 snprothrglycystyrserleuthrpheasnvalaspglusertrpleuglngluglyglnthrargi lei lepheaspglyvalasnseralaphehi 1401 CCTCTGGTGCAATGGCAGGTGGGTTGGCTATGGCCAAGACAGCAGGCTGCCCTCTGAGTTTGACCTCTCTGCCTTCCTCAGAGCTGGAGAGAACAGGCTG 146 sleutrpcysasnglyargtrpvalglytyrglyglnaspserargleuproserglupheaspleuseralapheleuargalaglygluasnargleu 1501 GCTGTCATGGTGCTCAGGTGGTCTGATGGCAGCTACCTGGAAGACCAAGACATGTGGAGGATGTCTGGCATCTTCAGGGATGTGAGCCTGCTGCACAAGC 180 AlaValMetValLeuArgTrpSerAspGlySerTyrLeuGluAspGlnAspMetTrpArgMetSerGlyI lepheargaspvalserleuleuhislysp 1601 CCACCACCCAGATTTCTGACTTCCATGTTGCCACCAGGTTCAATGATGACTTCAGCAGAGCTGTGCTGGAGGCTGAGGTGCAGATGTGTGGAGAACTCAG 213 rothrthrglni leseraspphehisvalalathrargpheasnaspasppheserargalavalleuglualagluvalglnmetcysglygluleuar 1701 AGACTACCTGAGAGTCACAGTGAGCCTCTGGCAAGGTGAGACCCAGGTGGCCTCTGGCACAGCCCCCTTTGGAGGAGAGATCATTGATGAGAGAGGAGGC 246 gasptyrleuargvalthrvalserleutrpglnglygluthrglnvalalaserglythralapropheglyglyglui lei leaspgluargglygly 1801 TATGCTGACAGAGTCACCCTGAGGCTCAATGTGGAGAACCCCAAGCTGTGGTCTGCTGAGATCCCCAACCTCTACAGGGCTGTTGTGGAGCTGCACACTG 280 TyrAlaAspArgValThrLeuArgLeuAsnValGluAsnProLysLeuTrpSerAlaGluI leproasnleutyrargalavalvalgluleuhisthra 1901 CTGATGGCACCCTGATTGAAGCTGAAGCCTGTGATGTTGGATTCAGAGAAGTCAGGATTGAGAATGGCCTGCTGCTGCTCAATGGCAAGCCTCTGCTCAT 313 laaspglythrleui leglualaglualacysaspvalglyphearggluvalargi legluasnglyleuleuleuleuasnglylysproleuleui l 2001 CAGGGGAGTCAACAGGCATGAGCACCACCCTCTGCATGGACAAGTGATGGATGAACAGACAATGGTGCAAGATATCCTGCTAATGAAGCAGAACAACTTC 346 eargglyvalasnarghisgluhishisproleuhisglyglnvalmetaspgluglnthrmetvalglnaspi leleuleumetlysglnasnasnphe 2101 AATGCTGTCAGGTGCTCTCACTACCCCAACCACCCTCTCTGGTACACCCTGTGTGACAGGTATGGCCTGTATGTTGTTGATGAAGCCAACATTGAGACAC 380 AsnAlaValArgCysSerHisTyrProAsnHisProLeuTrpTyrThrLeuCysAspArgTyrGlyLeuTyrValValAspGluAlaAsnI legluthrh 2201 ATGGCATGGTGCCCATGAACAGGCTCACAGATGACCCCAGGTGGCTGCCTGCCATGTCTGAGAGAGTGACCAGGATGGTGCAGAGAGACAGGAACCACCC 413 isglymetvalprometasnargleuthraspaspproargtrpleuproalametsergluargvalthrargmetvalglnargaspargasnhispr 2301 CTCTGTGATCATCTGGTCTCTGGGCAATGAGTCTGGACATGGAGCCAACCATGATGCTCTCTACAGGTGGATCAAGTCTGTTGACCCCAGCAGACCTGTG 446 oserval I lei letrpserleuglyasngluserglyhisglyalaasnhisaspalaleutyrargtrpi lelysservalaspproserargproval 2401 CAGTATGAAGGAGGTGGAGCAGACACCACAGCCACAGACATCATCTGCCCCATGTATGCCAGGGTTGATGAGGACCAGCCCTTCCCTGCTGTGCCCAAGT 480 GlnTyrGluGlyGlyGlyAlaAspThrThrAlaThrAspI lei lecyspromettyralaargvalaspgluaspglnpropheproalavalprolyst 2501 GGAGCATCAAGAAGTGGCTCTCTCTGCCTGGAGAGACCAGACCTCTGATCCTGTGTGAATATGCACATGCAATGGGCAACTCTCTGGGAGGCTTTGCCAA 513 rpseri lelyslystrpleuserleuproglygluthrargproleui leleucysglutyralahisalametglyasnserleuglyglyphealaly 2601 GTACTGGCAAGCCTTCAGACAGTACCCCAGGCTGCAAGGAGGATTTGTGTGGGACTGGGTGGACCAATCTCTCATCAAGTATGATGAGAATGGCAACCCC 546 styrtrpglnalapheargglntyrproargleuglnglyglyphevaltrpasptrpvalaspglnserleui lelystyraspgluasnglyasnpro 2701 TGGTCTGCCTATGGAGGAGACTTTGGTGACACCCCCAATGACAGGCAGTTCTGCATGAATGGCCTGGTCTTTGCAGACAGGACCCCTCACCCTGCCCTCA 580 TrpSerAlaTyrGlyGlyAspPheGlyAspThrProAsnAspArgGlnPheCysMetAsnGlyLeuValPheAlaAspArgThrProHisProAlaLeuT 2801 CAGAGGCCAAGCACCAGCAACAGTTCTTCCAGTTCAGGCTGTCTGGACAGACCATTGAGGTGACATCTGAGTACCTCTTCAGGCACTCTGACAATGAGCT 613 hrglualalyshisglnglnglnphepheglnpheargleuserglyglnthri legluvalthrserglutyrleuphearghisseraspasnglule 2901 CCTGCACTGGATGGTGGCCCTGGATGGCAAGCCTCTGGCTTCTGGTGAGGTGCCTCTGGATGTGGCCCCTCAAGGAAAGCAGCTGATTGAACTGCCTGAG 646 uleuhistrpmetvalalaleuaspglylysproleualaserglygluvalproleuaspvalalaproglnglylysglnleui legluleuproglu 3001 CTGCCTCAGCCAGAGTCTGCTGGACAACTGTGGCTAACAGTGAGGGTGGTTCAGCCCAATGCAACAGCTTGGTCTGAGGCAGGCCACATCTCTGCATGGC 680 LeuProGlnProGluSerAlaGlyGlnLeuTrpLeuThrValArgValValGlnProAsnAlaThrAlaTrpSerGluAlaGlyHisI leseralatrpg 3101 AGCAGTGGAGGCTGGCTGAGAACCTCTCTGTGACCCTGCCTGCTGCCTCTCATGCCATCCCTCACCTGACAACATCTGAAATGGACTTCTGCATTGAGCT 713 lnglntrpargleualagluasnleuservalthrleuproalaalaserhisalai leprohisleuthrthrserglumetaspphecysi leglule 3201 GGGCAACAAGAGATGGCAGTTCAACAGGCAGTCTGGCTTCCTGTCTCAGATGTGGATTGGAGACAAGAAGCAGCTCCTCACCCCTCTCAGGGACCAATTC 746 uglyasnlysargtrpglnpheasnargglnserglypheleuserglnmettrpi leglyasplyslysglnleuleuthrproleuargaspglnphe 3301 ACCAGGGCTCCTCTGGACAATGACATTGGAGTGTCTGAGGCCACCAGGATTGACCCAAATGCTTGGGTGGAGAGGTGGAAGGCTGCTGGACACTACCAGG 780 ThrArgAlaProLeuAspAsnAspI leglyvalserglualathrargi leaspproasnalatrpvalgluargtrplysalaalaglyhistyrglna 3401 CTGAGGCTGCCCTGCTCCAGTGCACAGCAGACACCCTGGCTGATGCTGTTCTGATCACCACAGCCCATGCTTGGCAGCACCAAGGCAAGACCCTGTTCAT 813 laglualaalaleuleuglncysthralaaspthrleualaaspalavalleui lethrthralahisalatrpglnhisglnglylysthrleuphei l 3501 CAGCAGAAAGACCTACAGGATTGATGGCTCTGGACAGATGGCAATCACAGTGGATGTGGAGGTTGCCTCTGACACACCTCACCCTGCAAGGATTGGCCTG 846 eserarglysthrtyrargi leaspglyserglyglnmetalai lethrvalaspvalgluvalalaseraspthrprohisproalaargi leglyleu 3601 AACTGTCAACTGGCACAGGTGGCTGAGAGGGTGAACTGGCTGGGCTTAGGCCCTCAGGAGAACTACCCTGACAGGCTGACAGCTGCCTGCTTTGACAGGT 880 AsnCysGlnLeuAlaGlnValAlaGluArgValAsnTrpLeuGlyLeuGlyProGlnGluAsnTyrProAspArgLeuThrAlaAlaCysPheAspArgT 3701 GGGACCTGCCTCTGTCTGACATGTACACCCCTTATGTGTTCCCTTCTGAGAATGGCCTGAGGTGTGGCACCAGGGAGCTGAACTATGGTCCTCACCAGTG 913 rpaspleuproleuseraspmettyrthrprotyrvalpheprosergluasnglyleuargcysglythrarggluleuasntyrglyprohisglntr

5 GAGGGGAGACTTCCAGTTCAACATCTCCAGGTACTCTCAGCAACAGCTCATGGAAACCTCTCACAGGCACCTGCTCCATGCAGAGGAGGGAACCTGGCTG pargglyasppheglnpheasni leserargtyrserglnglnglnleumetgluthrserhisarghisleuleuhisalaglugluglythrtrpleu AACATTGATGGCTTCCACATGGGCATTGGAGGAGATGACTCTTGGTCTCCTTCTGTGTCTGCTGAGTTCCAGTTATCTGCTGGCAGGTACCACTATCAGC AsnI leaspglyphehismetglyi leglyglyaspaspsertrpserproservalseralaglupheglnleuseralaglyargtyrhistyrglnl NheI (4028) TGGTGTGGTGCCAGAAGTAAACCTGAGCTAGCTGGCCAGACATGATAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAAAAATG euvaltrpcysglnlys CTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAG PacI (4272) GTTCAGGGGGAGGTGTGGGAGGTTTTTTAAAGCAAGTAAAACCTCTACAAATGTGGTATGGAAATGTTAATTAACTAGCCATGACCAAAATCCCTTAACG TGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAA AAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTG TTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAG TGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGC TTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAA GCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCG TCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCAC PacI (5012) ATGTTCTTAATTAAATTTTTCAAAAGTAGTTGACAATTAATCATCGGCATAGTATATCGGCATAGTATAATACGACTCACTATAGGAGGGCCACCATGAA 1 MetLy GAAACCTGAACTGACAGCAACTTCTGTTGAGAAGTTTCTCATTGAAAAATTTGATTCTGTTTCTGATCTCATGCAGCTGTCTGAAGGTGAAGAAAGCAGA slysprogluleuthralathrservalglulyspheleui leglulyspheaspservalseraspleumetglnleusergluglyglugluserarg GCCTTTTCTTTTGATGTTGGAGGAAGAGGTTATGTTCTGAGGGTCAATTCTTGTGCTGATGGTTTTTACAAAGACAGATATGTTTACAGACACTTTGCCT AlaPheSerPheAspValGlyGlyArgGlyTyrValLeuArgValAsnSerCysAlaAspGlyPheTyrLysAspArgTyrValTyrArgHisPheAlaS CTGCTGCTCTGCCAATTCCAGAAGTTCTGGACATTGGAGAATTTTCTGAATCTCTCACCTACTGCATCAGCAGAAGAGCACAAGGAGTCACTCTCCAGGA eralaalaleuproi leprogluvalleuaspi leglygluphesergluserleuthrtyrcysi leserargargalaglnglyvalthrleuglnas TCTCCCTGAAACTGAGCTGCCAGCTGTTCTGCAACCTGTTGCTGAAGCAATGGATGCCATTGCAGCAGCTGATCTGAGCCAAACCTCTGGATTTGGTCCT pleuprogluthrgluleuproalavalleuglnprovalalaglualametaspalai lealaalaalaaspleuserglnthrserglypheglypro TTTGGTCCCCAAGGCATTGGTCAGTACACCACTTGGAGGGATTTCATTTGTGCCATTGCTGATCCTCATGTCTATCACTGGCAGACTGTGATGGATGACA PheGlyProGlnGlyI leglyglntyrthrthrtrpargaspphei lecysalai lealaaspprohisvaltyrhistrpglnthrvalmetaspaspt CAGTTTCTGCTTCTGTTGCTCAGGCACTGGATGAACTCATGCTGTGGGCAGAAGATTGTCCTGAAGTCAGACACCTGGTCCATGCTGATTTTGGAAGCAA hrvalseralaservalalaglnalaleuaspgluleumetleutrpalagluaspcysprogluvalarghisleuvalhisalaasppheglyseras CAATGTTCTGACAGACAATGGCAGAATCACTGCAGTCATTGACTGGTCTGAAGCCATGTTTGGAGATTCTCAATATGAGGTTGCCAACATTTTTTTTTGG nasnvalleuthraspasnglyargi lethralaval I leasptrpserglualametpheglyaspserglntyrgluvalalaasni lephephetrp AGACCTTGGCTGGCTTGCATGGAACAACAAACAAGATATTTTGAAAGAAGACACCCAGAACTGGCTGGTTCCCCCAGACTGAGAGCCTACATGCTCAGAA ArgProTrpLeuAlaCysMetGluGlnGlnThrArgTyrPheGluArgArgHisProGluLeuAlaGlySerProArgLeuArgAlaTyrMetLeuArgI TTGGCCTGGACCAACTGTATCAATCTCTGGTTGATGGAAACTTTGATGATGCTGCTTGGGCACAAGGAAGATGTGATGCCATTGTGAGGTCTGGTGCTGG leglyleuaspglnleutyrglnserleuvalaspglyasnpheaspaspalaalatrpalaglnglyargcysaspalai levalargserglyalagl AACTGTTGGAAGAACTCAAATTGCAAGAAGGTCTGCTGCTGTTTGGACTGATGGATGTGTTGAAGTTCTGGCTGACTCTGGAAACAGGAGACCCTCCACA ythrvalglyargthrglni lealaargargseralaalavaltrpthraspglycysvalgluvalleualaaspserglyasnargargproserthr PacI (6179) AGACCCAGAGCCAAGGAATGAATATTAGCTAGGAGTTTCAGAAAAGGGGGCCTGAGTGGCCCCTTTTTTCAACTTAATTAACCTGCAGGGCCTGAAATAA ArgProArgAlaLysGlu CCTCTGAAAGAGGAACTTGGTTAGGTACCTTCTGAGGCTGAAAGAACCAGCTGTGGAATGTGTGTCAGTTAGGGTGTGGAAAGTCCCCAGGCTCCCCAGC AGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCAGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTC AATTAGTCAGCAACCATAGTCCCACTAGTTTCATCACCACCGCCACCCCCCCGCCCCCCCGCCATCTGAAAGGGTTCTAGGGGATTTGCAACCTCTCTCG TGTGTTTCTTCTTTCCGAGAAGCGCCGCCACACGAGAAAGCTGGCCGCGAAAGTCGTGCTGGAATCACTTCCAACGAAACCCCAGGCATAGATGGGAAAG BamHI (6660) GGTGAAGAACACGTTGTCATGGCTACCGTTTCCCCGGTCACGGAATAAACGCTCTCTAGGATCCGGAAGTAGTTCCGCCGCGACCTCTCTAAAAGGATGG ATGTGTTCTCTGCTTACATTCATTGGACGTTTTCCCTTAGAGGCCAAGGCCGCCCAGGCAAAGGGGCGGTCCCACGCGTGAGGGGCCCGCGGAGCCATTT GATTGGAGAAAAGCTGCAAACCCTGACCAATCGGAAGGAGCCACGCTTCGGGCATCGGTCACCGCACCTGGACAGCTCCGATTGGTGGACTTCCGCCCCC CCTCACGAATCCTCATTGGGTGCCGTGGGTGCGTGGTGCGGCGCGATTGGTGGGTTCATGTTTCCCGTCCCCCGCCCGCGAGAAGTGGGGGTGAAAAGCG BamHI (7052) GCCCGACCTGCTTGGGGTGTAGTGGGCGGACCGCGCGGCTGGAGGTGTGAGGATCCGAACCCAGGGGTGGGGGGTGGAGGCGGCTCCTGCGATCGAAGGG BspHI (7126) GACTTGAGACTCACCGGTCGCACGTCATGAGCAAGGGAGAAGAACTCTTTACTGGTGTTGTCCCAATTCTGGTTGAGCTGGATGGTGATGTGAATGGCCA 1 MetSerLysGlyGluGluLeuPheThrGlyValValProI leleuvalgluleuaspglyaspvalasnglyhi CAAATTCTCTGTGTCTGGTGAAGGTGAAGGAGATGCAACTTATGGAAAGCTGACTCTGAAGTTCATTTGTACAACAGGAAAGCTGCCAGTGCCTTGGCCA slyspheservalserglygluglygluglyaspalathrtyrglylysleuthrleulysphei lecysthrthrglylysleuprovalprotrppro ACTCTGGTGACCACCCTGACTTATGGTGTTCAATGTTTCAGCAGGTACCCTGACCACATGAAGCAGCATGACTTCTTTAAATCTGCAATGCCAGAAGGTT ThrLeuValThrThrLeuThrTyrGlyValGlnCysPheSerArgTyrProAspHisMetLysGlnHisAspPhePheLysSerAlaMetProGluGlyT

6 7401 ATGTTCAGGAGAGGACAATCTTCTTTAAGGATGATGGAAATTATAAGACAAGGGCAGAAGTGAAGTTTGAAGGTGATACACTGGTTAACAGAATTGAGCT 92 yrvalglngluargthri lephephelysaspaspglyasntyrlysthrargalagluvallysphegluglyaspthrleuvalasnargi leglule 7501 GAAAGGCATTGATTTTAAGGAAGATGGAAACATTCTGGGTCACAAGCTGGAGTACAACTATAATTCTCACAATGTTTACATTATGGCAGATAAGCAGAAG 125 ulysglyi leaspphelysgluaspglyasni leleuglyhislysleuglutyrasntyrasnserhisasnvaltyri lemetalaasplysglnlys 7601 AATGGAATTAAGGTTAATTTCAAGATTAGACACAACATTGAGGATGGATCTGTCCAACTGGCAGACCATTACCAGCAGAACACCCCTATTGGTGATGGCC 159 AsnGlyI lelysvalasnphelysi learghisasni legluaspglyservalglnleualaasphistyrglnglnasnthrproi leglyaspglyp 7701 CAGTTCTCCTCCCAGATAATCACTATCTCCGCACTCAATCTGCTCTGTCCAAAGACCCTAATGAGAAAAGAGACCACATGGTCCTCCTGGAGTTTGTGAC 192 rovalleuleuproaspasnhistyrleuargthrglnseralaleuserlysaspproasnglulysargasphismetvalleuleugluphevalth AvrII (7846) 7801 AGCAGCAGGAATTACTCTGGGAATGGATGAGCTGTACAAGTAAACCTAGGATTATCCCTAATACCTGCCACCCCACTCTTAATCAGTGGTGGAAGAACGG 225 ralaalaglyi lethrleuglymetaspgluleutyrlys 7901 TCTCAGAACTGTTTGTTTCAATTGGCCATTTAAGTTTAGTAGTAAAAGACTGGTTAATGATAACAATGCATCGTAAAACCTTCAGAAGGAAAGGAGAATG TTTTGTGGACCACTTTGGTTTTCTTTTTTGCGTGTGGCAGTTTTAAGTTATTAGTTTTTAAAATCAGTACTTTTTAATGGAAACAACTTGACCAAAAATT TGTCACAGAATTTTGAGACCCATTAAAAAAGTTAAATGAGAAACCTGTGTGTTCCTTTGGTCAACACCGAGACATTTAGGTGAAAGACATCTAATTCTGG TTTTACGAATCTGGAAACTTCTTGAAAATGTAATTCTTGAGTTAACACTTCTGGGTGGAGAATAGGGTTGTTTTCCCCCCACATAATTGGAAGGGGAAGG AATATCATTTAAAGCTATGGGAGGGTTTCTTTGATTACAACACTGGAGAGAAATGCAGCATGTTGCTGATTGCCTGTCACTAAAACAGGCCAAAAACTGA GTCCTTGGGTTGCATAGAAAGCTG