US007070959B1
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`(54) MODIFIED CHIMERIC POLYPEPTIDES
`WITH IMPROVED PHARMACOKINETIC
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`PROPERTIES
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`(75)
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`Inventors: Nicholas J. Papadopoulos,
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`Lagrangeville, NY (US); Samuel Davis.
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`New York, NY (US); George D.
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`Yancopoulos, Yorktown Heights, NY
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`(US)
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`WoO
`wo
`Wo
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`(*) Notice:
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`(73) Assignee: Regeneron Pharmaceuticals, Inc.,
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`Tarrytown, NY (US)
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`Subject to any disclaimer, the term of this
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`patent is extended or adjusted under 35
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`U.S.C. 154(b) by 0 days.
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`This patent is subject to a terminal dis-
`claimer.
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`(21) Appl. No.:
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`(22) PCT Filed:
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`(86) PCT No.:
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`10/009,852
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`May 23, 2000
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`PCT/US00/14142
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`§ 371 ©),
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`(2), (4) Date: Dec. 6, 2001
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`(87) PCT Pub. No.: WO00/75319
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`PCT Pub. Date: Dee. 14, 2000
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`Related U.S. Application Data
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`(60) Provisional application No. 60/138,133, filed on Jun.
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`8, 1999.
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`(51)
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`Int. Cl.
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`(2006.01)
`CI2N 15/62
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`(2006.01)
`CI2N 15/63
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`(2006.01)
`CO7H 21/04
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`(2006.01)
`CO7K 16/00
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`(2006.01)
`CO7K 14/00
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`(52) US. Ch coca 435/69.7; 435/70.1; 435/71.1;
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`435/320.1; 435/325; 435/252.3; 530/387.3,
`530/350; 536/23.4
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`(58) Field of Classification Search ............... 435/69.7,
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`435/320.1, 70.1, 71.1; 530/387.3
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`See application file for complete search history.
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`a2) United States Patent
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`US 7,070,959 B1
`(10) Patent No.:
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`*Jul. 4, 2006
`(45) Date of Patent:
`Papadopoulosetal.
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`
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`FOREIGN PATENT DOCUMENTS
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`
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`WO097/44453
`11/1997
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`4/1998
`WO098/13071
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`W099/03996
`1/1999
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`Ilerley et al. (1999), Biochem. Biophys. Res. Comm. 262:
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`
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`(Continued)
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`Primary Examiner—Lorraine Spector
`Assistant Examiner—Jon M Lockard
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`(74) Attorney, Agent, or Firm—vValeta Gregg, Esq.
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`(57)
`ABSTRACT
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`Modified chimeric polypeptides with improved pharmaco-
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`kinetics are disclosed. Specifically, modified chimeric Flt]
`
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`receplor polypeptides that have been modifiedin such a way
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`as to improve their pharmacokinetic profile are disclosed.
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`Also disclosed are methods of making and using the modi-
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`fied polypeptides including but not limited to using the
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`modified polypeptides to decrease or inhibit plasma leakage
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`and/or vascular permeability in a mammal.
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`(56)
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`References Cited
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`U.S. PATENT DOCUMENTS
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`1/1998 Kendall et al.
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`1/2000 Charnock-Jonesetal.
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`8/2000 Davis-Smyth et al.
`..... 435/69.7
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`5,712,380 A
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`6,011,003 A
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`6,100,071 A *
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`15 Claims, 55 Drawing Shects
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`CELLTRION - EXHIBIT 1023
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`CELLTRION - EXHIBIT 1023
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`US 7,070,959 B1
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`Page 2
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`OTHER PUBLICATIONS
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`Davis-Smyth,T., et al., 1996, “The second immunoglobulin-
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`like domain of the VEGF tyrosine kinase receptor Flt-1
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`determines
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`Chemistry, vol. 273, No. 6, pp. 3216-3222.
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`* cited by examiner
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`U.S. Patent
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`Jul. 4, 2006
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`Sheet 1 of 55
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`US 7,070,959 B1
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`Fig.1.
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`12 3 45 67 8
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`1
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`ug/ml
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`—f— 'TIE-2-Fe
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`confacetylated Fit-1(1-3)-Fe
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`----©--- unmodified Fit-1(1-3)-Fe
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`U.S. Patent
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`Jul. 4, 2006
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`Sheet 2 of 55
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`US 7,070,959 B1
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`Sheet6 of 55
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`Fig.7.
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`—{}— rTIiE-2-Fc
`cesoeeeeO--- unmodified Fit-1(1-3)-Fe
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`-~--O---: acetylated Fit-1(1-3)-Fe (10X)
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`------fy----- acetylated Fit-1(1-3)-Fe (20X)
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`---4#--- acetylated Fit-1(1-3)-Fe (30X)
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`Sheet7 of 55
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`US 7,070,959 B1
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`US 7,070,959 B1
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`Fig.10A.
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`
`Trp Ser Leu Pro Glu Met Val Ser Lys Glu Ser Glu Arg Leu Ser Ile Thr Lys Ser Ala>
`
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`
`
`
`
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`
`
`260
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`
`
`
`270
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`
`*
`
`
`*
`
`
`
`280
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`
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`
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`
`290
`t
`
`250
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`Cys Gly Arg Asn Gly Lys Gln Phe Cys Ser Thr Leu Thr Leu Asn Thr Ala Gin Ala Asn>
`
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`
`300
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`320
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`310
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`
`
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`
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`360
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`390
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`410
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`
`400
`380
`370
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`CTT AGA CGT TAG ATA TAT AAA TAA TCA CTA TGT CCA TCT GGA AAG CAT CTC TAC ATG TCA
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`Glu Ser Ala Ile Tyr Ile Phe Tle Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser>
`
`
`420
`*
`
`
`
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`
`430
`480
`440
`460
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`GAA ATC CCC GAA ATT ATA CAC ATG ACT GAA GGA AGG GAG CIC GIC ATT CCC TGC CGG GTT
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`Glu Ile Pro Glu Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val>
`
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`
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`470
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`
`-
`
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`4390
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`
`500
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`
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`510
`520
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`
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`ACG TCA CCT AAC ATC ACT GTT ACT TTA AAA AAG TIT CCA CTT GAC ACT T1G ATC CCT GAT
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`TGC AGT GGA TTG TAG TGA CAA TGA AAT TTT TTC AAA GGT GAA CTG TGA AAC TAG GGA CTA
`
`
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`Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr Leu Ile Pro Asp>
`
`*
`
`
`
`*
`
`
`
`530
`*
`
`540
`*
`
`

`

`
`U.S. Patent
`
`
`
`
`Jul. 4, 2006
`
`
`
`
`Sheet 10 of 55
`
`
`
`US 7,070,959 B1
`
`Fig.10B.
`
`6
`
`550
`*
`
`
`
`*
`
`
`560
`*
`
`
`
`*
`
`
`
`
`
`570
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`
`
`x
`
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`580
`¥
`
`
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`580
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`600
`¥
`
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`
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`
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`GGA AKA CGC ATA ATC TGG GAC AGT AGA AAG GGC TIC ATC ATA TCA AAT GCA ACG TAC AAA
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`CCT TIT GCG TAT TAG ACC CIG ICA TCT TIC CCG AAG TAG TAT AGT TTA CGT TGC ATG TIT
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`Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe Ile Ile Ser Asn Ala Thr Tyr Lys>
`
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`
`610
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`
`
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`
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`
`
`
`620
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`
`
`
`630
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`640
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`GAA ATA GGG CTT CTG ACC TGT GAA GCA ACA GTC AAT GGG CAT TTG TAT AAG ACA AAC TAT
`
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`CTT TAT CCC GAA GAC TGG ACA CTT CGT TGT CAG TTA CCC GTA AAC ATA TIC ToT TIG ATA
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`
`Glu Ile Gly Leu Leu Thr Cys Glu Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr>
`
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`
`
`
`670
`*
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`
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`
`
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`680
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`
`690
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`
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`
`700
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`710
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`729
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`
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`CTC ACA CAT CGA CAA ACC AAT ACA ATC ATA GAT GTC CAA ATA AGC ACA CCA CGC CCA GIC
`
`
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`
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`GAG TCT GTA CCT GTT TGG TIA TGT TAG TAT CTA CAG GTT TAT TOG TGT GGT GCG GGT CAG
`
`
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`
`Leu Thr His Arg Gln Thr Asn Thr Ile Ile Asp Val Gin Ile Ser Thr Pro Arg Pro Val>
`
`
`
`730
`*
`
`*
`
`
`*
`
`
`
`740
`*
`
`
`
`
`
`
`
`
`
`
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`
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`
`AAA TTA CIT AGA GGC Cat ACT CTT GTC CIC AAT TGT ACT GCT ACC ACT CCC TG AAC ACG
`
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`TTT AAT GAA TCT COG GTA TGA GAA CAG GAG TTA ACA TGA CGA TGG TGA GGG AAC TTG Tot
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`Lys Leu Leu Arg Gly His Thr Leu Val Leu Asn Cys Thr Ala Thr Thr Pro Leu Asn Thr>
`
`
`
`750
`*
`
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`
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`
`760
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`
`
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`
`«
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`
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`
`770
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`
`
`
`780
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`
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`
`
`730
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`
`*
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`800
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`
`
`
`
`810
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`*
`
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`
`*
`
`
`
`820
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`
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`
`
`
`830
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`
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`
`840
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`
`
`
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`
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`
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`AGA GTT CAA ATG ACC TGG AGT TAC CCT GAT GAA AAA AAT AAG AGA GCT TCC GTA AGG CGA
`
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`TCT CAA GIT TAC TGG ACC TCA ATG GGA CTA CTT TIT TIA TIC TCT CGA AGG CAT TCC CCT
`
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`
`Arg Val Gln Met Thr Trp Ser Tyr Pro Asp Glu Lys Asn Lys Arg Ala Ser Val Arg Arg>
`
`
`
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`
`
`
`850
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`
`
`x
`
`
`860
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`
`
`870
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`880
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`CGA ATT GAC CAA AGC AAT TCC CAT GCC AAC ATA TIC TAC AGT GTT CTT ACT ATT GAC AAA
`
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`
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`
`Arg Ile Asp Gin Ser Asn Ser His Ala Asn Ile Phe Tyr Ser Val Leu Thr Ile Asp Lys>
`
`*
`
`
`910
`4
`
`*
`
`
`920
`*
`
`«
`
`
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`
`930
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`
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`950
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`ATG CAG AAC ABA GAC AAA GGA CTT TAT ACT TGT CGT GTA AGG AGT GGA CCA TCA TIC AAA
`TAC GTC TTG TTT C1G TIT CCT GAA ATA TGA ACA GCA CAT TCC TCA CCT GGT AGT AAG TIT
`
`
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`
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`
`Met Gln Asn Lys Asp Lys Gly Leu Tyr Thr Cys Arg Val Arg Ser Gly Pro Ser Phe Lys>
`
`
`
`*
`
`
`
`970
`*
`
`*
`
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`
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`*
`
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`1000
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`TCT GTT AAC ACC TCA GIG CAT ATA TAT GAT AAA GCA GGC CCG GGC GAG CCC AAA TCT TGT
`
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`AGA CAA TIG IGG AGT CAC GTA TAT ATA CTA TIT CGT CCG GGC CCG CIC GGG TTT AGA ACA
`
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`
`
`
`
`Ser Val Asn Thr Ser Val His Ile Tyr Asp Lys Ala Gly Pro Gly Glu Pro Lys Ser Cys>
`
`
`
`*
`
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`
`1030
`*
`
`
`
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`
`1040
`*«
`
`
`
`
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`GAC AAA ACT CAC ACA TGC CCA CCG TGC CCA GCA CCT GAA CIC CTG GGG GGA COG TA GTC
`CTG TTT TGA GTG TGT ACG GOT GGC ACG GST CGT GGA CTT GAG GAC CCC CCT GOC AGT CAG
`
`
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`
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`
`
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`
`
`
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`
`Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val>
`
`*
`
`1050
`
`*
`
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`
`1060
`*€
`
`*
`
`1070
`*
`
`
`
`1080
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`
`
`
`*
`
`

`

`
`U.S. Patent
`
`
`
`
`
`
`
`Sheet 11 of 55
`
`
`
`US 7,070,959 B1
`
`
`Jul. 4, 2006
`
`
`Fig.10C.
`
`1090
`
`*
`*
`
`
`
`
`
`TN cre Tr ccc
`AAG GAG AAG GGG
`
`
`
`
`
`
`Phe Leu Phe Pro
`
`*
`
`
`
`1110
`*
`
`
`
`*
`
`1160
`*
`
`AAG
`
`
`
`
`
`
`
`CCA AAA CCC
`GAC ACC CTC
`TTC
`GGT TTT GGG
`CTG TGG GAG
`
`
`
`
`
`
`
`
`
`
`
`
`Pro Lys Pro
`Lys
`Asp Thr Leu
`
`*
`
`1120
`*
`
`
`
`
`
`
`ATG ATC TCC
`TAC TAG AGG
`
`
`
`
`
`
`Met Ile Ser
`
`1140
`*
`
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`
`1130
`*
`
`
`
`
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`
`
`CGG ACC CCT GAG GTC ACA
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`GCC IGG GGA CTC CAG TCT
`
`
`
`
`
`
`
`Arg Thr Pro Glu Val Thr>
`
`
`
`*
`
`1160
`*
`
`
`
`1150
`
`1176
`cy
`*
`*
`*
`
`
`
`
`GAA GAC CCT
`CAC
`
`
`
`
`
`
`
`
`
`
`
`
`
`GAC GIG AGC
`GAG GTC AAG
`TGC GIG GIG GIG
`CTC CAG TIC
`ACG CAC CAC CAC
`GTG
`
`
`
`
`
`
`
`
`
`
`
`CT CAC TCG
`CTT CTG GGA
`
`
`
`
`
`
`
`
`
`
`
`
`
`His
`Cys Val Val Val
`Glu Val Lys
`Glu Asp Pro
`Asp Val Ser
`
`*
`
`1180
`
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`
`1190
`*
`
`
`
`1200
`*
`
`
`
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`
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`
`TTC AAC TSG TAC GTG GAC
`
`
`
`
`
`
`
`AAG TIG ACC ATG CAC CTG
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Phe Asn Trp Tyr Val Asp>
`
`x
`
`
`
`1210
`*
`
`
`*
`
`1226
`
`*
`
`
`1230
`*
`
`
`
`*
`
`
`
`
`
`
`
`
`
`
`
`
`GSC GIG GAG GIG
`CAT AAT GCC
`AAG ACA AAG CCG
`cee cac CIC CAC
`
`
`
`
`
`
`
`
`TIC TT TH Ge
`GTA TTA CGG
`
`
`
`
`
`
`
`
`
`
`His Asn Ala
`Gly Val Glu Val
`Lys Thr Lys Pro
`
`*®
`
`1240
`*
`
`
`
`
`
`
`CGG GAG GAG
`ece cre cre
`
`
`
`
`
`
`Arg Glu Glu
`
`*
`
`1250
`*
`
`
`
`1260
`*
`
`
`
`«
`
`
`
`CAG TAC AAC AGC ACG TAC
`
`
`
`
`
`
`
`GTC ATG TG TcG TGC ATG
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Gln Tyr Asn Ser Thr Tyr>
`
`*
`
`1270
`*
`
`
`
`©
`
`
`1280
`*
`
`Gr
`GTtc cTc Acc
`
`
`
`
`
`
`
`
`CGT GIG GTC AGC
`CTG Cac CAG
`GAC GTG GIC
`GCA CAC CAG TCG
`
`
`
`
`
`
`
`
`
`
`CAG GAG TGG
`CAG
`
`
`
`
`
`
`
`
`
`
`Val
`Val Leu Thr
`Leu His Gin
`Arg Val Vai Ser
`
`1230
`.
`
`
`
`*
`
`*
`
`1300
`*
`
`
`
`
`
`
`GAC TGG CTG
`cTG ACC GAC
`
`
`
`
`
`
`Asp Trp Leu
`
`*
`
`1310
`*
`
`*
`
`1320
`
`
`*
`
`
`
`AAT GGC AAG GAG TAC AAG
`
`
`
`
`
`
`
`TTA CCG TIC CIC ATG TIC
`
`
`
`
`
`
`
`
`
`
`
`
`
`Asn Gly Lys Glu Tyr Lys>
`
`1370
`*
`
`
`
`1380
`*
`
`*
`
`*
`
`*
`
`1346
`1330
`
`
`*
`*
`
`
`CCA GC CCC
`TGC AAG GTC TCC
`eTe
`AAC ABA GCC
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`GGT CGG GGG
`GAG
`ACG TIC CAG AGG
`TTG TIT CGG
`
`
`
`
`
`
`
`
`
`
`Leu
`Pro Ala Pro
`Cys Lys Val Ser
`Asn Lys Ala
`
`1350
`
`*
`
`*
`
`*
`
`1360
`*
`
`
`
`ATC GAG AAA
`
`
`
`TAG CIC TIT
`
`
`
`
`
`
`Ile Glu Lys
`
`
`*
`
`
`ACC ATC TCC AAA GCC AAA
`
`
`
`
`
`
`
`TGG TAG AGG TIT CGG TIT
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Thr Ile Ser Lys Ala Lys>
`
`*
`
`1390
`«
`
`
`
`*
`
`1406
`*
`
`
`
`*
`
`1416
`*
`
`
`GGG CAG CCC CGA
`
`
`
`
`
`
`
`
`
`
`GTG
`GAA CCA CAG
`TAC ACC CIG
`ATG TGG GAC
`ccc GiIC GGG ccT
`Cac
`
`
`
`
`
`
`
`
`CTT Got Gtc
`
`
`
`
`
`
`
`
`
`
`Val
`Glu Pro Gin
`Tyr Thr Leu
`Gly Gin Pro Arg
`
`
`
`*
`
`1420
`*
`
`
`
`
`ece CCA TCC
`
`
`
`GGG GGT AGG
`
`
`
`
`
`Pre Pro Ser
`
`1440
`*
`
`
`
`*
`
`1430
`*
`
`*
`
`
`
`
`
`
`
`
`
`
`CGG GAT GAG CTG ACC AAG
`Gece CTA CTC GAC TGG TIC
`
`
`
`
`
`
`
`
`
`
`
`
`
`Arg Asp Glu Leu Thr Lys>
`
`*
`
`*
`
`1460
`1450
`
`
`*
`*
`
`
`AAC CAG GIC AGC
`CTG acc TSC
`
`
`
`
`TTG GTC CAG TCG
`GAC TGG ACG
`
`
`
`
`
`
`
`
`
`
`
`
`Asn Gin Val Ser
`Leu Thr Cys
`
`1470
`*
`
`*
`
`
`
`1480
`1490
`
`
`*
`*
`=
`¥
`*
`
`
`
`
`
`
`
`TTC TAP Ccc
`AGC GAC ATC GCC GTG GAG
`cis
`GTC AAA GCC
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`AAG ATA GGG
`CAG TTT CCG
`TOG CTG TAG CGG CAC CTC
`GAC
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Leu
`Val Lys Gly
`Phe Tyr Pro
`Ser Asp Ile Ala Val Glu>
`
`
`
`1500
`*
`
`*
`
`1520
`*
`
`*
`
`1516
`*
`
`
`
`
`
`GAG
`
`
`
`
`
`
`
`TSG GAG AGC AAT
`GGG CAG CCG
`ACC CTC TOG TTA
`
`
`
`
`
`
`
`ere
`ece Gt Got
`
`
`
`
`
`
`
`Glu
`fxp Glu Ser Asn
`Gly Gln Pro
`
`1530
`*
`
`
`
`*
`
`x
`
`1540
`*
`
`
`
`1550
`*
`
`
`
`*
`
`*
`
`cet ccc GIG CTG GAC TCC
`AAG ACC ACG
`AAC AAC TAC
`
`
`
`
`
`
`
`
`
`
`
`
`
`TIC TGG TGC
`GGA GGG CAC GAC CTIG AGG
`TTG TIG ATG
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Asn, Asn Tyr
`Pro Pro Val Leu Asp Ser>
`Lys Thr Thr
`
`1560
`*
`
`
`
`*
`
`
`
`*
`
`1580
`*
`
`
`
`1590
`*
`
`
`
`*
`
`1570
`*
`
`
`
`GAC GGC TCC Tr
`AGC
`AAG CIC ACC
`
`
`
`
`
`
`
`
`
`
`TI¢ cic TAC
`TCG
`AAG GAG ATG
`
`
`
`
`
`
`
`
`
`
`TIC GAG TGG
`CTG CCG AGG AAG
`
`
`
`
`
`
`
`
`
`
`ser
`Lys Leu Thr
`Asp Gly Ser Phe
`Phe Leu Tyr
`
`*
`
`1600
`*
`
`
`
`GTG GAC AAG
`
`
`
`CAC CTG TIC
`
`
`
`
`
`
`Val Asp Lys
`
`1620
`*
`
`*
`
`*
`
`1610
`*
`
`
`
`
`
`
`
`
`
`
`
`
`
`AGC AGG TGG CAG CAG GGG
`
`
`
`
`
`
`
`TOG TCC ACC GIc GTC ccc
`
`
`
`
`
`
`
`Ser Arg Trp Gin Gln Gly>
`
`

`

`
`U.S. Patent
`
`
`
`
`Jul. 4, 2006
`
`
`
`
`Sheet 12 of 55
`
`
`
`US 7,070,959 B1
`
`Fig.10D.
`
`
`
`*
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`AAC GTC TTC TCA TGC TCC GIG ATG CAT GAG GCT CTG CAC AAC CAC TAC ACG CAG AAG AGC
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`TTG CAG AAG AGT ACG AGG CAC TAC GTA CIC CGA GAC GIG TIG GIG ATG TCC GIC Tit TG
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gin Lys Ser>
`
`
`
`1660
`*€
`
`*
`
`
`
`
`
`1670
`x
`
`*
`
`
`
`1680
`*
`
`
`
`1630
`*
`
`*
`
`
`
`
`
`1640
`x
`
`*
`
`
`1656
`«x
`
`*
`
`*
`
`
`
`1700
`*
`
`
`
`*
`
`
`1696
`*
`
`
`
`
`
`
`crc ICC CIG TCT CCG GGT AAA TGA
`GAG AGG GAC AGA GGC CCA TIT ACT
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Leu Ser Leu Ser Pro Gly Lys ***>
`
`
`
`

`

`
`U.S. Patent
`
`
`
`
`
`
`
`“LEBI
`
`
`
`
`
`
`
`
`SSBuUlyJBIN|JedeJ}U|q.BulpuigpuebyJepnjjeoesyx3
`UIBWOPSULIQWSWSUB!SUIBWOP
`
`NZN9NSNvNeNe
`
`“1°dS}bussNZ
`“8dS}b-uasNG
`s™dS}b-u4SNv
`*°dS}b-l4SN9
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`MSSSSS§Syid5}bduewny
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`lL
`
` “5SSusSIdSIL-W4sNZGm8dS]b-ussNe
`
`
`
`Jul. 4, 2006
`
`Sheet 13 of 55
`
`
`
`US 7,070,959 B1
`
`
`
`
`
`OBZ.StelLSEL766926gle\6S40GZ289oggSPSreOE?LONploeOUlLuy
`
`
`
`
`
`
`

`

`
`U.S. Patent
`
`
`
`
`Jul. 4, 2006
`
`
`
`
`Sheet 14 of 55
`
`
`
`US 7,070,959 B1
`
`
`
`Fig.12A.
`
` .
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`SS8seseesssss
`
`
`
`
`OTON Ogio ysis
`
`AyorpydoupAyH
`
`CODD OOOO oOSCO
`
`SBO55 5000050
`
`
`
`LOT OA KONO TIO
`
`AyoupydoupAH
`
`

`

`
`U.S. Patent
`
`
`
`
`Jul. 4, 2006
`
`
`
`
`Sheet 15 of 55
`
`
`
`US 7,070,959 B1
`
`Fig.13A.
`
`
`
`*
`
`*
`
`
`
`*
`
`*
`
`*
`
`*
`
`*
`
`*
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`ATG GTC AGC TAC TGG GAC ACC GGG GTC CTG CIG TOC GCG CIG CTC acc ToT CIE CIT CH
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`TAC CAG TCG ATG ACC CTG TGG CCC CAG GAC GAC ACG CGC GAC GAG TCG ACA GAC GAA GAG
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Met Val Ser Tyr Trp Asp Thr Gly Val Leu Leu Cys Ala Leu Leu Ser Cys Leu Leu Leu>
`
`*
`
`*
`
`*
`
`*
`
`*
`
`
`70
`*
`
`*
`
`
`&O
`*
`
`
`
`
`
`
`100
`30
`*
`*
`*
`*
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`ACA GGA TCT AGT TCA GGT TCA AAA TTA ABA GAT CCT GAA CTG AGT TTA AAA GGC ACC CAG
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`TGT CCT AGA TCA AGT CCA AGT TTT AAT TTT CTA GGA CTT GAC TCA AAT TTP CCG IGG GTC
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Thr Gly Ser Ser Ser Gly Ser Lys Leu Lys Asp Pro Glu Leu Ser Leu Lys Gly Thr Gln>
`
`*
`
`
`
`110
`+t
`
`
`
`
`
`120
`*
`
`*
`
`*
`
`
`130
`*
`
`
`*
`
`
`
`140
`*
`
`
`250
`*
`
`*
`
`
`
`160
`*
`
`
`
`
`*
`
`
`170
`*
`
`
`*
`*
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`CAC ATC ATG CAA GCA GGC CAG ACA CTG CAT CTC CAA TGC AGG GGG GAA GCA GCC CAT AAA
`
`
`
`
`
`
`
`
`
`
`
`
`
`GIG TAG TAC GIT CGT CCG GTC TGT GAC GTA GAG GTT ACG TCC CCC CTT CGT CGG GTA TIT
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`His Ile Met Gln Ala Gly Gln Thr Leu His Leu Gln Cys Arg Gly Glu Ala Ala His Lys>
`
`
`
`180
`*
`
`
`*
`
`
`190
`*
`
`*
`
`
`200
`*
`
`
`
`210
`*
`
`*
`
`*
`
`220
`*
`
`
`
`
`*
`
`
`
`
`230
`*
`*
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`TGG TCT TTG CCT GAA ATG GTG AGT AAG GAA AGC GAA AGG CTG AGC ATA ACT AAA TCT Goce
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`ACC AGA AAC GGA CTT TAC CAC TCA TTC CTT TCG CTT TCC GAC TCG TAT TGA TIT AGA CGG
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Trp Ser Leu Pro Glu Met Val Ser Lys Glu Ser Glu Arg Leu Ser Ile Thr Lys Ser Ala>
`
`240
`*
`
`*
`
`
`
`*
`
`
`
`&
`
`
`260
`*
`
`*
`
`
`
`
`2S50
`300
`270
`*
`*
`*
`*
`*
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`TGT GGA AGA AAT GGC AAA CAA TTC TGC AGT ACT TTA ACC TTG AAC ACA GCT CAA GCA AAC
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`ACA CCT TCT TTA CCG TTT GTT AAG ACG TCA TGA AAT TGG AAC TIG TGT CGA GTT CGT THG
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Cys Gly Arg Asn Gly Lys Gln Phe Cys Ser Thr Leu Thr Leu Asn Thr Ala Gln Ala Asn>
`
`280
`*
`
`290
`¥
`
`*
`
`
`
`
`
`310
`*
`
`t
`
`
`320
`*
`
`
`
`340
`330
`*
`*
`*x
`*
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`CAC ACT GGC TTC TAC AGC TGC AAA TAT CTA GCT GTA CCT ACT TCA AAG AAG AAG GAA ACA
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`GTG TGA CCG AAG ATG TCG ACG TTT ATA GAT CGA CAT GGA TGA AGT TIC TIX Tic CTT Tet
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`His Thr Gly Phe Tyr Ser Cys tys Tyr Leu Ala Val Pro Thr Ser Lys Lys Lys Glu Thr>
`
`*
`
`
`
`*
`
`
`
`
`
`350
`*
`
`360
`*
`
`
`390
`370
`¥
`*
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`GAA TCT GCA ATC TAT ATA TTT ATT AGT GAT ACA GGT AGA CCT TIC GTA GAG ATG TAC AGT
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`CTT AGA CGT TAG ATA TAT ABA TAA TCA CTA TGT CCA TCT GGA AAG CAT CTC TAC ATG TCA
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Glu Ser Ala Iie Tyr Ile Phe Ile Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser>
`
`
`400
`*
`
`*
`
`
`
`*
`
`
`
`
`380
`*
`
`«=
`
`
`
`=
`
`*
`
`410
`*
`
`
`
`
`
`
`420
`*
`
`*
`
`
`*
`
`
`
`430
`*
`
`ca
`
`
`
`440°
`*
`*
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`GAA ATC CCC GAA ATT ATA CAC ATG ACT GAA GGA AGG GAG CTC GTC ATT CCC THC CGG GIT
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`CTT TAG GGG CTT TAA TAT GTG TAC TGA CTT CCT TCC CTC GAG CAG TAA GGG ACG GCC CAA
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Glu Tle Pro Glu Ile tle His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val>
`
`
`
`450
`oa
`
`«
`
`460
`4
`
`*
`
`
`
`470
`-
`
`
`
`480
`*
`
`*
`
`*
`
`
`
`490
`7
`
`
`500
`*
`
`*
`
`
`
`516
`cd
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`ACG TCA CCT AAC ATC ACT GTT ACT TTA AAA AAG TIT CCA CTT GAC ACT TTG ATC CCT GAT
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`TGC AGT GGA TIG TAG TGA CAA TGA AAT TTT TTC AAA GGT GAA CTG TGA AAC TAG GGA CTA
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Thr Ser Pro Asn Ile Thr Val Thr Lev Lys Lys Phe Pro Leu Asp Thr Leu Ile Pro Asp>
`
`*
`
`520
`*
`
`x
`
`§30
`*
`
`
`
`*
`
`
`
`540
`*
`
`

`

`
`U.S. Patent
`
`
`
`Jul. 4, 2006
`
`
`
`
`Sheet 16 of 55
`
`
`US 7,070,959 B1
`
`
`
`Fig.13B.
`
`
`
`*
`
`
`
`$50
`*
`
`*
`
`560
`*
`
`
`
`*
`
`
`
`570
`*
`
`*
`
`580
`*
`
`
`
`
`
`590
`*
`
`t
`
`
`
`600
`*
`
`*«
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`GGA AAA CGC ATA ATC TGG GAC AGT AGA AAG GGC TTC ATC ATA TCA AAT GCA ACG TAC AAA
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`CCT TPT GCG TAT TAG ACC CTG TCA TCT TIC CCG AAG TAG TAT AGT TTA CGT TCC ATG TIT
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe Ile Ile Ser Asn Ala Thr Tyr Lys>
`
`*
`
`
`
`610
`*
`
`*
`
`620
`
`*
`
`*&
`
`
`
`630
`*
`
`*
`
`640
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`
`
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`
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`
`
`
`650
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`
`
`
`660
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`
`*
`
`
`
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`
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`GAA ATA GGG CTT CTIG ACC IGT GAA GCA ACA GTC AAT GGG CAT TIG TAT AAG ACA AAC TAT
`
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`CTT TAT CCC GAA GAC TGG ACA CTT CGT TGT CAG TTA CCC GTA AAC ATA TIC TGT TIG ATA
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`Glu Ile Gly Leu Leu Thr Cys Glu Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr>
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`670
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`680
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`700
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`r
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`CTC ACA CAT CGA CAA ACC AAT ACA ATC ATA GAT GTC CAA ATA AGC ACA CCA CGC CCA GI
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`
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`Leu Thr His Arg Gin Thr Asn Thr Tle Ile Asp Val Gln Ile Ser Thr Pro Arg Pro Val>
`
`680
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`++
`
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`710
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`720
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`730
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`740
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`
`760
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`770
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`780
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`AAA TTA CTT AGA GGC CAT ACT CTT GTC CTC AAT TCT ACT GCT ACC ACT CCC TIG AAC ACG
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`TTT AAT GAA TCT COG GTA TGA GAA CAG GAG TTA ACA TGA CGA TGG TGA GGG AAC TTG TGC
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`Lys Leu Leu Arg Gly His Thr Leu Val Leu Asn Cys Thr Ala Thr Thr Pro Leu Asn Thr>
`
`
`
`
`
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`
`
`
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`
`840
`830
`810
`800
`790
`820
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`4 *.7* * * 7 * «x * * ¥
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`AGA GTT CAA ATG ACC IGG AGT TAC CCT GAT GAA ATT GAC CAA AGC AAT TCC CAT GCC AAC
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`TCT CAA GIT TAC TGG ACC TCA ATG GGA CTA CTT TAA CIG GTT TCG TTA AGG GTA CGG TIS
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`Arg Val Gln Met Thr Trp Ser Tyr Fro Asp Glu Ile Asp Gln Ser Asn Ser His Ala Asn>
`
`
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`
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`
`850
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`
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`860
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`
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`880
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`890
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`900
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`
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`
`870
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`
`
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`
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`
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`ATA TIC TAC AGT GTT CTT ACT ATT GAC AAA ATG CAG AAC AAA GAC AAA GGA CTT TAT ACT
`
`
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`
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`Ile Phe Tyr Ser Val Leu Thr Ile Asp Lys Met Gln Asn Lys Asp Lys Gly Leu Tyr Thr>
`
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`
`x
`
`
`
`920
`*
`
`
`
`$30
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`
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`$40
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`
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`950
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`
`
`
`960
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`
`
`910
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`
`
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`TGT CGT GTA AGG AGT GGA CCA TCA TIC AAA TCT GTT AAC ACC TCA GTG CAT ATA TAT GAT
`
`
`
`
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`ACA GCA CAT TCC TCA CCT GGT AGT AAG TTT AGA CAA TIG TGG AGT CAC GTA TAT ATA CTA
`
`
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`
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`
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`
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`
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`
`
`
`
`
`Cys Arg Val Arg Ser Gly Pro Ser Phe Lys Ser Val Asn Thr Ser Val His Iie Tyr Asp>
`
`*
`
`
`980
`*
`
`*
`
`
`
`990
`*
`
`*
`
`
`
`1060
`*
`
`
`1010
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`
`
`
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`
`
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`
`1020
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`
`970
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`
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`
`
`
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`AAA GCA GGC CCG GGC GAG CCC AAA TCT TGT GAC AAA ACT CAC ACA TGC CCA COG TGC CCA
`
`
`
`
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`
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`TIT CGT CCG GGC CCG CTC GGG TIT AGA ACA CTG TIT TGA GIG TGT ACG GGT GCC ACG GGT
`
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`
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`
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`
`
`
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`
`
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`
`
`
`
`
`
`
`
`Lys Ala Gly Pro Gly Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro>
`
`
`
`*
`
`1030
`*
`
`
`
`*
`
`1040
`
`*
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`
`
`
`
`
`
`GCA CCT GAA CTC CIG GGG GGA CCG TCA GTC TTC CIC TTC CCC CCA AAA CCC AAG GAC ACC
`
`
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`
`
`
`
`
`
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`
`
`
`
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`
`CGT GGA CIT GAG GAC CCC CCT GCC AGT CAG AAG GAG AAG GGG GOT TIT GGG TIC CIG TG
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr>
`
`1050
`*
`
`
`
`*
`
`*
`
`1060
`*
`
`
`
`1070
`x
`
`21080
`*
`
`
`
`*
`
`

`

`
`U.S. Patent
`
`
`
`
`Jul. 4, 2006
`
`
`
`
`Sheet 17 of 55
`
`
`
`US 7,070,959 B1
`
`Fig.13C.
`
`
`
`*
`
`
`
`1030
`*
`
`
`
`
`1100
`*
`
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`
`1110
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`
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`
`
`
`
`
`1120
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`
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`
`
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`
`1130
`*+
`
`
`
`12140
`*
`
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`
`
`
`
`
`
`
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`
`
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`
`
`
`
`
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`
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`
`CTC ATG ATC TCC CGS ACC CCT GAG GIC ACA IGC GIG GIG GTG GAC GIG AGC CAC GAA GAC
`
`
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`GAG TAC TAG AGG GCC TGG GGA CTC CAG TGT ACG CAC CAC CAC CTG CAC TCG GTG CTT CTG
`
`
`
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`
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`Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp>
`
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`
`1150
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`
`1160
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`
`1170
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`
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`CCT GAG GIC AAG TIC AAC TGG TAC GTG GAC GGC GTG GAG GTG CAT AAT GCC AAG ACA AAG
`
`
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`
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`
`
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`
`GGA CTC CAG TTC AAG TIG ACC ATG CAC CTG CCG CAC CIC CAC GTA TTA CGG TMC TCT TIC
`
`
`
`
`
`
`
`
`
`
`
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`
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`
`
`
`
`Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys>
`
`¥*
`
`1180
`*
`
`
`
`*
`
`1190
`*
`
`
`
`1200
`*
`
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`*
`
`
`
`1210
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`
`
`
`
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`
`1220
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`
`
`1230
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`
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`
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`
`1240
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`
`1250
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`
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`
`1260
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`
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`
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`COG CGG GAG GAG CAG TAC AAC AGC ACG TAC CGT GIG GTC AGC GTC Cit ACC GTC Cis CAC
`
`
`
`
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`
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`
`
`
`
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`
`
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`
`Goc Gce CIc ci GTC ATG TIG TCG TGC ATG GCA CAC CAG TCG CAG GAG TGG CAG GAC GTG
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`Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His>
`
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`‘*
`
`
`
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`
`1320
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`
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`
`1270
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`
`1280
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`
`1290
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`
`
`1300
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`
`
`1310
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`
`
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`
`CAG GAC TGG CTG AAT GGC AAG GAG TAC AAG TGC AAG GIC TCC AAC AAA GCC CTC CCA GCC
`
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`GTC CTG ACC GAC TTA COG TTC CI ATG TIC ACG TTC CAG AGG TIG TIT CGG GAG GGT CGG
`
`
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`
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`
`
`
`
`Gin Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala>
`
`
`
`
`
`
`
`1386
`1370
`1360
`1350
`1340
`1330
`
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`
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`
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`
`* «*S4* * * * * * * * *
`
`
`
`
`
`
`
`
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`
`
`
`
`
`
`
`
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`
`
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`
`CCC ATC GAG AAA ACC ATC TCC AAA GCC AAA GGG CAG CCC CGA GAA CCA CAG GIG TAC ACC
`GGG TAG CTC TIT TGS TAG AGG TTT CGG TTT CCC GH GGG GCT CTT GGT GIC CAC ATG TGG
`
`
`
`
`
`
`
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`
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`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr>
`
`*
`
`1390
`*
`
`*
`
`1400
`*
`
`
`
`1410
`*
`
`
`
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`
`
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`
`
`CTG CCC CCA TCC CGG GAT GAG CTG ACC AAG AAC CAG GIC AGC CTG ACC TGC CTG GTC AAA
`
`
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`