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1、.1/1722.The chemical tool of claim 21 wherein the restriction endonuclease for targeting DNA sequencesis selected from the group consisting of:Sequence cutEnzyme Name SEQ ID NO:AAR/AATTYAcsI53R/AATTYApoI53A/AGCTTHindIII54AA/CGTTPsp140 6I55AAT/ATTSspI56ACA/CRYGTAflIII57A/CCGGTAgeI58A/CATGTBspLU11I59A
2、CCTGC(4,8)BspMI60,61ACTGG(1,611)BsrI62,63R/CCGGYBsrFI64R/CCGGYCfr10I64A/CGTMaeII65A/CGCGTMluI66RCATG/YNspI67A/CCGGTPinAI58A/CCWGGTSexAI68A/CTAGTSpeI69AGAGG/CCTAatI70AG/CTAluI71A/GATCTBglII72R/GATCYBstYI73RG/GNCCYDraII74AGC/GCTEco47III75RG/GNCCYEcoO109I74RGCGC/YHaeII76RG/GWCCYPpuMI77AGT/ACTScaI78AGG/
3、CCTStuI70.2/17R/GATCYXhoII73ATAT/TAATAseI79AT/TAATAsnI79AT/CGATBspDI80AT/CGATClaI80ATGCA/TNsiI81ATTT/AAATSwaI82CACAGNNN/CTGAlwNI83CAC/GTGBbrPI84CACNNN/GTGDraIII85YAC/GTRBsaAI86C/AATTGMfeI87C/AATTGMunI87CA/TATGNdeI88CATG/NlaIII89CMG/CKGNspBII90CAC/GTGPmaCI84CAC/GTGPmlI84CAG/CTGPvuII91CR/CCGGYGSgrAI92
4、(13,9)CATCCFokI93,94CCC/CGCAciI95CC/TNAGGAocI96/CCWGGApyI97C/CTAGGAvrII98C/CTAGGBinI98C/CNNGGBsaJI99CCNNNNN/NNGGBsiYI23CCNNNNN/NNGGBslI23CCANNNNN/NTGGBssGI27CC/WGGBstNI100CCANNNNN/NTGGBstXI27CC/TNAGGBsu36I96C/CRYGGDsaI101.3/17C/YCGRGAvaI102CCTNN/NNNAGGEcoNI32/CCWGGEcoRII97C/CGGHpaII103CCGC/GGKspI104
5、CCTC(7,6)MnlI105,106C/CGGMspI103CC/TNAGGMstII96CC/WGGMvaI100CC/SGGNciI107C/CATGGNcoI108CMG/CKGNspBII90CCANNNN/NTGGPflMI44CCGC/GGSacII104CC/TNAGGSauI96CC/NGGScrFI109CCC/GGGSmaI110CCTGCA/GGSse8387I111CCGC/GGSstII104C/CWWGGStyI112CCANNNN/NTGGVan91I44CCANNNNN/NNNNTGGXcmI50C/CCGGGXmaI113C/CCGGGXmaCI113(1
6、,611)CCAGTBsrI114,115CG(10,12)CGANNNNNNTGC(12,10)BcgI116,117CGRY/CGBsiEI118C/GTACGBsiWI119CG/CGBstUI120C/GGCCGEagI121Y/GGCCRCfrI122Y/GGCCREaeI122C/GGCCGEclXI121CGTCTC(1,5)Esp3I123,124CG/CGFnuDII120CG/CGMvnI120CGAT/CGPvuI125CG/GWCCGRsrII126.4/17CR/CCGGYGSgrAI92CG/CGThaI120C/GGCCGXmaIII121CTC/TTAAGAfl
7、II127C/TAGBfaI128C/TTAAGBfrI127CTGGAG(16,14)BpmI129,130C/TNAGDdeI131CTCTTC(1,4)EarI132,133C/YCGRGAvaI102CTGAAG(16,14)Eco57I134,135CTGGAG(16,14)GsuI129,130C/TAGMaeI128C/TCGAGPaeR7I136CTGCA/GPstI137C/TAGRmaI128C/TRYAGSfcI138C/TCGAGXhoI136(14,16)CTCCAGBpmI139,140(14,16)CTGCACBsgI141,142(14,16)CTTCAGEco
8、57I143,144(14,16)CTCCAGGsuI139,140GAGACGT/CAatII145GACN/NNGTCAspI146GAANN/NNTTCAsp7008GACNNN/NNGTCAspEI9GAAGAC(2,6)BbsIBbsI147,148GAAGAC(2,6)BpuAI147,148GATNN/NNATCBsaBI20GAATGC(1,611)BsmI149,150GA/TCDpnI only if151G-Me/GATCDpnII151GACNNNN/NNGTCDrdI28GACNNN/NNGTCEam1105I9GAG/CTCEcl136II152.5/17R/AAT
9、TYAcsI53GR/CGYCAcyI153GR/CGYCAhaII153R/AATTYApoI53GWGCW/CAspHI154GRGCY/CBanII155GDGCH/CBmyI156GR/CGYCBsaHI153GDGCH/CBsp1286I156G/AATTCEcoRI157GAT/ATCEcoRV158GACGC(5,10)HgaI159,160,GWGCW/CHgiAI154G/ANTCHinfI161GATNN/NNATCMamI20/GATCMboI151GAAGA(8,7)MboII162,163/GATCNdeII151GDGCH/CNspII156GAGTC(4,5)Pl
10、eI164,165GAGCT/CSacI166/GATCSau3AI151GAGCT/CSstI166G/AWTCTfiI167GACN/NNGTCTth111I146GAANN/NNTTCXmnI8(9,5)GATGCSfaNI168,169(5,4)GATCCAlwI170,171(5,1)GAGACCBsaI172,173(5,1)GAGACBsmAI174,175(4,1)GAAGAGEarI176,177(5,1)GAGACGEsp3I178,179(6,7)GAGGMnlI180,181(5,4)GACTCPleI182,183GCGCAGC(8,12)BbvI184,185GCC
11、NNNN/NGGCBglI15GC/TNAGCBpu1102I186G/CGCGCBsePI187.6/17G/CGCGCBssHII187GC/TNAGCCelII186GCG/CCfoI188R/CCGGYBsrFI64R/CCGGYCfr10I64GC/TNAGCEspI186GC/NGCFnu4HI189GCG/CHhaI188G/CGCHinPI188GC/NGCItaI189GCC/GGCNaeI190G/CCGGCNgoMI191G/CTAGCNheI192GC/GGCCGCNotI193RCATG/YNspI67GCATC(5,9)SfaNI194,195GCATG/CSphI
12、196GCCC/GGGCSrfI197G/CGGAciI198(12,8)GCTGCBbvI199,200(10,12)GCANNNNNNTCG(12,10)BcgI201,202(1,1)GCATTCBsmI203,204(8,4)GCAGGTBspMI205,206(10,5)GCGTCHgaI207,208GGG/GTACCAcc65I209G/GWCCAflI210GGATC(4,5)AlwI211,212GGGCC/CApaI213GG/CGCGCCAscI214G/GTACCAsp718209G/GWCCAvaII210G/GATCCBamHI215G/GYRCCBanI216GG
13、TCTC(1,5)BsaI217,218G/GTNACCBstEII219R/GATCYBstYI73GR/CGYCAcyI153GR/CGYCAhaII153.7/17GRGCY/CBanII155GDGCH/CBmyI156GR/CGYCBsaHI153GDGCH/CBsp1286I156RG/GNCCYDraII74RG/GNCCYEcoO109I74GGATG(9,13)FokI220,221GGCCGG/CCFseI222RGCGC/YHaeII76GG/CCHaeIII223GGTGA(8,7)HphI224,225G/GCGCCKasI226GGTAC/CKpnI227GG/CG
14、CCNarI228GGN/NCCNlaIV229GDGCH/CNspII156RG/GWCCYPpuMI77G/GNCCSau96I230GGCCNNNN/NGGCCSfiI49R/GATCYXhoII73GTGTGTGT/MKACAccI231G/TGCACAlw44I232G/TGCACApaLI232GTGCAG(16,14)BsgI233,234GTCTC(1,5)BsmAI235,236GTA/TACBst1107I237GWGCW/CAspHI154GDGCH/CBmyI156GDGCH/CBsp1286I156GWGCW/CHgiAI154GTY/RACHincII238GTY/
15、RACHindII238GTT/AACHpaI239/GTNACMaeIII240GDGCH/CNspII156GTTT/AAACPmeI241GTGT/ACRsaI242G/TCGACSalI243.8/17G/TGCACSnoI232(6,2)GTCTTCBbsIBbsI244,245(6,2)GTCTTCBpuAI244,245TAYAC/GTRBsaAI86TAC/GTASnaBI246TCT/CCGGAAccII247T/CCGGABseAI247T/CCGGABspEI247T/CATGABspHI248T/CCGGAMroI247TCG/CGANruI249T/CATGARcaI
16、248T/CGATaqI250T/CTAGAXbaI251(7,8)TCACCHphI252,253(7,8)TCTTCMboII254,255TGTGC/GCAAosI256TGC/GCAAviII256TGG/CCABalI257T/GATCABclI258T/GTACABsp1407I259Y/GGCCRCfrI122Y/GGCCREaeI122TGC/GCAFspI256TGG/CCAMluNI257TGG/CCAMscI257TGC/GCAMstI256T/GTACASspBI259TTTTT/AAAAhaIII260TT/CGAAAsuII261TT/CGAABstBI261TTT
17、/AAADraI260.9/17T/TAAMseI262TT/CGAANspV261TTAAT/TAAPacI263TT/CGAASfuI261T/TAATru9I262去掉切口位置的酶切位点去掉切口位置的酶切位点Sequence cutEnzyme Name SEQ ID NO:AARAATTYAcsI53RAATTYApoI53AAGCTTHindIII54AACGTTPsp140 6I55AATATTSspI56ACACRYGTAflIII57ACCGGTAgeI58ACATGTBspLU11I59ACCTGC(4,8)BspMI60,61ACTGG(1,611)BsrI62,63RCC
18、GGYBsrFI64RCCGGYCfr10I64ACGTMaeII65ACGCGTMluI66RCATGYNspI67ACCGGTPinAI58ACCWGGTSexAI68ACTAGTSpeI69AGAGGCCTAatI70AGCTAluI71AGATCTBglII72RGATCYBstYI73.10/17RGGNCCYDraII74AGCGCTEco47III75RGGNCCYEcoO109I74RGCGCYHaeII76RGGWCCYPpuMI77AGTACTScaI78AGGCCTStuI70RGATCYXhoII73ATATTAATAseI79ATTAATAsnI79ATCGATBsp
19、DI80ATCGATClaI80ATGCATNsiI81ATTTAAATSwaI82CACAGNNNCTGAlwNI83CACGTGBbrPI84CACNNNGTGDraIII85YACGTRBsaAI86CAATTGMfeI87CAATTGMunI87CATATGNdeI88CATGNlaIII89CMGCKGNspBII90CACGTGPmaCI84CACGTGPmlI84CAGCTGPvuII91CRCCGGYGSgrAI92(13,9)CATCCFokI93,94CCCCGCAciI95CCTNAGGAocI96CCWGGApyI97CCTAGGAvrII98CCTAGGBinI98C
20、CNNGGBsaJI99.11/17CCNNNNNNNGGBsiYI23CCNNNNNNNGGBslI23CCANNNNNNTGGBssGI27CCWGGBstNI100CCANNNNNNTGGBstXI27CCTNAGGBsu36I96CCRYGGDsaI101CYCGRGAvaI102CCTNNNNNAGGEcoNI32CCWGGEcoRII97CCGGHpaII103CCGCGGKspI104CCTC(7,6)MnlI105,106CCGGMspI103CCTNAGGMstII96CCWGGMvaI100CCSGGNciI107CCATGGNcoI108CMGCKGNspBII90CCA
21、NNNNNTGGPflMI44CCGCGGSacII104CCTNAGGSauI96CCNGGScrFI109CCCGGGSmaI110CCTGCAGGSse8387I111CCGCGGSstII104CCWWGGStyI112CCANNNNNTGGVan91I44CCANNNNNNNNNTGGXcmI50CCCGGGXmaI113CCCGGGXmaCI113(1,611)CCAGTBsrI114,115CG(10,12)CGANNNNNNTGC(12,10)BcgI116,117CGRYCGBsiEI118CGTACGBsiWI119CGCGBstUI120CGGCCGEagI121YGGC
22、CRCfrI122.12/17YGGCCREaeI122CGGCCGEclXI121CGTCTC(1,5)Esp3I123,124CGCGFnuDII120CGCGMvnI120CGATCGPvuI125CGGWCCGRsrII126CRCCGGYGSgrAI92CGCGThaI120CGGCCGXmaIII121CTCTTAAGAflII127CTAGBfaI128CTTAAGBfrI127CTGGAG(16,14)BpmI129,130CTNAGDdeI131CTCTTC(1,4)EarI132,133CYCGRGAvaI102CTGAAG(16,14)Eco57I134,135CTGGA
23、G(16,14)GsuI129,130CTAGMaeI128CTCGAGPaeR7I136CTGCAGPstI137CTAGRmaI128CTRYAGSfcI138CTCGAGXhoI136(14,16)CTCCAGBpmI139,140(14,16)CTGCACBsgI141,142(14,16)CTTCAGEco57I143,144(14,16)CTCCAGGsuI139,140GAGACGTCAatII145GACNNNGTCAspI146GAANNNNTTCAsp7008GACNNNNNGTCAspEI9GAAGAC(2,6)BbsIBbsI147,148GAAGAC(2,6)BpuA
24、I147,148GATNNNNATCBsaBI20.13/17GAATGC(1,611)BsmI149,150GATCDpnI only if151G-MeGATCDpnII151GACNNNNNNGTCDrdI28GACNNNNNGTCEam1105I9GAGCTCEcl136II152RAATTYAcsI53GRCGYCAcyI153GRCGYCAhaII153RAATTYApoI53GWGCWCAspHI154GRGCYCBanII155GDGCHCBmyI156GRCGYCBsaHI153GDGCHCBsp1286I156GAATTCEcoRI157GATATCEcoRV158GACG
25、C(5,10)HgaI159,160,GWGCWCHgiAI154GANTCHinfI161GATNNNNATCMamI20GATCMboI151GAAGA(8,7)MboII162,163GATCNdeII151GDGCHCNspII156GAGTC(4,5)PleI164,165GAGCTCSacI166GATCSau3AI151GAGCTCSstI166GAWTCTfiI167GACNNNGTCTth111I146GAANNNNTTCXmnI8(9,5)GATGCSfaNI168,169(5,4)GATCCAlwI170,171(5,1)GAGACCBsaI172,173(5,1)GAG
26、ACBsmAI174,175(4,1)GAAGAGEarI176,177(5,1)GAGACGEsp3I178,179(6,7)GAGGMnlI180,181.14/17(5,4)GACTCPleI182,183GCGCAGC(8,12)BbvI184,185GCCNNNNNGGCBglI15GCTNAGCBpu1102I186GCGCGCBsePI187GCGCGCBssHII187GCTNAGCCelII186GCGCCfoI188RCCGGYBsrFI64RCCGGYCfr10I64GCTNAGCEspI186GCNGCFnu4HI189GCGCHhaI188GCGCHinPI188GC
27、NGCItaI189GCCGGCNaeI190GCCGGCNgoMI191GCTAGCNheI192GCGGCCGCNotI193RCATGYNspI67GCATC(5,9)SfaNI194,195GCATGCSphI196GCCCGGGCSrfI197GCGGAciI198(12,8)GCTGCBbvI199,200(10,12)GCANNNNNNTCG(12,10)BcgI201,202(1,1)GCATTCBsmI203,204(8,4)GCAGGTBspMI205,206(10,5)GCGTCHgaI207,208GGGGTACCAcc65I209GGWCCAflI210GGATC(4
28、,5)AlwI211,212GGGCCCApaI213GGCGCGCCAscI214GGTACCAsp718209GGWCCAvaII210.15/17GGATCCBamHI215GGYRCCBanI216GGTCTC(1,5)BsaI217,218GGTNACCBstEII219RGATCYBstYI73GRCGYCAcyI153GRCGYCAhaII153GRGCYCBanII155GDGCHCBmyI156GRCGYCBsaHI153GDGCHCBsp1286I156RGGNCCYDraII74RGGNCCYEcoO109I74GGATG(9,13)FokI220,221GGCCGGCC
29、FseI222RGCGCYHaeII76GGCCHaeIII223GGTGA(8,7)HphI224,225GGCGCCKasI226GGTACCKpnI227GGCGCCNarI228GGNNCCNlaIV229GDGCHCNspII156RGGWCCYPpuMI77GGNCCSau96I230GGCCNNNNNGGCCSfiI49RGATCYXhoII73GTGTGTGTMKACAccI231GTGCACAlw44I232GTGCACApaLI232GTGCAG(16,14)BsgI233,234GTCTC(1,5)BsmAI235,236GTATACBst1107I237GWGCWCAs
30、pHI154GDGCHCBmyI156GDGCHCBsp1286I156GWGCWCHgiAI154GTYRACHincII238.16/17GTYRACHindII238GTTAACHpaI239GTNACMaeIII240GDGCHCNspII156GTTTAAACPmeI241GTGTACRsaI242GTCGACSalI243GTGCACSnoI232(6,2)GTCTTCBbsIBbsI244,245(6,2)GTCTTCBpuAI244,245TAYACGTRBsaAI86TACGTASnaBI246TCTCCGGAAccII247TCCGGABseAI247TCCGGABspEI
31、247TCATGABspHI248TCCGGAMroI247TCGCGANruI249TCATGARcaI248TCGATaqI250TCTAGAXbaI251(7,8)TCACCHphI252,253(7,8)TCTTCMboII254,255TGTGCGCAAosI256TGCGCAAviII256TGGCCABalI257TGATCABclI258TGTACABsp1407I259YGGCCRCfrI122YGGCCREaeI122TGCGCAFspI256TGGCCAMluNI257TGGCCAMscI257TGCGCAMstI256.17/17TGTACASspBI259TTTTTAAAAhaIII260TTCGAAAsuII261TTCGAABstBI261TTTAAADraI260TTAAMseI262TTCGAANspV261TTAATTAAPacI263TTCGAASfuI261TTAATru9I262