| 质粒名称: | pDicAID_nCas9-PmCDA-2A-NptII_ETR |
|---|---|
| 出品公司: | 爱迪基因 |
| 目录编号: | 91695 |
| 存储实验室: | Akihiko Kondo |
|
载体骨架基本信息 |
|
| 载体名称: | pZD_OsU3gYSA_HolgerCas9_NPTII |
| 载体抗性: | 大观霉素 |
| 空载体大小: | 17766 bp |
| 载体修饰: | 引入Cas9（D10A）突变。IV2内含子插入Cas9的5′。 |
| 载体类型: | 植物表达 |
| 筛选标记: | 卡那霉素 |
| 生长条件: | 37 ℃ |
|
插入基因信息 |
|
| 插入基因名称: | SpCas9 |
| 物种来源: | Streptococcus pyogenes |
| 基因突变: | D10A for nickase Cas9 |
| 启动子 | PcUbi |
| 标签/融合蛋白: | PmCDA1（插入件上的C端子） |
| 克隆方法: | Gibson克隆 |
| 5' 测序引物 | GTGCATAGATCTGGATTACATG |
| 3' 测序引物 | GGTCTTGACAAAAAGAACCG |
|
文献引用方法 |
|
| 材料和方法部分: | pDicAID_nCas9-PmCDA-2A-NptII_ETR was a gift from Akihiko Kondo (Addgene plasmid # 91695 ; http://n2t.net/addgene:91695 ; RRID:Addgene_91695) |
| 参考文献部分: | Targeted base editing in rice and tomato using a CRISPR-Cas9 cytidine deaminase fusion. Shimatani Z, Kashojiya S, Takayama M, Terada R, Arazoe T, Ishii H, Teramura H, Yamamoto T, Komatsu H, Miura K, Ezura H, Nishida K, Ariizumi T, Kondo A. Nat Biotechnol. 2017 Mar 27. doi: 10.1038/nbt.3833. 10.1038/nbt.3833 PubMed 28346401 |
买家导航
载体基本信息
载体图谱质粒图谱和多克隆位点信息
载体简介
载体序列
LOCUS pDicAID_nCas9-PmCDA-2A-NptII_ETR 16623 bp ds-DNA circular SYN 06-AUG-2018
DEFINITION Dicot Target-AID vector expressing dicot-optimized
nCas9-PmCDA1-2A-NptII with sgRNA targeting SlEtr.
ACCESSION .
VERSION .
KEYWORDS pDicAID_nCas9-PmCDA-2A-NptII_ETR
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 16623)
AUTHORS Shimatani Z, Kashojiya S, Takayama M, Terada R, Arazoe T, Ishii H,
Teramura H, Yamamoto T, Komatsu H, Miura K, Ezura H, Nishida K,
Ariizumi T, Kondo A
TITLE Targeted base editing in rice and tomato using a CRISPR-Cas9
cytidine deaminase fusion.
JOURNAL Nat Biotechnol. 2017 Mar 27. doi: 10.1038/nbt.3833.
PUBMED 28346401
REFERENCE 2 (bases 1 to 16623)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Aug 6, 2018 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..16623
/organism="synthetic DNA construct"
/mol_type="other DNA"
misc_feature 113..137
/label=RB T-DNA repeat
/note="right border repeat from nopaline C58 T-DNA"
protein_bind 295..316
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 331..361
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 369..385
/label=lac operator
/bound_moiety="lac repressor encoded by lacI"
/note="The lac repressor binds to the lac operator to
inhibit transcription in E. coli. This inhibition can be
relieved by adding lactose or
isopropyl-beta-D-thiogalactopyranoside (IPTG)."
primer_bind 374..396
/label=M13/pUC Reverse
/note="In lacZ gene"
primer_bind 393..409
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind 393..409
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
protein_bind 485..509
/gene="mutant version of attB"
/label=attB1
/bound_moiety="BP Clonase(TM)"
/note="recombination site for the Gateway(R) BP reaction"
primer_bind complement(559..575)
/label=KS primer
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(560..576)
/label=pBluescriptKS
/note="For pBluescript vector"
protein_bind 596..695
/gene="mutant version of attL"
/label=attL1
/bound_moiety="LR Clonase(TM)"
/note="recombination site for the Gateway(R) LR reaction"
misc_RNA 1104..1179
/label=gRNA scaffold
/note="guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system"
protein_bind complement(1199..1223)
/gene="mutant version of attB"
/label=attB2
/bound_moiety="BP Clonase(TM)"
/note="recombination site for the Gateway(R) BP reaction"
protein_bind complement(1229..1328)
/gene="mutant version of attL"
/label=attL2
/bound_moiety="LR Clonase(TM)"
/note="recombination site for the Gateway(R) LR reaction"
protein_bind complement(1451..1575)
/gene="mutant version of attR"
/label=attR4
/bound_moiety="LR Clonase(TM)"
/note="recombination site for the Gateway(R) LR reaction"
CDS 2513..2560
/codon_start=1
/product="bipartite nuclear localization signal from
nucleoplasmin"
/label=nucleoplasmin NLS
/translation="KRPAATKKAGQAKKKK"
intron 2561..2749
/label=IV2 intron
/note="modified second intron of the potato ST-LS1 gene
(Vancanneyt et al., 1990)"
CDS 2765..6865
/codon_start=1
/product="nickase mutant of the Cas9 endonuclease from the
Streptococcus pyogenes Type II CRISPR/Cas system"
/label=Cas9(D10A)
/note="generates RNA-guided single strand nicks in DNA due
to the D10A mutation in the RuvC catalytic domain"
/translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
ATLIHQSITGLYETRIDLSQLGGD"
CDS 6878..6898
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 7091..7156
/codon_start=1
/product="three tandem FLAG(R) epitope tags, followed by an
enterokinase cleavage site"
/label=3xFLAG
/translation="DYKDHDGDYKDHDIDYKDDDDK"
CDS 7163..7786
/codon_start=1
/product="cytidine deaminase from the sea lamprey
Petromyzon marinus (Rogozin et al., 2007)"
/label=PmCDA1
/translation="MTDAEYVRIHEKLDIYTFKKQFFNNKKSVSHRCYVLFELKRRGER
RACFWGYAVNKPQSGTERGIHAEIFSIRKVEEYLRDNPGQFTINWYSSWSPCADCAEKI
LEWYNQELRGNGHTLKIWACKLYYEKNARNQIGLWNLRDNGVGLNVMVSEHYQCCRKIF
IQSSHNQLNENRWLEKTLKRAEKRRSELSIMIQVKILHTTKSPAV"
CDS 7790..7810
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 7883..8680
/codon_start=1
/gene="aph(3')-II (or nptII)"
/product="aminoglycoside phosphotransferase from Tn5"
/label=NeoR/KanR
/note="confers resistance to neomycin, kanamycin, and G418
(Geneticin(R))"
/translation="MGIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGR
PVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLL
SSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEH
QGLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDI
ALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
primer_bind complement(7940..7959)
/label=Neo-R
/note="Neomycin resistance gene, reverse primer"
primer_bind 8550..8569
/label=Neo-F
/note="Neomycin resistance gene, forward primer"
protein_bind complement(9832..9956)
/gene="mutant version of attR"
/label=attR2
/bound_moiety="LR Clonase(TM)"
/note="recombination site for the Gateway(R) LR reaction"
primer_bind complement(9973..9990)
/label=M13 Forward
/note="In lacZ gene. Also called M13-F20 or M13 (-21)
Forward"
primer_bind complement(9973..9989)
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(9982..10004)
/label=M13/pUC Forward
/note="In lacZ gene"
misc_feature 10204..10228
/label=LB T-DNA repeat
/note="left border repeat from nopaline C58 T-DNA"
CDS 10749..11540
/codon_start=1
/gene="aadA"
/product="aminoglycoside adenylyltransferase (Murphy,
1985)"
/label=SmR
/note="confers resistance to spectinomycin and
streptomycin"
/translation="MGEAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
rep_origin 11786..12374
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 12275..12294
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
primer_bind 12528..12545
/label=L4440
/note="L4440 vector, forward primer"
misc_feature 12560..12700
/label=bom
/note="basis of mobility region from pBR322"
primer_bind complement(12626..12645)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 12786..12808
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
rep_origin 13044..13238
/label=pVS1 oriV
/note="origin of replication for the Pseudomonas plasmid
pVS1 (Heeb et al., 2000)"
CDS complement(13304..14377)
/codon_start=1
/product="replication protein from the Pseudomonas plasmid
pVS1 (Heeb et al., 2000)"
/label=pVS1 RepA
/translation="MSGRKPSGPVQIGAALGDDLVEKLKAAQAAQRQRIEAEARPGESW
QAAADRIRKESRQPPAAGAPSIRKPPKGDEQPDFFVPMLYDVGTRDSRSIMDVAVFRLS
KRDRRAGEVIRYELPDGHVEVSAGPAGMASVWDYDLVLMAVSHLTESMNRYREGKGDKP
GRVFRPHVADVLKFCRRADGGKQKDDLVETCIRLNTTHVAMQRTKKAKNGRLVTVSEGE
ALISRYKIVKSETGRPEYIEIELADWMYREITEGKNPDVLTVHPDYFLIDPGIGRFLYR
LARRAAGKAEARWLFKTIYERSGSAGEFKKFCFTVRKLIGSNDLPEYDLKEEAGQAGPI
LVMRYRNLIEGEASAGS"
CDS complement(14806..15435)
/codon_start=1
/product="stability protein from the Pseudomonas plasmid
pVS1 (Heeb et al., 2000)"
/label=pVS1 StaA
/translation="MKVIAVLNQKGGSGKTTIATHLARALQLAGADVLLVDSDPQGSAR
DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP
VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI
LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI"
ORIGIN
1 gatcccgagg ggaaccctgt ggttggcatg cacatacaaa tggacgaacg gataaacctt
61 ttcacgccct tttaaatatc cgattattct aataaacgct cttttctctt aggtttaccc
121 gccaatatat cctgtcaaac actgatagtt taaactgaag gcgggaaacg acaatctgat
181 ccaagctcaa gctccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg
241 cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt
301 gagttagctc actcattagg caccccaggc tttacacttt atgcttccgg ctcgtatgtt
361 gtgtggaatt gtgagcggat aacaatttca cacaggaaac agctatgaca tgattacgaa
421 tttcaggtga ctgatagtga cctgttcgtt gcaacaaatt gatgagcaat gcttttttat
481 aatgccaact ttgtacaaaa aagcaggcgg cgcgtcctgc aggataggga taacagggta
541 atggcgcgcc aagcttatcg ataccgtcga cctcgagcgg ccgcgggaat tcgatcaaat
601 aatgatttta ttttgactga tagtgacctg ttcgttgcaa caaattgatg agcaatgctt
661 ttttataatg ccaactttgt acaaaaaagc aggctctttt tttcttcttc ttcgttcata
721 cagttttttt ttgtttatca gcttacattt tcttgaaccg tagctttcgt tttcttcttt
781 ttaactttcc attcggagtt tttgtatctt gtttcatagt ttgtcccagg attagaatga
841 ttaggcatcg aaccttcaag aatttgattg aataaaacat cttcattctt aagatatgaa
901 gataatcttc aaaaggcccc tgggaatctg aaagaagaga agcaggccca tttatatggg
961 aaagaacaat agtatttctt atataggccc atttaagttg aaaacaatct tcaaaagtcc
1021 cacatcgctt agataagaaa acgaagctga gtttatatac agctagagtc gaagtagtga
1081 ttgtgcacaa gaacccatct atagttttag agctagaaat agcaagttaa aataaggcta
1141 gtccgttatc aacttgaaaa agtggcaccg agtcggtgct ttttttacgc gtttctagac
1201 ccagctttct tgtacaaagt tggcattaac ccagctttct tgtacaaagt tggcattata
1261 aaaaataatt gctcatcaat ttgttgcaac gaacaggtca ctatcagtca aaataaaatc
1321 attatttgat cactagtgga tccgagctcg gtaccttaat taaattaccc tgttatccct
1381 atcctgcagg attaacaccc aacttttcta tggcccctac aggtcactaa taccatctaa
1441 gtagttgatt catagtgact ggatatgttg tgttttacag tattatgtag tctgtttttt
1501 atgcaaaatc taatttaata tattgatatt tatatcattt tacgtttctc gttcaacttt
1561 tctatacaaa gttgaagatc tgactagcaa cgattgtaca attgcttctt taaaaaagga
1621 agaaagaaag aaagaaaaga atcaacatca gcgttaacaa acggccccgt tacggcccaa
1681 acggtcatat agagtaacgg cgttaagcgt tgaaagactc ctatcgaaat acgtaaccgc
1741 aaacgtgtca tagtcagatc ccctcttcct tcaccgcctc aaacacaaaa ataatcttct
1801 acagcctata tatacaaccc ccccttctat ctctcctttc tcacaattca tcatctttct
1861 ttctctaccc ccaattttaa gaaatcctct cttctcctct tcattttcaa ggtaaatctc
1921 tctctctctc tctctctctg ttattccttg ttttaattag gtatgtatta ttgctagttt
1981 gttaatctgc ttatcttatg tatgccttat gtgaatatct ttatcttgtt catctcatcc
2041 gtttagaagc tataaatttg ttgatttgac tgtgtatcta cacgtggtta tgtttatatc
2101 taatcagata tgaatttctt catattgttg cgtttgtgtg taccaatccg aaatcgttga
2161 tttttttcat ttaatcgtgt agctaattgt acgtatacat atggatctac gtatcaattg
2221 ttcatctgtt tgtgtttgta tgtatacaga tctgaaaaca tcacttctct catctgattg
2281 tgttgttaca tacatagata tagatctgtt atatcatttt ttttattaat tgtgtatata
2341 tatatgtgca tagatctgga ttacatgatt gtgattattt acatgatttt gttatttacg
2401 tatgtatata tgtagatctg gactttttgg agttgttgac ttgattgtat ttgtgtgtgt
2461 atatgtgtgt tctgatcttg atatgttatg tatgtgcagc gaattcacca tgaagcgtcc
2521 agctgctacc aagaaagctg gacaggctaa gaagaagaag gtaagtttct gcttctacct
2581 ttgatatata tataataatt atcattaatt agtagtaata taatatttca aatatttttt
2641 tcaaaataaa agaatgtagt atatagcaat tgcttttctg tagtttataa gtgtgtatat
2701 tttaatttat aacttttcta atatatgacc aaaatttgtt gatgtgcagg gccggcgcgc
2761 aacggataag aagtactcta tcggactcgc tatcggaact aactctgtgg gatgggctgt
2821 gatcaccgat gagtacaagg tgccatctaa gaagttcaag gttctcggaa acaccgatag
2881 gcactctatc aagaaaaacc ttatcggtgc tctcctcttc gattctggtg aaactgctga
2941 ggctaccaga ctcaagagaa ccgctagaag aaggtacacc agaagaaaga acaggatctg
3001 ctacctccaa gagatcttct ctaacgagat ggctaaagtg gatgattcat tcttccacag
3061 gctcgaagag tcattcctcg tggaagaaga taagaagcac gagaggcacc ctatcttcgg
3121 aaacatcgtt gatgaggtgg cataccacga gaagtaccct actatctacc acctcagaaa
3181 gaagctcgtt gattctactg ataaggctga tctcaggctc atctacctcg ctctcgctca
3241 catgatcaag ttcagaggac acttcctcat cgagggtgat ctcaaccctg ataactctga
3301 tgtggataag ttgttcatcc agctcgtgca gacctacaac cagcttttcg aagagaaccc
3361 tatcaacgct tcaggtgtgg atgctaaggc tatcctctct gctaggctct ctaagtcaag
3421 aaggcttgag aacctcattg ctcagctccc tggtgagaag aagaacggac ttttcggaaa
3481 cttgatcgct ctctctctcg gactcacccc taacttcaag tctaacttcg atctcgctga
3541 ggatgcaaag ctccagctct caaaggatac ctacgatgat gatctcgata acctcctcgc
3601 tcagatcgga gatcagtacg ctgatttgtt cctcgctgct aagaacctct ctgatgctat
3661 cctcctcagt gatatcctca gagtgaacac cgagatcacc aaggctccac tctcagcttc
3721 tatgatcaag agatacgatg agcaccacca ggatctcaca cttctcaagg ctcttgttag
3781 acagcagctc ccagagaagt acaaagagat tttcttcgat cagtctaaga acggatacgc
3841 tggttacatc gatggtggtg catctcaaga agagttctac aagttcatca agcctatcct
3901 cgagaagatg gatggaaccg aggaactcct cgtgaagctc aatagagagg atcttctcag
3961 aaagcagagg accttcgata acggatctat ccctcatcag atccacctcg gagagttgca
4021 cgctatcctt agaaggcaag aggatttcta cccattcctc aaggataaca gggaaaagat
4081 tgagaagatt ctcaccttca gaatccctta ctacgtggga cctctcgcta gaggaaactc
4141 aagattcgct tggatgacca gaaagtctga ggaaaccatc accccttgga acttcgaaga
4201 ggtggtggat aagggtgcta gtgctcagtc tttcatcgag aggatgacca acttcgataa
4261 gaaccttcca aacgagaagg tgctccctaa gcactctttg ctctacgagt acttcaccgt
4321 gtacaacgag ttgaccaagg ttaagtacgt gaccgaggga atgaggaagc ctgctttttt
4381 gtcaggtgag caaaagaagg ctatcgttga tctcttgttc aagaccaaca gaaaggtgac
4441 cgtgaagcag ctcaaagagg attacttcaa gaaaatcgag tgcttcgatt cagttgagat
4501 ttctggtgtt gaggataggt tcaacgcatc tctcggaacc taccacgatc tcctcaagat
4561 cattaaggat aaggatttct tggataacga ggaaaacgag gatatcttgg aggatatcgt
4621 tcttaccctc accctctttg aagatagaga gatgattgaa gaaaggctca agacctacgc
4681 tcatctcttc gatgataagg tgatgaagca gttgaagaga agaagataca ctggttgggg
4741 aaggctctca agaaagctca ttaacggaat cagggataag cagtctggaa agacaatcct
4801 tgatttcctc aagtctgatg gattcgctaa cagaaacttc atgcagctca tccacgatga
4861 ttctctcacc tttaaagagg atatccagaa ggctcaggtt tcaggacagg gtgatagtct
4921 ccatgagcat atcgctaacc tcgctggatc tcctgcaatc aagaagggaa tcctccagac
4981 tgtgaaggtt gtggatgagt tggtgaaggt gatgggaagg cataagcctg agaacatcgt
5041 gatcgaaatg gctagagaga accagaccac tcagaaggga cagaagaact ctagggaaag
5101 gatgaagagg atcgaggaag gtatcaaaga gcttggatct cagatcctca aagagcaccc
5161 tgttgagaac actcagctcc agaatgagaa gctctacctc tactacctcc agaacggaag
5221 ggatatgtat gtggatcaag agttggatat caacaggctc tctgattacg atgttgatca
5281 tatcgtgcca cagtcattct tgaaggatga ttctatcgat aacaaggtgc tcaccaggtc
5341 tgataagaac aggggtaaga gtgataacgt gccaagtgaa gaggttgtga agaaaatgaa
5401 gaactattgg aggcagctcc tcaacgctaa gctcatcact cagagaaagt tcgataactt
5461 gactaaggct gagaggggag gactctctga attggataag gcaggattca tcaagaggca
5521 gcttgtggaa accaggcaga tcactaagca cgttgcacag atcctcgatt ctaggatgaa
5581 caccaagtac gatgagaacg ataagttgat cagggaagtg aaggttatca ccctcaagtc
5641 aaagctcgtg tctgatttca gaaaggattt ccaattctac aaggtgaggg aaatcaacaa
5701 ctaccaccac gctcacgatg cttaccttaa cgctgttgtt ggaaccgctc tcatcaagaa
5761 gtatcctaag ctcgagtcag agttcgtgta cggtgattac aaggtgtacg atgtgaggaa
5821 gatgatcgct aagtctgagc aagagatcgg aaaggctacc gctaagtatt tcttctactc
5881 taacatcatg aatttcttca agaccgagat taccctcgct aacggtgaga tcagaaagag
5941 gccactcatc gagacaaacg gtgaaacagg tgagatcgtg tgggataagg gaagggattt
6001 cgctaccgtt agaaaggtgc tctctatgcc acaggtgaac atcgttaaga aaaccgaggt
6061 gcagaccggt ggattctcta aagagtctat cctccctaag aggaactctg ataagctcat
6121 tgctaggaag aaggattggg accctaagaa atacggtggt ttcgattctc ctaccgtggc
6181 ttactctgtt ctcgttgtgg ctaaggttga gaagggaaag agtaagaagc tcaagtctgt
6241 taaggaactt ctcggaatca ctatcatgga aaggtcatct ttcgagaaga acccaatcga
6301 tttcctcgag gctaagggat acaaagaggt taagaaggat ctcatcatca agctcccaaa
6361 gtactcactc ttcgaactcg agaacggtag aaagaggatg ctcgcttctg ctggtgagct
6421 tcaaaaggga aacgagcttg ctctcccatc taagtacgtt aactttcttt acctcgcttc
6481 tcactacgag aagttgaagg gatctccaga agataacgag cagaagcaac ttttcgttga
6541 gcagcacaag cactacttgg atgagatcat cgagcagatc tctgagttct ctaaaagggt
6601 gatcctcgct gatgcaaacc tcgataaggt gttgtctgct tacaacaagc acagagataa
6661 gcctatcagg gaacaggcag agaacatcat ccatctcttc acccttacca acctcggtgc
6721 tcctgctgct ttcaagtact tcgatacaac catcgatagg aagagataca cctctaccaa
6781 agaagtgctc gatgctaccc tcatccatca gtctatcact ggactctacg agactaggat
6841 cgatctctca cagctcggtg gtgattcaag ggctgatcct aagaagaaga ggaaggttgg
6901 aggagggcca ggagcagagt atgttagagc gttgtttgat tttaacggta acgatgaaga
6961 ggatttaccc tttaagaaag gcgacattct caggattagg gataaacctg aagagcaatg
7021 gtggaatgct gaggatagtg aaggcaaacg aggaatgatt ttagtgccgt atgtggagaa
7081 atattcgggt gactacaaag atcatgatgg tgattacaaa gaccatgaca tcgactacaa
7141 ggatgatgat gataagtcag ggatgacaga tgctgaatat gtcagaatcc acgaaaagtt
7201 ggacatttac acgtttaaga agcagttctt caacaacaag aaatctgttt cgcataggtg
7261 ctatgtgctt ttcgaactaa aacgtcgtgg agaaagacgg gcttgctttt ggggttacgc
7321 ggttaacaaa ccacaatcag gtactgaacg aggaatacac gctgaaatct tttctatccg
7381 aaaggttgag gaatatctac gtgacaatcc tggacagttc actatcaatt ggtattctag
7441 ctggtcacca tgtgcagatt gtgctgagaa gattctcgaa tggtacaatc aagagcttag
7501 aggcaatgga catacattga aaatatgggc atgcaagctc tactacgaaa agaatgccag
7561 aaaccaaatt gggctttgga acttgaggga taatggagtt gggcttaatg tcatggtttc
7621 tgagcactat caatgttgtc ggaagatctt catacaaagt tcccataacc agttgaatga
7681 gaacagatgg ttagagaaaa cccttaaaag agccgagaag agaagatccg aactgagcat
7741 tatgatacag gtcaaaattc tgcataccac taagagtcca gctgtaggtc ctaagaagaa
7801 acgtaaagta gggcccggac aacttctcaa ctttgacttg ctaaatggtg atgttgaatc
7861 taatcctgga ccaggttcta gaatggggat tgaacaagat ggattgcacg caggttctcc
7921 ggccgcttgg gtggagaggc tattcggcta tgactgggca caacagacaa tcggctgctc
7981 tgatgccgcc gtgtttcggc tgtcagcgca ggggcgcccg gttctttttg tcaagaccga
8041 cctgtccggt gccctgaatg aactccagga cgaggcagcg cggctatcgt ggctggccac
8101 gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa gggactggct
8161 gctattgggc gaagtgccgg ggcaggatct cctgtcatct caccttgctc ctgccgagaa
8221 agtatccatc atggctgatg caatgcggcg gctgcatacg cttgatccgg ctacctgccc
8281 attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg aagccggtct
8341 tgtcgatcag gatgatctgg acgaagagca tcaggggctc gcgccagccg aactgttcgc
8401 caggctcaag gcgcgcatgc ccgacggcga ggatctcgtc gtgacacatg gcgatgcctg
8461 cttgccgaat atcatggtgg aaaatggccg cttttctgga ttcatcgact gtggccggct
8521 gggtgtggcg gaccgctatc aggacatagc gttggctacc cgtgatattg ctgaagagct
8581 tggcggcgaa tgggctgacc gcttcctcgt gctttacggt atcgccgctc ccgattcgca
8641 gcgcatcgcc ttctatcgcc ttctcgacga gttcttctga gagctctggg ctaatctaaa
8701 acgatttatc tgtggcttca agtgtatcga tcacttatgt gaggtgtaat tactggtgtt
8761 tttggtgtgc tctggttcct ttcaagtgtg ttgttgccgc tcgaactact ccgctatgta
8821 aaacggtaaa acctgttgtc tcattatgaa agtgaactat attatgttct actactactc
8881 tacttagtca attttcttca ccttgattag tgtaaatatg aatcttatat tcttatgtct
8941 taagaaatta gcacatgtga agcctccaag tgcatatttt ctcgatcgcg agacgcacaa
9001 tgcgtgagaa attcagctgg ttatactcaa atatattaat atatctagca gcagctcatg
9061 gagattcagg aaacttggca tccctaatcc ctaccatttc cattcttccg agattgacag
9121 ttcaatacaa gtacagtaat ctcctggtaa gtttcttatt aacttgacat gtagtagtaa
9181 taatttgtac gtagcataga tacatagaca caaaaatgtc ctccccattg agctagccga
9241 ttggagccga acaccgcagg aatgaattta cataatctgc aaagaataaa tggaatgtgc
9301 ctccacagga aaaccagcgg cagtgtggcg ttttcaagag cagccgtaag tcgaagccta
9361 ttctgaatcg tagaaatcac tggggcatgg tgtaattaca tccgactcca acatctgtac
9421 caccctgtgc attgtaggcc gctcctctgg caaagaactt acacattgtt tagcaagaga
9481 aagtagagca tccaaggtct caatctgcac tccctcacaa tatggatcga caatttccct
9541 ctcccgattc tcaccgacca ggaaattcaa ctgccacaga gcaagtagat tatttcaaga
9601 atacattaaa tcaattgaag gcatacgtaa ttcatatcag aaaactgtgg atatgaaatg
9661 gaaggacata aaggtcatac atacccatcc aacaatgttc aatccctttt caataaatga
9721 tgcatcagta ggtcgttttc cgcttagtat ttcaagtagc aaaactccaa aactgtagac
9781 gtcagtcttt tcggtggctc tgccactttg catatactcc tgcaggtcga ccatagtgac
9841 tggatatgtt gtgttttaca gtattatgta gtctgttttt tatgcaaaat ctaatttaat
9901 atattgatat ttatatcatt ttacgtttct cgttcagctt tcttgtacaa agtggtgata
9961 tcccagcttg gcactggccg tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac
10021 ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata gcgaagaggc
10081 ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatgct agagcaattc
10141 ggcgttaatt cagtacatta aaaacgtccg caatgtgtta ttaagttgtc taagcgtcaa
10201 tttgtttaca ccacaatata tcctgccacc agccagccaa cagctccccg accggcagct
10261 cggcacaaaa tcaccactcg atacaggcag cccatcagtc cgggacggcg tcagcgggag
10321 agccgttgta aggcggcaga ctttgctcat gttaccgatg ctattcggaa gaacggcaac
10381 taagctgccg ggtttgaaac acggatgatc tcgcggaggg tagcatgttg attgtaacga
10441 tgacagagcg ttgctgcctg tgatcaattc gggcacgaac ccagtggaca taagcctgtt
10501 cggttcgtaa gctgtaatgc aagtagcgta tgcgctcacg caactggtcc agaaccttga
10561 ccgaacgcag cggtggtaac ggcgcagtgg cggttttcat ggcttgttat gactgttttt
10621 ttggggtaca gtctatgcct cgggcatcca agcagcaagc gcgttacgcc gtgggtcgat
10681 gtttgatgtt atggagcagc aacgatgtta cgcagcaggg cagtcgccct aaaacaaagt
10741 taaacatcat gggggaagcg gtgatcgccg aagtatcgac tcaactatca gaggtagttg
10801 gcgtcatcga gcgccatctc gaaccgacgt tgctggccgt acatttgtac ggctccgcag
10861 tggatggcgg cctgaagcca cacagtgata ttgatttgct ggttacggtg accgtaaggc
10921 ttgatgaaac aacgcggcga gctttgatca acgacctttt ggaaacttcg gcttcccctg
10981 gagagagcga gattctccgc gctgtagaag tcaccattgt tgtgcacgac gacatcattc
11041 cgtggcgtta tccagctaag cgcgaactgc aatttggaga atggcagcgc aatgacattc
11101 ttgcaggtat cttcgagcca gccacgatcg acattgatct ggctatcttg ctgacaaaag
11161 caagagaaca tagcgttgcc ttggtaggtc cagcggcgga ggaactcttt gatccggttc
11221 ctgaacagga tctatttgag gcgctaaatg aaaccttaac gctatggaac tcgccgcccg
11281 actgggctgg cgatgagcga aatgtagtgc ttacgttgtc ccgcatttgg tacagcgcag
11341 taaccggcaa aatcgcgccg aaggatgtcg ctgccgactg ggcaatggag cgcctgccgg
11401 cccagtatca gcccgtcata cttgaagcta gacaggctta tcttggacaa gaagaagatc
11461 gcttggcctc gcgcgcagat cagttggaag aatttgtcca ctacgtgaaa ggcgagatca
11521 ccaaggtagt cggcaaataa tgtctagcta gaaattcgtt caagccgacg ccgcttcgcg
11581 gcgcggctta actcaagcgt tagatgcact aagcacataa ttgctcacag ccaaactatc
11641 aggtcaagtc tgcttttatt atttttaagc gtgcataata agccctacac aaattgggag
11701 atatatcatg catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
11761 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
11821 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
11881 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag
11941 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
12001 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
12061 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
12121 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
12181 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
12241 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
12301 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
12361 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
12421 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
12481 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga
12541 gcgaggaagc ggaagagcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt
12601 cacaccgcat atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt
12661 atacactccg ctatcgctac gtgactgggt catggctgcg ccccgacacc cgccaacacc
12721 cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac
12781 cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca
12841 gggtgccttg atgtgggcgc cggcggtcga gtggcgacgg cgcggcttgt ccgcgccctg
12901 gtagattgcc tggccgtagg ccagccattt ttgagcggcc agcggccgcg ataggccgac
12961 gcgaagcggc ggggcgtagg gagcgcagcg accgaagggt aggcgctttt tgcagctctt
13021 cggctgtgcg ctggccagac agttatgcac aggccaggcg ggttttaaga gttttaataa
13081 gttttaaaga gttttaggcg gaaaaatcgc cttttttctc ttttatatca gtcacttaca
13141 tgtgtgaccg gttcccaatg tacggctttg ggttcccaat gtacgggttc cggttcccaa
13201 tgtacggctt tgggttccca atgtacgtgc tatccacagg aaagagacct tttcgacctt
13261 tttcccctgc tagggcaatt tgccctagca tctgctccgt acattaggaa ccggcggatg
13321 cttcgccctc gatcaggttg cggtagcgca tgactaggat cgggccagcc tgccccgcct
13381 cctccttcaa atcgtactcc ggcaggtcat ttgacccgat cagcttgcgc acggtgaaac
13441 agaacttctt gaactctccg gcgctgccac tgcgttcgta gatcgtcttg aacaaccatc
13501 tggcttctgc cttgcctgcg gcgcggcgtg ccaggcggta gagaaaacgg ccgatgccgg
13561 gatcgatcaa aaagtaatcg gggtgaaccg tcagcacgtc cgggttcttg ccttctgtga
13621 tctcgcggta catccaatca gctagctcga tctcgatgta ctccggccgc ccggtttcgc
13681 tctttacgat cttgtagcgg ctaatcaagg cttcaccctc ggataccgtc accaggcggc
13741 cgttcttggc cttcttcgta cgctgcatgg caacgtgcgt ggtgtttaac cgaatgcagg
13801 tttctaccag gtcgtctttc tgctttccgc catcggctcg ccggcagaac ttgagtacgt
13861 ccgcaacgtg tggacggaac acgcggccgg gcttgtctcc cttcccttcc cggtatcggt
13921 tcatggattc ggttagatgg gaaaccgcca tcagtaccag gtcgtaatcc cacacactgg
13981 ccatgccggc cggccctgcg gaaacctcta cgtgcccgtc tggaagctcg tagcggatca
14041 cctcgccagc tcgtcggtca cgcttcgaca gacggaaaac ggccacgtcc atgatgctgc
14101 gactatcgcg ggtgcccacg tcatagagca tcggaacgaa aaaatctggt tgctcgtcgc
14161 ccttgggcgg cttcctaatc gacggcgcac cggctgccgg cggttgccgg gattctttgc
14221 ggattcgatc agcggccgct tgccacgatt caccggggcg tgcttctgcc tcgatgcgtt
14281 gccgctgggc ggcctgcgcg gccttcaact tctccaccag gtcatcaccc agcgccgcgc
14341 cgatttgtac cgggccggat ggtttgcgac cgctcacgcc gattcctcgg gcttgggggt
14401 tccagtgcca ttgcagggcc ggcagacaac ccagccgctt acgcctggcc aaccgcccgt
14461 tcctccacac atggggcatt ccacggcgtc ggtgcctggt tgttcttgat tttccatgcc
14521 gcctccttta gccgctaaaa ttcatctact catttattca tttgctcatt tactctggta
14581 gctgcgcgat gtattcagat agcagctcgg taatggtctt gccttggcgt accgcgtaca
14641 tcttcagctt ggtgtgatcc tccgccggca actgaaagtt gacccgcttc atggctggcg
14701 tgtctgccag gctggccaac gttgcagcct tgctgctgcg tgcgctcgga cggccggcac
14761 ttagcgtgtt tgtgcttttg ctcattttct ctttacctca ttaactcaaa tgagttttga
14821 tttaatttca gcggccagcg cctggacctc gcgggcagcg tcgccctcgg gttctgattc
14881 aagaacggtt gtgccggcgg cggcagtgcc tgggtagctc acgcgctgcg tgatacggga
14941 ctcaagaatg ggcagctcgt acccggccag cgcctcggca acctcaccgc cgatgcgcgt
15001 gcctttgatc gcccgcgaca cgacaaaggc cgcttgtagc cttccatccg tgacctcaat
15061 gcgctgctta accagctcca ccaggtcggc ggtggcccat atgtcgtaag ggcttggctg
15121 caccggaatc agcacgaagt cggctgcctt gatcgcggac acagccaagt ccgccgcctg
15181 gggcgctccg tcgatcacta cgaagtcgcg ccggccgatg gccttcacgt cgcggtcaat
15241 cgtcgggcgg tcgatgccga caacggttag cggttgatct tcccgcacgg ccgcccaatc
15301 gcgggcactg ccctggggat cggaatcgac taacagaaca tcggccccgg cgagttgcag
15361 ggcgcgggct agatgggttg cgatggtcgt cttgcctgac ccgcctttct ggttaagtac
15421 agcgataacc ttcatgcgtt ccccttgcgt atttgtttat ttactcatcg catcatatac
15481 gcagcgaccg catgacgcaa gctgttttac tcaaatacac atcacctttt tagacggcgg
15541 cgctcggttt cttcagcggc caagctggcc ggccaggccg ccagcttggc atcagacaaa
15601 ccggccagga tttcatgcag ccgcacggtt gagacgtgcg cgggcggctc gaacacgtac
15661 ccggccgcga tcatctccgc ctcgatctct tcggtaatga aaaacggttc gtcctggccg
15721 tcctggtgcg gtttcatgct tgttcctctt ggcgttcatt ctcggcggcc gccagggcgt
15781 cggcctcggt caatgcgtcc tcacggaagg caccgcgccg cctggcctcg gtgggcgtca
15841 cttcctcgct gcgctcaagt gcgcggtaca gggtcgagcg atgcacgcca agcagtgcag
15901 ccgcctcttt cacggtgcgg ccttcctggt cgatcagctc gcgggcgtgc gcgatctgtg
15961 ccggggtgag ggtagggcgg gggccaaact tcacgcctcg ggccttggcg gcctcgcgcc
16021 cgctccgggt gcggtcgatg attagggaac gctcgaactc ggcaatgccg gcgaacacgg
16081 tcaacaccat gcggccggcc ggcgtggtgg tgtcggccca cggctctgcc aggctacgca
16141 ggcccgcgcc ggcctcctgg atgcgctcgg caatgtccag taggtcgcgg gtgctgcggg
16201 ccaggcggtc tagcctggtc actgtcacaa cgtcgccagg gcgtaggtgg tcaagcatcc
16261 tggccagctc cgggcggtcg cgcctggtgc cggtgatctt ctcggaaaac agcttggtgc
16321 agccggccgc gtgcagttcg gcccgttggt tggtcaagtc ctggtcgtcg gtgctgacgc
16381 gggcatagcc cagcaggcca gcggcggcgc tcttgttcat ggcgtaatgt ctccggttct
16441 agtcgcaagt attctacttt atgcgactaa aacacgcgac aagaaaacgc caggaaaagg
16501 gcagggcggc agcctgtcgc gtaacttagg acttgtgcga catgtcgttt tcagaagacg
16561 gctgcactga acgtcagaag ccgactgcac tatagcagcg gaggggttgg atcaaagtac
16621 ttt
//
