| 质粒名称: | pCSD_2XNLS-SpCas9WT-NLS-3xHA-2XNLS-NmCas9WT-2XNLS |
|---|---|
| 出品公司: | 爱迪基因 |
| 目录编号: | 107324 |
| 存储实验室: | Scot Wolfe |
|
载体骨架基本信息 |
|
| 载体名称: | pCSDest2 |
| 载体抗性: | 氨苄青霉素 |
| 完整质粒大小: | 11837 bp |
| 载体类型: | 哺乳细胞表达,CRISPR |
| 高拷贝/低拷贝: | 高拷贝 |
| 生长条件: | 37 ℃ |
|
插入基因信息 |
|
| 插入基因名称: | SpCas9-NmCas9 fusion |
| 物种来源: | Synthetic; S. pyogenes, N. meningitidis |
| 插入大小: | 7743 bp |
| 基因突变: | SpCas9 is fused to NmCas9 |
| 启动子 | CMV IE94 |
| 标签/融合蛋白: |
NLS（插入件上的N端子）
NLS（插入件上的C端子）
|
| 克隆方法: | Gibson克隆 |
| 5' 测序引物 | Sp6 |
| 3' 测序引物 | T3 |
|
文献引用方法 |
|
| 材料和方法部分: | pCSD_2XNLS-SpCas9WT-NLS-3xHA-2XNLS-NmCas9WT-2XNLS was a gift from Scot Wolfe (Addgene plasmid # 107324 ; http://n2t.net/addgene:107324 ; RRID:Addgene_107324) |
| 参考文献部分: | Orthogonal Cas9-Cas9 chimeras provide a versatile platform for genome editing. Bolukbasi MF, Liu P, Luk K, Kwok SF, Gupta A, Amrani N, Sontheimer EJ, Zhu LJ, Wolfe SA. Nat Commun. 2018 Nov 19;9(1):4856. doi: 10.1038/s41467-018-07310-x. 10.1038/s41467-018-07310-x PubMed 30451839 |
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 pCSD_2XNLS-SpCas9WT-NLS-3xHA-2XNLS-NmCas9WT-2XNLS 11834 bp ds-DNA circular SYN 12-MAY-2021
DEFINITION Expresses SpCas9 fused to NmCas9 in mammalian cells.
ACCESSION .
VERSION .
KEYWORDS pCSD_2XNLS-SpCas9WT-NLS-3xHA-2XNLS-NmCas9WT-2XNLS
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 11834)
AUTHORS Bolukbasi MF, Liu P, Luk K, Kwok SF, Gupta A, Amrani N, Sontheimer
EJ, Zhu LJ, Wolfe SA
TITLE Orthogonal Cas9-Cas9 chimeras provide a versatile platform for
genome editing.
JOURNAL Nat Commun. 2018 Nov 19;9(1):4856. doi: 10.1038/s41467-018-07310-x.
PUBMED 30451839
REFERENCE 2 (bases 1 to 11834)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..11834
/organism="synthetic DNA construct"
/mol_type="other DNA"
promoter 33..51
/label=SP6 promoter
/note="promoter for bacteriophage SP6 RNA polymerase"
primer_bind 33..50
/label=SP6
/note="SP6 promoter, forward primer"
CDS 101..148
/codon_start=1
/product="bipartite nuclear localization signal from
nucleoplasmin"
/label=nucleoplasmin NLS
/translation="KRPAATKKAGQAKKKK"
CDS 167..187
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 194..4294
/codon_start=1
/product="Cas9 (Csn1) endonuclease from the Streptococcus
pyogenes Type II CRISPR/Cas system"
/label=Cas9
/note="generates RNA-guided double strand breaks in DNA"
/translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
ATLIHQSITGLYETRIDLSQLGGD"
CDS 4307..4327
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 4328..4417
/codon_start=1
/product="three tandem HA epitope tags"
/label=3xHA
/translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
CDS 4430..4456
/codon_start=1
/product="nuclear localization signal (Makkerh et al.,
1996)"
/label=NLS
/translation="PAAKKKKLD"
CDS 4472..4492
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 4493..7735
/codon_start=1
/product="Cas9 endonuclease from the Neisseria meningitidis
Type II CRISPR/Cas system (Esvelt et al., 2013)"
/label=NmCas9
/note="generates RNA-guided double strand breaks in DNA"
/translation="AAFKPNPINYILGLDIGIASVGWAMVEIDEDENPICLIDLGVRVF
ERAEVPKTGDSLAMARRLARSVRRLTRRRAHRLLRARRLLKREGVLQAADFDENGLIKS
LPNTPWQLRAAALDRKLTPLEWSAVLLHLIKHRGYLSQRKNEGETADKELGALLKGVAD
NAHALQTGDFRTPAELALNKFEKESGHIRNQRGDYSHTFSRKDLQAELILLFEKQKEFG
NPHVSGGLKEGIETLLMTQRPALSGDAVQKMLGHCTFEPAEPKAAKNTYTAERFIWLTK
LNNLRILEQGSERPLTDTERATLMDEPYRKSKLTYAQARKLLGLEDTAFFKGLRYGKDN
AEASTLMEMKAYHAISRALEKEGLKDKKSPLNLSPELQDEIGTAFSLFKTDEDITGRLK
DRIQPEILEALLKHISFDKFVQISLKALRRIVPLMEQGKRYDEACAEIYGDHYGKKNTE
EKIYLPPIPADEIRNPVVLRALSQARKVINGVVRRYGSPARIHIETAREVGKSFKDRKE
IEKRQEENRKDREKAAAKFREYFPNFVGEPKSKDILKLRLYEQQHGKCLYSGKEINLGR
LNEKGYVEIDHALPFSRTWDDSFNNKVLVLGSENQNKGNQTPYEYFNGKDNSREWQEFK
ARVETSRFPRSKKQRILLQKFDEDGFKERNLNDTRYVNRFLCQFVADRMRLTGKGKKRV
FASNGQITNLLRGFWGLRKVRAENDRHHALDAVVVACSTVAMQQKITRFVRYKEMNAFD
GKTIDKETGEVLHQKTHFPQPWEFFAQEVMIRVFGKPDGKPEFEEADTPEKLRTLLAEK
LSSRPEAVHEYVTPLFVSRAPNRKMSGQGHMETVKSAKRLDEGVSVLRVPLTQLKLKDL
EKMVNREREPKLYEALKARLEAHKDDPAKAFAEPFYKYDKAGNRTQQVKAVRVEQVQKT
GVWVRNHNGIADNATMVRVDVFEKGDKYYLVPIYSWQVAKGILPDRAVVQGKDEEDWQL
IDDSFNFKFSLHPNDLVEVITKKARMFGYFASCHRGTGNINIRIHDLDHKIGKNGILEG
IGVKTALSFQKYQIDELGKEIRPCRLKKRPPVR"
regulatory 7322..7331
/regulatory_class="other"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
CDS 7748..7768
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 7784..7831
/codon_start=1
/product="bipartite nuclear localization signal from
nucleoplasmin"
/label=nucleoplasmin NLS
/translation="KRPAATKKAGQAKKKK"
promoter complement(7862..7879)
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
polyA_signal 7884..8018
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(7908..7927)
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
primer_bind 7962..7981
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
primer_bind complement(8131..8151)
/label=T3
/note="T3 promoter, forward primer"
promoter complement(8131..8149)
/label=T3 promoter
/note="promoter for bacteriophage T3 RNA polymerase"
primer_bind complement(8170..8186)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(8170..8186)
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind complement(8183..8205)
/label=M13/pUC Reverse
/note="In lacZ gene"
protein_bind 8194..8210
/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)."
promoter complement(8218..8248)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 8263..8284
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(8401..8418)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(8572..9160)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(8652..8671)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS complement(9331..10191)
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/label=AmpR
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
primer_bind 9954..9973
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
promoter complement(10192..10296)
/gene="bla"
/label=AmpR promoter
rep_origin complement(10322..10777)
/direction=LEFT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(10459..10480)
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind 10671..10690
/label=F1ori-R
/note="F1 origin, reverse primer"
promoter 10855..11832
/label=CMV IE94 promoter
/note="enhancer/promoter region of simian cytomegalovirus
major immediate early transcription unit IE94"
ORIGIN
1 ccattctgcc tggggacgtc ggagcaagct tgatttaggt gacactatag aatacaagct
61 acttgttctt tttgcaggat cccatcgatt cgaattcatg aaaaggccgg cggccacgaa
121 aaaggccggc caggcaaaaa agaaaaagct ggacggcagc ggaagcccca agaagaagag
181 gaaggtacca tgggacaaga agtactccat tgggctcgat atcggtacca acagcgtcgg
241 ctgggccgtc attacggacg agtacaaggt gccgagcaaa aaattcaaag ttctgggcaa
301 taccgatcgc cacagcataa agaagaacct cattggagcc ctcctgttcg actccgggga
361 gacggccgaa gccacgcggc tcaaaagaac agcacggcgc agatataccc gcagaaagaa
421 tcggatctgc tacctgcagg agatctttag taatgagatg gctaaggtgg atgactcttt
481 cttccatagg ctggaggagt cctttttggt ggaggaggat aaaaagcacg agcgccaccc
541 aatctttggc aatatcgtgg acgaggtggc gtaccatgaa aagtacccaa ccatatatca
601 tctgaggaag aagctggtag acagtactga taaggctgac ttgcggttga tctatctcgc
661 gctggcgcac atgatcaaat ttcggggaca cttcctcatc gagggggacc tgaacccaga
721 caacagcgat gtcgacaaac tctttatcca actggttcag acttacaatc agcttttcga
781 ggagaacccg atcaacgcat ccggcgttga cgccaaagca atcctgagcg ctaggctgtc
841 caaatcccgg cggctcgaaa acctcatcgc acagctccct ggggagaaga agaacggcct
901 gtttggtaat cttatcgccc tgtcactcgg gctgaccccc aactttaaat ctaacttcga
961 cctggccgaa gatgccaagc tgcaactgag caaagacacc tacgatgatg atctcgacaa
1021 tctgctggcc cagatcggcg accagtacgc agaccttttt ttggcggcaa agaacctgtc
1081 agacgccatt ctgctgagtg atattctgcg agtgaacacg gagatcacca aagctccgct
1141 gagcgctagt atgatcaagc gctatgatga gcaccaccaa gacttgactt tgctgaaggc
1201 ccttgtcaga cagcaactgc ctgagaagta caaggaaatt ttcttcgatc agtctaaaaa
1261 tggctacgcc ggatacattg acggcggagc aagccaggag gaattttaca aatttattaa
1321 gcccatcttg gaaaaaatgg acggcaccga ggagctgctg gtaaagctga acagagaaga
1381 tctgttgcgc aaacagcgca ctttcgacaa tggaagcatc ccccaccaga ttcacctggg
1441 cgaactgcac gctatcctca ggcggcaaga ggatttctac ccctttttga aagataacag
1501 ggaaaagatt gagaaaatcc tcacatttcg gataccctac tatgtaggcc ccctcgctcg
1561 gggaaattcc agattcgcgt ggatgactcg caaatcagaa gagaccatca ctccctggaa
1621 cttcgaggaa gtcgtggata agggggcctc tgcccagtcc ttcatcgaaa ggatgactaa
1681 ctttgataaa aatctgccta acgaaaaggt gcttcctaaa cactctctgc tgtacgagta
1741 cttcacagtt tataacgagc tcaccaaggt caaatacgtc acagaaggga tgagaaagcc
1801 agcattcctg tctggagagc agaagaaagc tatcgtggac ctcctcttca agacgaaccg
1861 gaaagttacc gtgaaacagc tcaaagaaga ctatttcaaa aagattgaat gtttcgactc
1921 tgttgaaatc agcggagtgg aggatcgctt caacgcatcc ctgggaacgt atcacgatct
1981 cctgaaaatc attaaagaca aggacttcct ggacaatgag gagaacgagg acattcttga
2041 ggacattgtc ctcaccctta cgttgtttga agatagggag atgattgaag aacgcttgaa
2101 aacttacgct catctcttcg acgacaaagt catgaaacag ctcaagagac gccgatatac
2161 aggatggggg cggctgtcaa gaaaactgat caatggcatc cgagacaagc agagtggaaa
2221 gacaatcctg gattttctta agtccgatgg atttgccaac cggaacttca tgcagttgat
2281 ccatgatgac tctctcacct ttaaggagga catccagaaa gcacaagttt ctggccaggg
2341 ggacagtctt cacgagcaca tcgctaatct tgcaggtagc ccagctatca aaaagggaat
2401 actgcagacc gttaaggtcg tggatgaact cgtcaaagta atgggaaggc ataagcccga
2461 gaatatcgtt atcgagatgg cccgagagaa ccaaactacc cagaagggac agaagaacag
2521 tagggaaagg atgaagagga ttgaagaggg tataaaagaa ctggggtccc aaatccttaa
2581 ggaacaccca gttgaaaaca cccagcttca gaatgagaag ctctacctgt actacctgca
2641 gaacggcagg gacatgtacg tggatcagga actggacatc aaccggttgt ccgactacga
2701 cgtggatcat atcgtgcccc aaagctttct caaagatgat tctattgata ataaagtgtt
2761 gacaagatcc gataaaaata gagggaagag tgataacgtc ccctcagaag aagttgtcaa
2821 gaaaatgaaa aattattggc ggcagctgct gaacgccaaa ctgatcacac aacggaagtt
2881 cgataatctg actaaggctg aacgaggtgg cctgtctgag ttggataaag ccggcttcat
2941 caaaaggcag cttgttgaga cacgccagat caccaagcac gtggcccaaa ttctcgattc
3001 acgcatgaac accaagtacg atgaaaatga caaactgatt cgagaggtga aagttattac
3061 tctgaagtct aagctggtct cagatttcag aaaggacttt cagttttata aggtgagaga
3121 gatcaacaat taccaccatg cgcatgatgc ctacctgaat gcagtggtag gcactgcact
3181 tatcaaaaaa tatcccaagc tggaatctga atttgtttac ggagactata aagtgtacga
3241 tgttaggaaa atgatcgcaa agtctgagca ggaaataggc aaggccaccg ctaagtactt
3301 cttttacagc aatattatga attttttcaa gaccgagatt acactggcca atggagagat
3361 tcggaagcga ccacttatcg aaacaaacgg agaaacagga gaaatcgtgt gggacaaggg
3421 tagggatttc gcgacagtcc gcaaggtcct gtccatgccg caggtgaaca tcgttaaaaa
3481 gaccgaagta cagaccggag gcttctccaa ggaaagtatc ctcccgaaaa ggaacagcga
3541 caagctgatc gcacgcaaaa aagattggga ccccaagaaa tacggcggat tcgattctcc
3601 tacagtcgct tacagtgtac tggttgtggc caaagtggag aaagggaagt ctaaaaaact
3661 caaaagcgtc aaggaactgc tgggcatcac aatcatggag cgatccagct tcgagaaaaa
3721 ccccatcgac tttctcgaag cgaaaggata taaagaggtc aaaaaagacc tcatcattaa
3781 gctgcccaag tactctctct ttgagcttga aaacggccgg aaacgaatgc tcgctagtgc
3841 gggcgagctg cagaaaggta acgagctggc actgccctct aaatacgtta atttcttgta
3901 tctggccagc cactatgaaa agctcaaagg gtctcccgaa gataatgagc agaagcagct
3961 gttcgtggaa caacacaaac actaccttga tgagatcatc gagcaaataa gcgagttctc
4021 caaaagagtg atcctcgccg acgctaacct cgataaggtg ctttctgctt acaataagca
4081 cagggataag cccatcaggg agcaggcaga aaacattatc cacttgttta ctctgaccaa
4141 cttgggcgcg cctgcagcct tcaagtactt cgacaccacc atagacagaa agcggtacac
4201 ctctacaaag gaggtcctgg acgccacact gattcatcag tcaattacgg ggctctatga
4261 aacaagaatc gacctctctc agctcggtgg agacggcacc ggcgggccca agaagaagag
4321 gaaggtatac ccatacgatg ttcctgacta tgcgggctat ccctatgacg tcccggacta
4381 tgcaggatcg tatccttatg acgttccaga ttacgctgga tccgccgctc cggcagctaa
4441 gaaaaagaaa ctggatttcg aatccggaat gcccaagaag aagaggaagg tggccgcctt
4501 caagcccaac cccatcaact acatcctggg cctggacatc ggcatcgcca gcgtgggctg
4561 ggccatggtg gagatcgacg aggacgagaa ccccatctgc ctgatcgacc tgggtgtgcg
4621 cgtgttcgag cgcgctgagg tgcccaagac tggtgacagt ctggctatgg ctcgccggct
4681 tgctcgctct gttcggcgcc ttactcgccg gcgcgctcac cgccttctgc gcgctcgccg
4741 cctgctgaag cgcgagggtg tgctgcaggc tgccgacttc gacgagaacg gcctgatcaa
4801 gagcctgccc aacactcctt ggcagctgcg cgctgccgct ctggaccgca agctgactcc
4861 tctggagtgg agcgccgtgc tgctgcacct gatcaagcac cgcggctacc tgagccagcg
4921 caagaacgag ggcgagaccg ccgacaagga gctgggtgct ctgctgaagg gcgtggccga
4981 caacgcccac gccctgcaga ctggtgactt ccgcactcct gctgagctgg ccctgaacaa
5041 gttcgagaag gagagcggcc acatccgcaa ccagcgcggc gactacagcc acaccttcag
5101 ccgcaaggac ctgcaggccg agctgatcct gctgttcgag aagcagaagg agttcggcaa
5161 cccccacgtg agcggcggcc tgaaggaggg catcgagacc ctgctgatga cccagcgccc
5221 cgccctgagc ggcgacgccg tgcagaagat gctgggccac tgcaccttcg agccagccga
5281 gcccaaggcc gccaagaaca cctacaccgc cgagcgcttc atctggctga ccaagctgaa
5341 caacctgcgc atcctggagc agggcagcga gcgccccctg accgacaccg agcgcgccac
5401 cctgatggac gagccctacc gcaagagcaa gctgacctac gcccaggccc gcaagctgct
5461 gggtctggag gacaccgcct tcttcaaggg cctgcgctac ggcaaggaca acgccgaggc
5521 cagcaccctg atggagatga aggcctacca cgccatcagc cgcgccctgg agaaggaggg
5581 cctgaaggac aagaagagtc ctctgaacct gagccccgag ctgcaggacg agatcggcac
5641 cgccttcagc ctgttcaaga ccgacgagga catcaccggc cgcctgaagg accgcatcca
5701 gcccgagatc ctggaggccc tgctgaagca catcagcttc gacaagttcg tgcagatcag
5761 cctgaaggcc ctgcgccgca tcgtgcccct gatggagcag ggcaagcgct acgacgaggc
5821 ctgcgccgag atctacggcg accactacgg caagaagaac accgaggaga agatctacct
5881 gcctcctatc cccgccgacg agatccgcaa ccccgtggtg ctgcgcgccc tgagccaggc
5941 ccgcaaggtg atcaacggcg tggtgcgccg ctacggcagc cccgcccgca tccacatcga
6001 gaccgcccgc gaggtgggca agagcttcaa ggaccgcaag gagatcgaga agcgccagga
6061 ggagaaccgc aaggaccgcg agaaggccgc cgccaagttc cgcgagtact tccccaactt
6121 cgtgggcgag cccaagagca aggacatcct gaagctgcgc ctgtacgagc agcagcacgg
6181 caagtgcctg tacagcggca aggagatcaa cctgggccgc ctgaacgaga agggctacgt
6241 ggagatcgac catgccctgc ccttcagccg cacctgggac gacagcttca acaacaaggt
6301 gctggtgctg ggcagcgaga atcagaacaa gggcaaccag accccctacg agtacttcaa
6361 cggcaaggac aacagccgcg agtggcagga gttcaaggcc cgcgtggaga ccagccgctt
6421 cccccgcagc aagaagcagc gcatcctgct gcagaagttc gacgaggacg gcttcaagga
6481 gcgcaacctg aacgacaccc gctacgtgaa ccgcttcctg tgccagttcg tggccgaccg
6541 catgcgcctg accggcaagg gcaagaagcg cgtgttcgcc agcaacggcc agatcaccaa
6601 cctgctgcgc ggcttctggg gcctgcgcaa ggtgcgcgcc gagaacgacc gccaccacgc
6661 cctggacgcc gtggtggtgg cctgcagcac cgtggccatg cagcagaaga tcacccgctt
6721 cgtgcgctac aaggagatga acgccttcga cggtaaaacc atcgacaagg agaccggcga
6781 ggtgctgcac cagaagaccc acttccccca gccctgggag ttcttcgccc aggaggtgat
6841 gatccgcgtg ttcggcaagc ccgacggcaa gcccgagttc gaggaggccg acacccccga
6901 gaagctgcgc accctgctgg ccgagaagct gagcagccgc cctgaggccg tgcacgagta
6961 cgtgactcct ctgttcgtga gccgcgcccc caaccgcaag atgagcggtc agggtcacat
7021 ggagaccgtg aagagcgcca agcgcctgga cgagggcgtg agcgtgctgc gcgtgcccct
7081 gacccagctg aagctgaagg acctggagaa gatggtgaac cgcgagcgcg agcccaagct
7141 gtacgaggcc ctgaaggccc gcctggaggc ccacaaggac gaccccgcca aggccttcgc
7201 cgagcccttc tacaagtacg acaaggccgg caaccgcacc cagcaggtga aggccgtgcg
7261 cgtggagcag gtgcagaaga ccggcgtgtg ggtgcgcaac cacaacggca tcgccgacaa
7321 cgccaccatg gtgcgcgtgg acgtgttcga gaagggcgac aagtactacc tggtgcccat
7381 ctacagctgg caggtggcca agggcatcct gcccgaccgc gccgtggtgc agggcaagga
7441 cgaggaggac tggcagctga tcgacgacag cttcaacttc aagttcagcc tgcaccccaa
7501 cgacctggtg gaggtgatca ccaagaaggc ccgcatgttc ggctacttcg ccagctgcca
7561 ccgcggcacc ggcaacatca acatccgcat ccacgacctg gaccacaaga tcggcaagaa
7621 cggcatcctg gagggcatcg gcgtgaagac cgccctgagc ttccagaagt accagatcga
7681 cgagctgggc aaggagatcc gcccctgccg cctgaagaag cgccctcctg tgcgcagcag
7741 ggctgacccc aagaagaaga ggaaggtgga ggccagcctc gagaaaaggc cggcggccac
7801 gaaaaaggcc ggccaggcaa aaaagaaaaa gggcagttaa tgatctagta atgatctaga
7861 actatagtga gtcgtattac gtagatccag acatgataag atacattgat gagtttggac
7921 aaaccacaac tagaatgcag tgaaaaaaat gctttatttg tgaaatttgt gatgctattg
7981 ctttatttgt aaccattata agctgcaata aacaagttaa caacaacaat tgcattcatt
8041 ttatgtttca ggttcagggg gaggtgtggg aggtttttta attcgcggcc gcggcgccaa
8101 tgcattgggc ccggtaccca gcttttgttc cctttagtga gggttaattg cgcgcttggc
8161 gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa
8221 catacgagcc gggagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac
8281 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
8341 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc
8401 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
8461 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
8521 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
8581 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
8641 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
8701 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
8761 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
8821 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
8881 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
8941 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
9001 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
9061 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
9121 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
9181 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
9241 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
9301 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
9361 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
9421 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg
9481 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
9541 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
9601 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
9661 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
9721 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
9781 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
9841 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
9901 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
9961 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
10021 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
10081 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
10141 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct
10201 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
10261 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc
10321 taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca
10381 ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag
10441 atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc
10501 aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc
10561 taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc
10621 ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa
10681 gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc
10741 acacccgccg cgcttaatgc gccgctacag ggcgcgtccc attcgccatt caggctgcgc
10801 aactgttggg aagggcgatc ggtgcgggcc tcttcgctat tacgccagtc gaccgccaat
10861 tcaatatggc gtatatggac tcatgccaat tcaatatggt ggatctggac ctgtgccaat
10921 tcaatatggc gtatatggac tcgtgccaat tcaatatggt ggatctggac cccagccaat
10981 tcaatatggc ggacttggca ccatgccaat tcaatatggc ggacctggca ctgtgccaac
11041 tggggagggg tctacttggc acggtgccaa gtttgaggag gggtcttggc cctgtgccaa
11101 gtccgccata ttgaattggc atggtgccaa taatggcggc catattggct atatgccagg
11161 atcaatatat aggcaatatc caatatggcc ctatgccaat atggctattg gccaggttca
11221 atactatgta ttggccctat gccatatagt attccatata tgggttttcc tattgacgta
11281 gatagcccct cccaatgggc ggtcccatat accatatatg gggcttccta ataccgccca
11341 tagccactcc cccattgacg tcaatggtct ctatatatgg tctttcctat tgacgtcata
11401 tgggcggtcc tattgacgta tatggcgcct cccccattga cgtcaattac ggtaaatggc
11461 ccgcctggct caatgcccat tgacgtcaat aggaccaccc accattgacg tcaatgggat
11521 ggctcattgc ccattcatat ccgttctcac gccccctatt gacgtcaatg acggtaaatg
11581 gcccacttgg cagtacatca atatctatta atagtaactt ggcaagtaca ttactattgg
11641 aagtacgcca gggtacattg gcagtactcc cattgacgtc aatggcggta aatggcccgc
11701 gatggctgcc aagtacatcc ccattgacgt caatggggag gggcaatgac gcaaatgggc
11761 gttccattga cgtaaatggg cggtaggcgt gcctaatggg aggtctatat aagcaatgct
11821 cgtttaggga accg
//
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