| 质粒名称: | pEJS578_DD-dSpyCas9-mCherry-APEX2 |
|---|---|
| 出品公司： | addgene |
| 目录编号: | 108570 |
| 存储实验室: | Erik Sontheimer |
|
载体骨架基本信息 |
|
| 载体名称: | pHAGE-DEST |
| 空载体大小: | 6000bp |
| 完整质粒大小: | 12774bp |
| 载体类型: | 哺乳细胞表达 |
| 载体抗性: | 氨苄霉素 |
| 生长温度: | 37 ℃ |
| 宿主菌: | NEB Stable |
| 拷贝数: | 高拷贝 |
|
插入基因信息 |
|
| 插入基因名称: | dSpyCas9 |
| 物种来源: | Synthetic |
| 插入大小: | 4113 bp |
| 标签/融合蛋白: |
(C terminal on insert)
APEX2 (C terminal on insert)
|
| 克隆方法: | 限制性内切酶 |
| 5’克隆位点: | MluI(未破坏) |
| 3’克隆位点: | SpeI(未破坏) |
| 5’测序引物 | cgcaaatgggcggtaggcgtg |
|
文献引用方法 |
|
| 材料和方法部分: | pEJS578_DD-dSpyCas9-mCherry-APEX2 was a gift from Erik Sontheimer (Addgene plasmid # 108570 ; http://n2t.net/addgene:108570 ; RRID:Addgene_108570) |
| 参考文献部分: | C-BERST: defining subnuclear proteomic landscapes at genomic elements with dCas9–APEX2. Gao , Xin D., Li-Chun Tu, Aamir Mir, Tomás Rodriguez, Yuehe Ding, John Leszyk, Job Dekker, Scott A. Shaffer, Lihua Julie Zhu, Scot A. Wolfe & Erik J. Sontheimer. Nature Methods (2018) 10.1038/s4159 |
买家导航
pEJS578_DD-dSpyCas9-mCherry-APEX2载体质粒基本信息
pEJS578_DD-dSpyCas9-mCherry-APEX2质粒图谱载体图谱和pEJS578_DD-dSpyCas9-mCherry-APEX2载体序列质粒序列多克隆位点信息
pEJS578_DD-dSpyCas9-mCherry-APEX2质粒载体简介
pEJS578_DD-dSpyCas9-mCherry-APEX2质粒序列载体序列
LOCUS pEJS578_DD-dSpyCas9-mCherry-APEX2 12774 bp ds-DNA circular SYN 12-MAY-2021
DEFINITION Expression of drug-inducible dSpyCas9-mCherry-APEX2 for
biotin-labeling genomic element-associated protein factors.
ACCESSION .
VERSION .
KEYWORDS pEJS578_DD-dSpyCas9-mCherry-APEX2
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 12774)
AUTHORS Gao , Xin D., Li-Chun Tu, Aamir Mir, Tomas Rodriguez, Yuehe Ding,
John Leszyk, Job Dekker, Scott A. Shaffer, Lihua Julie Zhu, Scot A.
TITLE C-BERST: defining subnuclear proteomic landscapes at genomic
elements with dCas9–APEX2
JOURNAL Nature Methods (2018)
REFERENCE 2 (bases 1 to 12774)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58
http://www.biofeng.com
FEATURES Location/Qualifiers
source 1..12774
/organism="synthetic DNA construct"
/mol_type="other DNA"
LTR 1..634
/label=3' LTR
/note="3' long terminal repeat (LTR) from HIV-1"
misc_feature 681..806
/label=HIV-1 Psi
/note="packaging signal of human immunodeficiency virus
type 1"
misc_feature 1303..1536
/label=RRE
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
CDS 1721..1765
/codon_start=1
/product="antigenic peptide corresponding to amino acids
655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik
et al., 2013)"
/label=gp41 peptide
/note="recognized by the 2H10 single-chain llama nanobody"
/translation="KNEQELLELDKWASL"
misc_feature 2058..2175
/label=cPPT/CTS
/note="central polypurine tract and central termination
sequence of HIV-1"
enhancer 2299..2602
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 2603..2806
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
primer_bind 2757..2777
/label=CMV-F
/note="Human CMV immediate early promoter, forward primer"
protein_bind 2808..2826
/gene="tetO"
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O2 for the tetR and tetA genes"
protein_bind 2829..2847
/gene="tetO"
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O2 for the tetR and tetA genes"
primer_bind 2857..2881
/label=LNCX
/note="Human CMV promoter, forward primer"
regulatory 2965..2974
/regulatory_class="other"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
CDS 2977..2997
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 3004..3051
/codon_start=1
/product="bipartite nuclear localization signal from
nucleoplasmin"
/label=nucleoplasmin NLS
/translation="KRPAATKKAGQAKKKK"
CDS 3064..3384
/codon_start=1
/product="destabilization domain that can be stabilized by
Shield1 in the ProteoTuner(TM) system"
/label=DD
/note="L106P mutant of FKBP12"
/translation="GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKP
FKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELL
KPE"
CDS 3409..7509
/codon_start=1
/product="catalytically dead mutant of the Cas9
endonuclease from the Streptococcus pyogenes Type II
CRISPR/Cas system"
/label=dCas9
/note="RNA-guided DNA-binding protein that lacks
endonuclease activity due to the D10A mutation in the RuvC
catalytic domain and the H840A mutation in the HNH
catalytic domain"
/translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
ATLIHQSITGLYETRIDLSQLGGD"
CDS 7528..7548
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 7561..8268
/codon_start=1
/product="monomeric derivative of DsRed fluorescent protein
(Shaner et al., 2004)"
/label=mCherry
/note="mammalian codon-optimized"
/translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
EGRHSTGGMDELYK"
primer_bind complement(7706..7724)
/label=mCherry-R
/note="mCherry, reverse primer"
primer_bind 7974..7993
/label=mCherry-F
/note="mCherry, forward primer"
CDS 8296..8319
/codon_start=1
/product="FLAG(R) epitope tag, followed by an enterokinase
cleavage site"
/label=FLAG
/translation="DYKDDDDK"
CDS 8320..9066
/codon_start=1
/product="soybean ascorbate peroxidase, engineered to serve
as a monomeric tag for electron microscopy and live-cell
proteomics (Lam et al., 2015)"
/label=APEX2
/note="more active than the original APEX due to the A134P
mutation"
/translation="GKSYPTVSADYQDAVEKAKKKLRGFIAEKRCAPLMLRLAFHSAGT
FDKGTKTGGPFGTIKHPAELAHSANNGLDIAVRLLEPLKAEFPILSYADFYQLAGVVAV
EVTGGPKVPFHPGREDKPEPPPEGRLPDPTKGSDHLRDVFGKAMGLTDQDIVALSGGHT
IGAAHKERSGFEGPWTSNPLIFDNSYFTELLSGEKEGLLQLPSDKALLSDPVFRPLVDK
YAADEDAFFADYAEAHQKLSELGFADA"
CDS 9073..9093
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 9100..9147
/codon_start=1
/product="bipartite nuclear localization signal from
nucleoplasmin"
/label=nucleoplasmin NLS
/translation="KRPAATKKAGQAKKKK"
misc_feature 9164..9752
/label=WPRE
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
primer_bind complement(9217..9237)
/label=WPRE-R
/note="WPRE, reverse primer"
CDS complement(9635..9646)
/codon_start=1
/product="Factor Xa recognition and cleavage site"
/label=Factor Xa site
/translation="IEGR"
LTR 9827..10060
/label=3' LTR (Delta-U3)
/note="self-inactivating 3' long terminal repeat (LTR) from
HIV-1"
primer_bind complement(10179..10197)
/label=pBRforEco
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
promoter 10265..10369
/gene="bla"
/label=AmpR promoter
CDS 10370..11230
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/label=AmpR
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
primer_bind complement(10588..10607)
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
rep_origin 11401..11989
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 11890..11909
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
primer_bind 12143..12160
/label=L4440
/note="L4440 vector, forward primer"
primer_bind 12252..12271
/label=SV40pro-F
/note="SV40 promoter/origin, forward primer"
protein_bind 12400..12421
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 12436..12466
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 12474..12490
/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 12479..12501
/label=M13/pUC Reverse
/note="In lacZ gene"
primer_bind 12498..12514
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind 12498..12514
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind 12564..12583
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
ORIGIN
1 tggaagggct aattcactcc caaagaagac aagatatcct tgatctgtgg atctaccaca
61 cacaaggcta cttccctgat tagcagaact acacaccagg gccaggggtc agatatccac
121 tgacctttgg atggtgctac aagctagtac cagttgagcc agataaggta gaagaggcca
181 ataaaggaga gaacaccagc ttgttacacc ctgtgagcct gcatgggatg gatgacccgg
241 agagagaagt gttagagtgg aggtttgaca gccgcctagc atttcatcac gtggcccgag
301 agctgcatcc ggagtacttc aagaactgct gatatcgagc ttgctacaag ggactttccg
361 ctggggactt tccagggagg cgtggcctgg gcgggactgg ggagtggcga gccctcagat
421 cctgcatata agcagctgct ttttgcctgt actgggtctc tctggttaga ccagatctga
481 gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct
541 tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc
601 agaccctttt agtcagtgtg gaaaatctct agcagtggcg cccgaacagg gacttgaaag
661 cgaaagggaa accagaggag ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg
721 caagaggcga ggggcggcga ctggtgagta cgccaaaaat tttgactagc ggaggctaga
781 aggagagaga tgggtgcgag agcgtcagta ttaagcgggg gagaattaga tcgcgatggg
841 aaaaaattcg gttaaggcca gggggaaaga aaaaatataa attaaaacat atagtatggg
901 caagcaggga gctagaacga ttcgcagtta atcctggcct gttagaaaca tcagaaggct
961 gtagacaaat actgggacag ctacaaccat cccttcagac aggatcagaa gaacttagat
1021 cattatataa tacagtagca accctctatt gtgtgcatca aaggatagag ataaaagaca
1081 ccaaggaagc tttagacaag atagaggaag agcaaaacaa aagtaagacc accgcacagc
1141 aagcggccgg ccgctgatct tcagacctgg aggaggagat atgagggaca attggagaag
1201 tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac ccaccaaggc
1261 aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt tgttccttgg
1321 gttcttggga gcagcaggaa gcactatggg cgcagcgtca atgacgctga cggtacaggc
1381 cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg ctattgaggc
1441 gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg caagaatcct
1501 ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt gctctggaaa
1561 actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat ctctggaaca
1621 gatttggaat cacacgacct ggatggagtg ggacagagaa attaacaatt acacaagctt
1681 aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac aagaattatt
1741 ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt ggctgtggta
1801 tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag tttttgctgt
1861 actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc agacccacct
1921 cccaaccccg aggggacccg acaggcccga aggaatagaa gaagaaggtg gagagagaga
1981 cagagacaga tccattcgat tagtgaacgg atctcgacgg tatcgccgaa ttgacaaatg
2041 gcagtattca tccacaattt taaaagaaaa ggggggattg gggggtacag tgcaggggaa
2101 agaatagtag acataatagc aacagacata caaactaaag aattacaaaa acaaattaca
2161 aaaattcaaa attttcgggt ttattacagg gacagcagag atccagtttg gactagcgat
2221 gctagctgat gcgggcccta gttattaata gtaatcaatt acggggtcat tagttcatag
2281 cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc
2341 caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg
2401 gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca
2461 tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc
2521 ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt
2581 attagtcatc gctattacca tggtgatgcg gttttggcag tacatcaatg ggcgtggata
2641 gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt
2701 ttggcaccaa aatcaacggg actttccaaa atgtcgtaac aactccgccc cattgacgca
2761 aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agagctctcc ctatcagtga
2821 tagagatctc cctatcagtg atagagatcg tcgacgagct cgtttagtga accgtcagat
2881 cgcctggaga cgccatccac gctgttttga cctccataga agacaccggg accgatccag
2941 cctaccggtt aaggtgaacg cgtggccacc atggccccta aaaagaaacg caaggtcgaa
3001 gataaacgcc cagcagctac aaagaaggca ggtcaagcca agaaaaagaa ggaagatgca
3061 tgcggagtgc aggtggaaac catctcccca ggagacgggc gcaccttccc caagcgcggc
3121 cagacctgtg tggtgcacta caccgggatg cttgaagatg gaaagaaagt cgattcctcc
3181 cgggacagaa acaagccctt taagtttatg ctaggcaagc aggaggtgat ccgaggctgg
3241 gaagaagggg ttgcccagat gagtgtgggt cagagagcca aactgactat atctccagat
3301 tatgcctatg gtgccactgg gcacccaggc atcatcccac cacatgccac tctcgtcttc
3361 gatgtggagc ttctaaaacc ggaaggcagt gaattcggaa gcggaagcga caagaagtac
3421 tccattgggc tcgctatcgg taccaacagc gtcggctggg ccgtcattac ggacgagtac
3481 aaggtgccga gcaaaaaatt caaagttctg ggcaataccg atcgccacag cataaagaag
3541 aacctcattg gagccctcct gttcgactcc ggggagacgg ccgaagccac gcggctcaaa
3601 agaacagcac ggcgcagata tacccgcaga aagaatcgga tctgctacct gcaggagatc
3661 tttagtaatg agatggctaa ggtggatgac tctttcttcc ataggctgga ggagtccttt
3721 ttggtggagg aggataaaaa gcacgagcgc cacccaatct ttggcaatat cgtggacgag
3781 gtggcgtacc atgaaaagta cccaaccata tatcatctga ggaagaagct ggtagacagt
3841 actgataagg ctgacttgcg gttgatctat ctcgcgctgg cgcacatgat caaatttcgg
3901 ggacacttcc tcatcgaggg ggacctgaac ccagacaaca gcgatgtcga caaactcttt
3961 atccaactgg ttcagactta caatcagctt ttcgaggaga acccgatcaa cgcatccggc
4021 gttgacgcca aagcaatcct gagcgctagg ctgtccaaat cccggcggct cgaaaacctc
4081 atcgcacagc tccctgggga gaagaagaac ggcctgtttg gtaatcttat cgccctgtca
4141 ctcgggctga cccccaactt taaatctaac ttcgacctgg ccgaagatgc caagctgcaa
4201 ctgagcaaag acacctacga tgatgatctc gacaatctgc tggcccagat cggcgaccag
4261 tacgcagacc tttttttggc ggcaaagaac ctgtcagacg ccattctgct gagtgatatt
4321 ctgcgagtga acacggagat caccaaagct ccgctgagcg ctagtatgat caagcgctat
4381 gatgagcacc accaagactt gactttgctg aaggcccttg tcagacagca actgcctgag
4441 aagtacaagg aaattttctt cgatcagtct aaaaatggct acgccggata cattgacggc
4501 ggagcaagcc aggaggaatt ttacaaattt attaagccca tcttggaaaa aatggacggc
4561 accgaggagc tgctggtaaa gctgaacaga gaagatctgt tgcgcaaaca gcgcactttc
4621 gacaatggaa gcatccccca ccagattcac ctgggcgaac tgcacgctat cctcaggcgg
4681 caagaggatt tctacccctt tttgaaagat aacagggaaa agattgagaa aatcctcaca
4741 tttcggatac cctactatgt aggccccctc gctcggggaa attccagatt cgcgtggatg
4801 actcgcaaat cagaagagac catcactccc tggaacttcg aggaagtcgt ggataagggg
4861 gcctctgccc agtccttcat cgaaaggatg actaactttg ataaaaatct gcctaacgaa
4921 aaggtgcttc ctaaacactc tctgctgtac gagtacttca cagtttataa cgagctcacc
4981 aaggtcaaat acgtcacaga agggatgaga aagccagcat tcctgtctgg agagcagaag
5041 aaagctatcg tggacctcct cttcaagacg aaccggaaag ttaccgtgaa acagctcaaa
5101 gaagactatt tcaaaaagat tgaatgtttc gactctgttg aaatcagcgg agtggaggat
5161 cgcttcaacg catccctggg aacgtatcac gatctcctga aaatcattaa agacaaggac
5221 ttcctggaca atgaggagaa cgaggacatt cttgaggaca ttgtcctcac ccttacgttg
5281 tttgaagata gggagatgat tgaagaacgc ttgaaaactt acgctcatct cttcgacgac
5341 aaagtcatga aacagctcaa gagacgccga tatacaggat gggggcggct gtcaagaaaa
5401 ctgatcaatg gcatccgaga caagcagagt ggaaagacaa tcctggattt tcttaagtcc
5461 gatggatttg ccaaccggaa cttcatgcag ttgatccatg atgactctct cacctttaag
5521 gaggacatcc agaaagcaca agtttctggc cagggggaca gtcttcacga gcacatcgct
5581 aatcttgcag gtagcccagc tatcaaaaag ggaatactgc agaccgttaa ggtcgtggat
5641 gaactcgtca aagtaatggg aaggcataag cccgagaata tcgttatcga gatggcccga
5701 gagaaccaaa ctacccagaa gggacagaag aacagtaggg aaaggatgaa gaggattgaa
5761 gagggtataa aagaactggg gtcccaaatc cttaaggaac acccagttga aaacacccag
5821 cttcagaatg agaagctcta cctgtactac ctgcagaacg gcagggacat gtacgtggat
5881 caggaactgg acatcaaccg gttgtccgac tacgacgtgg atgctatcgt gccccaaagc
5941 tttctcaaag atgattctat tgataataaa gtgttgacaa gatccgataa aaatagaggg
6001 aagagtgata acgtcccctc agaagaagtt gtcaagaaaa tgaaaaatta ttggcggcag
6061 ctgctgaacg ccaaactgat cacacaacgg aagttcgata atctgactaa ggctgaacga
6121 ggtggcctgt ctgagttgga taaagccggc ttcatcaaaa ggcagcttgt tgagacacgc
6181 cagatcacca agcacgtggc ccaaattctc gattcacgca tgaacaccaa gtacgatgaa
6241 aatgacaaac tgattcgaga ggtgaaagtt attactctga agtctaagct ggtctcagat
6301 ttcagaaagg actttcagtt ttataaggtg agagagatca acaattacca ccatgcgcat
6361 gatgcctacc tgaatgcagt ggtaggcact gcacttatca aaaaatatcc caagctggaa
6421 tctgaatttg tttacggaga ctataaagtg tacgatgtta ggaaaatgat cgcaaagtct
6481 gagcaggaaa taggcaaggc caccgctaag tacttctttt acagcaatat tatgaatttt
6541 ttcaagaccg agattacact ggccaatgga gagattcgga agcgaccact tatcgaaaca
6601 aacggagaaa caggagaaat cgtgtgggac aagggtaggg atttcgcgac agtccgcaag
6661 gtcctgtcca tgccgcaggt gaacatcgtt aaaaagaccg aagtacagac cggaggcttc
6721 tccaaggaaa gtatcctccc gaaaaggaac agcgacaagc tgatcgcacg caaaaaagat
6781 tgggacccca agaaatacgg cggattcgat tctcctacag tcgcttacag tgtactggtt
6841 gtggccaaag tggagaaagg gaagtctaaa aaactcaaaa gcgtcaagga actgctgggc
6901 atcacaatca tggagcgatc cagcttcgag aaaaacccca tcgactttct cgaagcgaaa
6961 ggatataaag aggtcaaaaa agacctcatc attaagctgc ccaagtactc tctctttgag
7021 cttgaaaacg gccggaaacg aatgctcgct agtgcgggcg agctgcagaa aggtaacgag
7081 ctggcactgc cctctaaata cgttaatttc ttgtatctgg ccagccacta tgaaaagctc
7141 aaagggtctc ccgaagataa tgagcagaag cagctgttcg tggaacaaca caaacactac
7201 cttgatgaga tcatcgagca aataagcgag ttctccaaaa gagtgatcct cgccgacgct
7261 aacctcgata aggtgctttc tgcttacaat aagcacaggg ataagcccat cagggagcag
7321 gcagaaaaca ttatccactt gtttactctg accaacttgg gcgcgcctgc agccttcaag
7381 tacttcgaca ccaccataga cagaaagcgg tacacctcta caaaggaggt cctggacgcc
7441 acactgattc atcagtcaat tacggggctc tatgaaacaa gaatcgacct ctctcagctc
7501 ggtggagacg gctctactag tggctctccc aagaagaaga ggaaggtagg atccggaagt
7561 atggtgagca agggcgagga ggataacatg gccatcatca aggagttcat gcgcttcaag
7621 gtgcacatgg agggctccgt gaacggccac gagttcgaga tcgagggcga gggcgagggc
7681 cgcccctacg agggcaccca gaccgccaag ctgaaggtga ccaagggtgg ccccctgccc
7741 ttcgcctggg acatcctgtc ccctcagttc atgtacggct ccaaggccta cgtgaagcac
7801 cccgccgaca tccccgacta cttgaagctg tccttccccg agggcttcaa gtgggagcgc
7861 gtgatgaact tcgaggacgg cggcgtggtg accgtgaccc aggactcctc actgcaggac
7921 ggcgagttca tctacaaggt gaagctgcgc ggcaccaact tcccctccga cggccccgta
7981 atgcagaaga agaccatggg ctgggaggcc tcctccgagc ggatgtaccc cgaggacggc
8041 gccctgaagg gcgagatcaa gcagaggctg aagctgaagg acggcggcca ctacgacgct
8101 gaggtcaaga ccacctacaa ggccaagaag cccgtgcagc tgcccggcgc ctacaacgtc
8161 aacatcaagt tggacatcac ctcccacaac gaggactaca ccatcgtgga acagtacgaa
8221 cgcgctgagg gccgccactc caccggcggc atggacgagc tgtacaagtc tggcggccgc
8281 ggcggtggag gtagcgacta caaggatgac gacgataagg gaaagtctta cccaactgtg
8341 agtgctgatt accaggacgc cgttgagaag gcgaagaaga agctcagagg cttcatcgct
8401 gagaagagat gcgctcctct aatgctccgt ttggcattcc actctgctgg aacctttgac
8461 aagggcacga agaccggtgg acccttcgga accatcaagc accctgccga actggctcac
8521 agcgctaaca acggtcttga catcgctgtt aggcttttgg agccactcaa ggcggagttc
8581 cctattttga gctacgccga tttctaccag ttggctggcg ttgttgccgt tgaggtcacg
8641 ggtggaccta aggttccatt ccaccctgga agagaggaca agcctgagcc accaccagag
8701 ggtcgcttgc ccgatcccac taagggttct gaccatttga gagatgtgtt tggcaaagct
8761 atggggctta ctgaccaaga tatcgttgct ctatctgggg gtcacactat tggagctgca
8821 cacaaggagc gttctggatt tgagggtccc tggacctcta atcctcttat tttcgacaac
8881 tcatacttca cggagttgtt gagtggtgag aaggaaggtc tccttcagct accttctgac
8941 aaggctcttt tgtctgaccc tgtattccgc cctctcgttg acaaatatgc agcggacgaa
9001 gatgccttct ttgctgatta cgctgaggct caccaaaagc tttccgagct tgggtttgct
9061 gatgccctcg agcctaaaaa gaaacgcaag gtcgaagata aacgcccagc agctacaaag
9121 aaggcaggtc aagccaagaa aaagaagggc tcttaatcta gataatcaac ctctggatta
9181 caaaatttgt gaaagattga ctggtattct taactatgtt gctcctttta cgctatgtgg
9241 atacgctgct ttaatgcctt tgtatcatgc tattgcttcc cgtatggctt tcattttctc
9301 ctccttgtat aaatcctggt tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca
9361 acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc actggttggg gcattgccac
9421 cacctgtcag ctcctttccg ggactttcgc tttccccctc cctattgcca cggcggaact
9481 catcgccgcc tgccttgccc gctgctggac aggggctcgg ctgttgggca ctgacaattc
9541 cgtggtgttg tcggggaaat catcgtcctt tccttggctg ctcgcctgtg ttgccacctg
9601 gattctgcgc gggacgtcct tctgctacgt cccttcggcc ctcaatccag cggaccttcc
9661 ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt cttcgccttc gccctcagac
9721 gagtcggatc tccctttggg ccgcctcccc gcctgagatc ctttaagacc aatgacttac
9781 aaggcagctg tagatcttag ccacttttta aaagaaaagg ggggactgga agggctaatt
9841 cactcccaac gaagacaaga tctgcttttt gcttgtactg ggtctctctg gttagaccag
9901 atctgagcct gggagctctc tggctaacta gggaacccac tgcttaagcc tcaataaagc
9961 ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg taactagaga
10021 tccctcagac ccttttagtc agtgtggaaa atctctagca gtagtagttc atgtcatctt
10081 attattcagt atttataact tgcaaagaaa tgaatatcag agagtgagag gcccgggtta
10141 attaaggaaa gggctagatc attcttgaag acgaaagggc ctcgtgatac gcctattttt
10201 ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa
10261 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
10321 gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca
10381 acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca
10441 cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta
10501 catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt
10561 tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc gtgttgacgc
10621 cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc
10681 accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc
10741 cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa
10801 ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga
10861 accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat
10921 ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca
10981 attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc
11041 ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat
11101 tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag
11161 tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa
11221 gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca
11281 tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc
11341 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
11401 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
11461 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
11521 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
11581 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
11641 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
11701 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
11761 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
11821 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
11881 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
11941 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
12001 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
12061 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
12121 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat
12181 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagcaagc tcatggctga
12241 ctaatttttt ttatttatgc agaggccgag gccgcctcgg cctctgagct attccagaag
12301 tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa agctccccgt ggcacgacag
12361 gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca
12421 ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag
12481 cggataacaa tttcacacag gaaacagcta tgacatgatt acgaatttca caaataaagc
12541 atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt
12601 ctggatcaac tggataactc aagctaacca aaatcatccc aaacttccca ccccataccc
12661 tattaccact gccaattacc tgtggtttca tttactctaa acctgtgatt cctctgaatt
12721 attttcattt taaagaaatt gtatttgtta aatatgtact acaaacttag tagt
//
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