| 质粒名称: | pAAVS1-NDi-CRISPRi (Gen1) |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 73497 |
| 存储实验室: | Bruce Conklin |
|
载体骨架基本信息 |
|
| 载体名称: | pAAVS1 |
| 载体出品公司: | Clontech |
| 空载体大小: | 3356bp |
| 完整质粒大小: | 13834bp |
| 载体类型: | 哺乳细胞表达、CRISPR |
| 筛选标记: | 新霉素（G418） |
| 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1: | dCas9-KRAB-P2A-mCherry |
| 物种来源: | Synthetic; S. pyogenes |
| 插入大小: | 5255bp |
| 基因突变: | D10A, H840A (catalytically deactivated Cas9) |
| 克隆方法: | TRE3G |
| 标签/融合蛋白: |
KRAB (N terminal on insert)
NLS (C terminal on insert)
HA (C terminal on insert)
|
| 克隆方法: | 限制性内切酶 |
| 5’克隆位点: | PacI (not destroyed) |
| 3’克隆位点: | AgeI (not destroyed) |
| 5’测序引物 | GCTCGTTTAGTGAACCGTCAG |
| 3’测序引物 | TGTGGAATTGTGAGCGGATA |
| 插入基因名称2: | rtTA |
| 物种来源: | Synthetic |
| 插入大小: | 853bp |
| 克隆方法: | CAG |
| 克隆方法: | Restriction Enzyme |
| 5’克隆位点: | XbaI (not destroyed) |
| 3’克隆位点: | MluI (not destroyed) |
| 5’测序引物 | CTCTAGAGCCTCTGCTAACC |
| 3’测序引物 | CAGAGGGAAAAAGATCTCAGT |
|
文献引用方法 |
|
| 材料和方法部分: | |
| 参考文献部分: | |
买家导航
pAAVS1-NDi-CRISPRi (Gen1)载体质粒基本信息
pAAVS1-NDi-CRISPRi (Gen1)质粒图谱载体图谱和pAAVS1-NDi-CRISPRi (Gen1)载体序列质粒序列多克隆位点信息
质粒图谱.png)
pAAVS1-NDi-CRISPRi (Gen1)质粒载体简介
pAAVS1-NDi-CRISPRi (Gen1)质粒序列载体序列
LOCUS pAAVS1-NDi-CRISPRi (Gen1) 13833 bp DNA circular SYN 25-AUG-2024
DEFINITION Dox-inducible CRISPR interference (CRISPRi) knock in construct into
the AAVS1 locus with mCherry marker.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 13833)
AUTHORS Mandegar MA, Huebsch N, Frolov EB, Shin E, Truong A, Olvera MP, Chan
AH, Miyaoka Y, Holmes K, Spencer CI, Judge LM, Gordon DE, Eskildsen
TV, Villalta JE, Horlbeck MA, Gilbert LA, Krogan NJ, Sheikh SP,
Weissman JS, Qi LS, So PL, Conklin BR
TITLE CRISPR Interference Efficiently Induces Specific and Reversible Gene
Silencing in Human iPSCs.
JOURNAL Cell Stem Cell. 2016 Apr 7;18(4):541-53. doi:
10.1016/j.stem.2016.01.022. Epub 2016 Mar 10.
PUBMED 26971820
REFERENCE 2 (bases 1 to 13833)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Aug 25, 2024 from SnapGene Server 7.0.3
https://www.biofeng.com
COMMENT SGRef: number: 1; type: "Journal Article"; doi:
"10.1016/j.stem.2016.01.022"; journalName: "Cell Stem Cell"; date:
"2016-04-7- 7"; volume: "18"; issue: "4"; pages: "541-53"
FEATURES Location/Qualifiers
source 1..13833
/mol_type="other DNA"
/organism="synthetic DNA construct"
CDS 54..914
/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 complement(272..291)
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
rep_origin 1085..1673
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 1574..1593
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
misc_feature 1721..2557
/label=HA-R
/note="right homology arm from the adeno-associated virus
integration site (AAVS1) within intron 1 of the human
PPP1R12C gene"
protein_bind 2590..2608
/gene="tetO"
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O2 for the tetR and tetA genes"
protein_bind 2626..2644
/gene="tetO"
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O2 for the tetR and tetA genes"
protein_bind 2662..2680
/gene="tetO"
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O2 for the tetR and tetA genes"
protein_bind 2698..2716
/gene="tetO"
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O2 for the tetR and tetA genes"
protein_bind 2734..2752
/gene="tetO"
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O2 for the tetR and tetA genes"
protein_bind 2770..2788
/gene="tetO"
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O2 for the tetR and tetA genes"
protein_bind 2806..2824
/gene="tetO"
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O2 for the tetR and tetA genes"
primer_bind 2869..2893
/label=LNCX
/note="Human CMV promoter, forward primer"
regulatory 2977..2986
/label=Kozak sequence
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
/regulatory_class="other"
CDS 3013..3198
/codon_start=1
/product="Kruppel-associated box (KRAB) transcriptional
repression domain from the human zinc finger protein ZNF10
(Margolin et al., 1994)"
/label=KRAB
/translation="RTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLENYKNLVSLGY
QLTKPDVILRLEKGEEP"
CDS 3220..7323
/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="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
DATLIHQSITGLYETRIDLSQLGGD"
CDS 3220..7323
/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="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
DATLIHQSITGLYETRIDLSQLGGD"
CDS 7327..7353
/codon_start=1
/product="HA (human influenza hemagglutinin) epitope tag"
/label=HA
/translation="YPYDVPDYA"
CDS 7333..7359
/codon_start=1
/product="HA (human influenza hemagglutinin) epitope tag"
/label=HA
/note="This is a variant of the standard HA tag, which has
sequence YPYDVPDYA. Some anti-HA antibodies may not
recognize this variant."
/translation="YDVPDYASL"
CDS 7372..7392
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 7399..7419
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 7459..7515
/codon_start=1
/product="2A peptide from porcine teschovirus-1
polyprotein"
/label=P2A
/note="Eukaryotic ribosomes fail to insert a peptide bond
between the Gly and Pro residues, yielding separate
polypeptides."
/translation="ATNFSLLKQAGDVEENPGP"
CDS 7516..8226
/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(7661..7679)
/label=mCherry-R
/note="mCherry, reverse primer"
primer_bind 7929..7948
/label=mCherry-F
/note="mCherry, forward primer"
polyA_signal 8258..8379
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(8295..8314)
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
primer_bind 8349..8368
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
primer_bind complement(8428..8444)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(8428..8444)
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind complement(8441..8463)
/label=M13/pUC Reverse
/note="In lacZ gene"
protein_bind 8452..8468
/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(8476..8506)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 8521..8542
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(8659..8676)
/label=L4440
/note="L4440 vector, forward primer"
protein_bind 8732..8765
/label=loxP
/bound_moiety="Cre recombinase"
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (ATGTATGC) (Shaw et al., 2021)."
primer_bind 9096..9115
/label=rbglobpA-R
/note="Rabbit beta-globin polyA, reverse primer. Also
called rb-glob-pA-term-R"
polyA_signal 9114..9169
/label=beta-globin poly(A) signal
/note="rabbit beta-globin polyadenylation signal (Gil and
Proudfoot, 1987)"
primer_bind 9215..9234
/label=Bglob-pA-R
/note="Rabbit beta-globin polyA region, reverse primer"
CDS complement(9325..10071)
/codon_start=1
/product="modified rtTA protein that binds tightly to
promoters containing the tet operator in the presence of
doxycycline"
/label=Tet-On(R) 3G
/translation="MSRLDKSKVINSALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV
KNKRALLDALPIEMLDRHHTHSCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTR
PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
DSMPPLLKQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPA
DALDDFDLDMLPADALDDFDLDMLPG"
primer_bind complement(10103..10122)
/label=pCAG-F
/note="Rabbit beta-globin intron, for pCAG plasmids,
forward primer"
intron 10130..11146
/label=chimeric intron
/note="chimera between introns from chicken beta-actin and
rabbit beta-globin"
primer_bind complement(10184..10206)
/label=pCAGGS-5
/note="Chimeric intron in CAG promoter, forward primer"
promoter complement(11147..11424)
/label=chicken beta-actin promoter
enhancer 11426..11805
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
polyA_signal 11811..12035
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
primer_bind 12024..12041
/label=BGH-rev
/note="Bovine growth hormone terminator, reverse primer.
Also called BGH reverse"
CDS complement(12061..12864)
/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="MGSAIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQ
GRPVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQD
LLSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE
EHQGLAPAELFARLKARMPDGDDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQ
DIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
primer_bind complement(12172..12191)
/label=Neo-F
/note="Neomycin resistance gene, forward primer"
primer_bind 12782..12801
/label=Neo-R
/note="Neomycin resistance gene, reverse primer"
regulatory 12861..12870
/label=Kozak sequence
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
/regulatory_class="other"
CDS complement(12886..12939)
/codon_start=1
/product="2A peptide from Thosea asigna virus capsid
protein"
/label=T2A
/note="Eukaryotic ribosomes fail to insert a peptide bond
between the Gly and Pro residues, yielding separate
polypeptides."
/translation="EGRGSLLTCGDVEENPGP"
misc_feature 12963..12988
/label=SA
/note="splice acceptor site"
protein_bind 12995..13028
/label=loxP
/bound_moiety="Cre recombinase"
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (ATGTATGC) (Shaw et al., 2021)."
misc_feature 13030..13833
/label=HA-L
/note="left homology arm from the adeno-associated virus
integration site (AAVS1) within intron 1 of the human
PPP1R12C gene"
ORIGIN
1 aagggcgaat tcaaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
61 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
121 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
181 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
241 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg
301 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
361 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
421 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
481 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
541 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag
601 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
661 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
721 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta
781 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
841 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
901 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
961 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
1021 tcccttaacg ggagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
1081 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
1141 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
1201 gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc
1261 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
1321 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
1381 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
1441 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
1501 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
1561 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
1621 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
1681 gcaacgcggc ctttttacgg ttcctggcga attcgccctt agagcagagc caggaacccc
1741 tgtagggaag gggcaggaga gccaggggca tgagatggtg gacgaggaag ggggacaggg
1801 aagcctgagc gcctctcctg ggcttgccaa ggactcaaac ccagaagccc agagcagggc
1861 cttagggaag cgggaccctg ctctgggcgg aggaatatgt cccagatagc actggggact
1921 ctttaaggaa agaaggatgg agaaagagaa agggagtaga ggcggccacg acctggtgaa
1981 cacctaggac gcaccattct cacaaaggga gttttccaca cggacacccc cctcctcacc
2041 acagccctgc caggacgggg ctggctactg gccttatctc acaggtaaaa ctgacgcacg
2101 gaggaacaat ataaattggg gactagaaag gtgaagagcc aaagttagaa ctcaggacca
2161 acttattctg attttgtttt tccaaactgc ttctcctctt gggaagtgta aggaagctgc
2221 agcaccagga tcagtgaaac gcaccagaca gccgcgtcag agcagctcag gttctgggag
2281 agggtagcgc agggtggcca ctgagaaccg ggcaggtcac gcatcccccc cttccctccc
2341 accccctgcc aagctctccc tcccaggatc ctctctggct ccatcgtaag caaaccttag
2401 aggttctggc aaggagagag atggctccag gaaatggggg tgtgtcacca gataaggaat
2461 ctgcctaaca ggaggtgggg gttagaccca atatcaggag actaggaagg aggaggccta
2521 aggatggggc ttttctgtca ccaatcctgt ccctagtaaa gctgatatct ccaccccaca
2581 gtggtttact ccctatcagt gatagagaac gtatgaagag tttactccct atcagtgata
2641 gagaacgtat gcagacttta ctccctatca gtgatagaga acgtataagg agtttactcc
2701 ctatcagtga tagagaacgt atgaccagtt tactccctat cagtgataga gaacgtatct
2761 acagtttact ccctatcagt gatagagaac gtatatccag tttactccct atcagtgata
2821 gagaacgtat aagctttagg cgtgtacggt gggcgcctat aaaagcagag ctcgtttagt
2881 gaaccgtcag atcgcctgga gcaattccac aacacttttg tcttatacca actttccgta
2941 ccacttccta ccctcgtaaa cttaaggtta attaacgcca ccatggatgc taagtcacta
3001 actgcctggt cccggacact ggtgaccttc aaggatgtat ttgtggactt caccagggag
3061 gagtggaagc tgctggacac tgctcagcag atcgtgtaca gaaatgtgat gctggagaac
3121 tataagaacc tggtttcctt gggttatcag cttactaagc cagatgtgat cctccggttg
3181 gagaagggag aagagcccgg cggttccggc ggagggtcga tggacaagaa gtacagcatc
3241 ggcctggcca tcggcaccaa ctctgtgggc tgggccgtga tcaccgacga gtacaaggtg
3301 cccagcaaga aattcaaggt gctgggcaac accgaccggc acagcatcaa gaagaacctg
3361 atcggcgccc tgctgttcga cagcggagaa acagccgagg ccacccggct gaagagaacc
3421 gccagaagaa gatacaccag acggaagaac cggatctgct atctgcaaga gatcttcagc
3481 aacgagatgg ccaaggtgga cgacagcttc ttccacagac tggaagagtc cttcctggtg
3541 gaagaggata agaagcacga gcggcacccc atcttcggca acatcgtgga cgaggtggcc
3601 taccacgaga agtaccccac catctaccac ctgagaaaga aactggtgga cagcaccgac
3661 aaggccgacc tgcggctgat ctatctggcc ctggcccaca tgatcaagtt ccggggccac
3721 ttcctgatcg agggcgacct gaaccccgac aacagcgacg tggacaagct gttcatccag
3781 ctggtgcaga cctacaacca gctgttcgag gaaaacccca tcaacgccag cggcgtggac
3841 gccaaggcca tcctgtctgc cagactgagc aagagcagac ggctggaaaa tctgatcgcc
3901 cagctgcccg gcgagaagaa gaatggcctg ttcggcaacc tgattgccct gagcctgggc
3961 ctgaccccca acttcaagag caacttcgac ctggccgagg atgccaaact gcagctgagc
4021 aaggacacct acgacgacga cctggacaac ctgctggccc agatcggcga ccagtacgcc
4081 gacctgtttc tggccgccaa gaacctgtcc gacgccatcc tgctgagcga catcctgaga
4141 gtgaacaccg agatcaccaa ggcccccctg agcgcctcta tgatcaagag atacgacgag
4201 caccaccagg acctgaccct gctgaaagct ctcgtgcggc agcagctgcc tgagaagtac
4261 aaagagattt tcttcgacca gagcaagaac ggctacgccg gctacatcga tggcggagcc
4321 agccaggaag agttctacaa gttcatcaag cccatcctgg aaaagatgga cggcaccgag
4381 gaactgctcg tgaagctgaa cagagaggac ctgctgcgga agcagcggac cttcgacaac
4441 ggcagcatcc cccaccagat ccacctggga gagctgcacg ccattctgcg gcggcaggaa
4501 gatttttacc cattcctgaa ggacaaccgg gaaaagatcg agaagatcct gaccttccgc
4561 atcccctact acgtgggccc tctggccagg ggaaacagca gattcgcctg gatgaccaga
4621 aagagcgagg aaaccatcac cccctggaac ttcgaggaag tggtggacaa gggcgccagc
4681 gcccagagct tcatcgagcg gatgaccaac ttcgataaga acctgcccaa cgagaaggtg
4741 ctgcccaagc acagcctgct gtacgagtac ttcaccgtgt acaacgagct gaccaaagtg
4801 aaatacgtga ccgagggaat gagaaagccc gccttcctga gcggcgagca gaaaaaagcc
4861 atcgtggacc tgctgttcaa gaccaaccgg aaagtgaccg tgaagcagct gaaagaggac
4921 tacttcaaga aaatcgagtg cttcgactcc gtggaaatct ccggcgtgga agatcggttc
4981 aacgcctccc tgggcacata ccacgatctg ctgaaaatta tcaaggacaa ggacttcctg
5041 gacaatgagg aaaacgagga cattctggaa gatatcgtgc tgaccctgac actgtttgag
5101 gacagagaga tgatcgagga acggctgaaa acctatgccc acctgttcga cgacaaagtg
5161 atgaagcagc tgaagcggcg gagatacacc ggctggggca ggctgagccg gaagctgatc
5221 aacggcatcc gggacaagca gtccggcaag acaatcctgg atttcctgaa gtccgacggc
5281 ttcgccaaca gaaacttcat gcagctgatc cacgacgaca gcctgacctt taaagaggac
5341 atccagaaag cccaggtgtc cggccagggc gatagcctgc acgagcacat tgccaatctg
5401 gccggcagcc ccgccattaa gaagggcatc ctgcagacag tgaaggtggt ggacgagctc
5461 gtgaaagtga tgggccggca caagcccgag aacatcgtga tcgaaatggc cagagagaac
5521 cagaccaccc agaagggaca gaagaacagc cgcgagagaa tgaagcggat cgaagagggc
5581 atcaaagagc tgggcagcca gatcctgaaa gaacaccccg tggaaaacac ccagctgcag
5641 aacgagaagc tgtacctgta ctacctgcag aatgggcggg atatgtacgt ggaccaggaa
5701 ctggacatca accggctgtc cgactacgat gtggacgcta tcgtgcctca gagctttctg
5761 aaggacgact ccatcgataa caaagtgctg actcggagcg acaagaaccg gggcaagagc
5821 gacaacgtgc cctccgaaga ggtcgtgaag aagatgaaga actactggcg ccagctgctg
5881 aatgccaagc tgattaccca gaggaagttc gacaatctga ccaaggccga gagaggcggc
5941 ctgagcgaac tggataaggc cggcttcatc aagagacagc tggtggaaac ccggcagatc
6001 acaaagcacg tggcacagat cctggactcc cggatgaaca ctaagtacga cgagaacgac
6061 aaactgatcc gggaagtgaa agtgatcacc ctgaagtcca agctggtgtc cgatttccgg
6121 aaggatttcc agttttacaa agtgcgcgag atcaacaact accaccacgc ccacgacgcc
6181 tacctgaacg ccgtcgtggg aaccgccctg atcaaaaagt accctaagct ggaaagcgag
6241 ttcgtgtacg gcgactacaa ggtgtacgac gtgcggaaga tgatcgccaa gagcgagcag
6301 gaaatcggca aggctaccgc caagtacttc ttctacagca acatcatgaa ctttttcaag
6361 accgagatta ccctggccaa cggcgagatc cggaagcggc ctctgatcga gacaaacggc
6421 gaaacaggcg agatcgtgtg ggataagggc cgggactttg ccaccgtgcg gaaagtgctg
6481 tctatgcccc aagtgaatat cgtgaaaaag accgaggtgc agacaggcgg cttcagcaaa
6541 gagtctatcc tgcccaagag gaacagcgac aagctgatcg ccagaaagaa ggactgggac
6601 cctaagaagt acggcggctt cgacagcccc accgtggcct attctgtgct ggtggtggcc
6661 aaagtggaaa agggcaagtc caagaaactg aagagtgtga aagagctgct ggggatcacc
6721 atcatggaaa gaagcagctt cgagaagaat cccatcgact ttctggaagc caagggctac
6781 aaagaagtga aaaaggacct gatcatcaag ctgcctaagt actccctgtt cgagctggaa
6841 aacggccgga agagaatgct ggcctctgcc ggcgaactgc agaagggaaa cgaactggcc
6901 ctgccctcca aatatgtgaa cttcctgtac ctggccagcc actatgagaa gctgaagggc
6961 tcccccgagg ataatgagca gaaacagctg tttgtggaac agcacaaaca ctacctggac
7021 gagatcatcg agcagatcag cgagttctcc aagagagtga tcctggccga cgctaatctg
7081 gacaaggtgc tgagcgccta caacaagcac agagacaagc ctatcagaga gcaggccgag
7141 aatatcatcc acctgtttac cctgaccaat ctgggagccc ctgccgcctt caagtacttt
7201 gacaccacca tcgaccggaa gaggtacacc agcaccaaag aggtgctgga cgccaccctg
7261 atccaccaga gcatcaccgg cctgtacgag acacggatcg acctgtctca gctgggaggc
7321 gacgcctatc cctatgacgt gcccgattat gccagcctgg gcagcggctc ccccaagaaa
7381 aaacgcaagg tggaagatcc taagaaaaag cggaaagtgg acggcattgg tagtgggagc
7441 aacggcagca gcggatccgc tactaacttc agcctgctga agcaggctgg agacgtggag
7501 gagaaccctg gacctatggt gagcaagggc gaggaggata acatggccat catcaaggag
7561 ttcatgcgct tcaaggtgca catggagggc tccgtgaacg gccacgagtt cgagatcgag
7621 ggcgagggcg agggccgccc ctacgagggc acccagaccg ccaagctgaa ggtgaccaag
7681 ggtggccccc tgcccttcgc ctgggacatc ctgtcccctc agttcatgta cggctccaag
7741 gcctacgtga agcaccccgc cgacatcccc gactacttga agctgtcctt ccccgagggc
7801 ttcaagtggg agcgcgtgat gaacttcgag gacggcggcg tggtgaccgt gacccaggac
7861 tcctccctgc aggacggcga gttcatctac aaggtgaagc tgcgcggcac caacttcccc
7921 tccgacggcc ccgtaatgca gaagaagacc atgggctggg aggcctcctc cgagcggatg
7981 taccccgagg acggcgccct gaagggcgag atcaagcaga ggctgaagct gaaggacggc
8041 ggccactacg acgctgaggt caagaccacc tacaaggcca agaagcccgt gcagctgccc
8101 ggcgcctaca acgtcaacat caagttggac atcacctccc acaacgagga ctacaccatc
8161 gtggaacagt acgaacgcgc cgagggccgc cactccaccg gcggcatgga cgagctgtac
8221 aagtaaaccg gtgcggccgc aatcgatcgc ccaccccaac ttgtttattg cagcttataa
8281 tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca
8341 ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgta taccgtcgac
8401 ctctagctag agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc
8461 gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta
8521 atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
8581 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
8641 tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg
8701 agcggtatca gctcactcaa aggacggccg cataacttcg tataatgtat gctatacgaa
8761 gttataagct tgggctgcag gtcgagggat ctccataaga gaagagggac agctatgact
8821 gggagtagtc aggagaggag gaaaaatctg gctagtaaaa catgtaagga aaattttagg
8881 gatgttaaag aaaaaaataa cacaaaacaa aatataaaaa aaatctaacc tcaagtcaag
8941 gcttttctat ggaataagga atggacagca gggggctgtt tcatatactg atgacctctt
9001 tatagccaac ctttgttcat ggcagccagc atatgggcat atgttgccaa actctaaacc
9061 aaatactcat tctgatgttt taaatgattt gccctcccat atgtccttcc gagtgagaga
9121 cacaaaaaat tccaacacac tattgcaatg aaaataaatt tcctttatta gccagaagtc
9181 agatgctcaa ggggcttcat gatgtcccca taatttttgg cagagggaaa aagatctcag
9241 tggtatttgt gagccagggc attggccaca ccagccacca ccttctgata ggcagcctgc
9301 acctgaggag tgaattcacg cgttttagcc tggcagcata tcgagatcga agtcatcgag
9361 agcgtcagca gggagcatat ccagatcaaa atcatccagg gcatcggcgg gcagcatgtc
9421 caggtcgaag tcgtccaggg cgtcggtggg tccgccgctc tcgcacttca gctgcttctc
9481 gaggccgcag atgatcagtt ccaggccgaa caggaaggcg ggctcggcgc cctgtctgtc
9541 gaacagctcg atggcctgct tcagcagggg gggcatgctg tcggtggtgg gtgtctctct
9601 ttcctctttg gcgacctggt gttcctgttc ttccagcacg cagcccaggg tgaagtggcc
9661 cacggcgctc agggcgtaca gggcgttttc caggctgaag ccctgctggc acaggaaggc
9721 cagctggttt tccagtgtct cgtactgctt ctcggtgggt ctggtgccca ggtgcacttt
9781 ggcgccgtcc cggtggctca gcagggcgca tctgtagctc ttggcgttgt tccgcaggaa
9841 gtcctgccag ctctcgcctt ccagagggca gctgtgggtg tggtgccggt ccagcatctc
9901 gatgggcagg gcgtccagca gggcccgctt gttcttcacg tgccagtaca gggtgggctg
9961 ttccacgccc agtttctggg ccagcttccg ggtggtcagg ccctcgatgc ccacgccgtt
10021 cagcagttcc agggcgctgt tgatcacttt gctcttgtcc agccggctca tgtcgacatt
10081 ctttgccaaa atgatgagac agcacaataa ccagcacgtt gcccaggagc tgtaggaaaa
10141 agaagaaggc atgaacatgg ttagcagagg ctctagagcc gccggtcaca cgccagaagc
10201 cgaaccccgc cctgccccgt cccccccgaa ggcagccgtc cccccgcgga cagccccgag
10261 gctggagagg gagaagggga cggcggcgcg gcgacgcacg aaggccctcc ccgcccattt
10321 ccttcctgcc ggcgccgcac cgcttcgccc cgcgcccgct agagggggtg cggcggcgcc
10381 tcccagattt cggctccgca cagatttggg acaaaggaag tccctgcgcc ctctcgcacg
10441 attaccataa aaggcaatgg ctgcggctcg ccgcgcctcg acagccgccg gcgctccggg
10501 ggccgccgcg cccctccccc gagccctccc cggcccgagg cggccccgcc ccgcccggca
10561 cccccacctg ccgccacccc ccgcccggca cggcgagccc cgcgccacgc cccgtacgga
10621 gccccgcacc cgaagccggg ccgtgctcag caactcgggg aggggggtgc agggggggtt
10681 gcagcccgac cgacgcgccc acaccccctg ctcacccccc cacgcacaca ccccgcacgc
10741 agcctttgtt cccctcgcag ccccccccgc accgcggggc accgcccccg gccgcgctcc
10801 cctcgcgcac actgcggagc gcacaaagcc ccgcgccgcg cccgcagcgc tcacagccgc
10861 cgggcagcgc ggagccgcac gcggcgctcc ccacgcacac acacacgcac gcaccccccg
10921 agccgctccc cccgcacaaa gggccctccc ggagcccctc aaggctttca cgcagccaca
10981 gaaaagaaac aagccgtcat taaaccaagc gctaattaca gcccggagga gaagggccgt
11041 cccgcccgct cacctgtggg agtaacgcgg tcagtcagag ccggggcggg cggcgcgagg
11101 cggcggcgga gcggggcacg gggcgaaggc agcgcgcagc gactcccgcc cgccgcgcgc
11161 ttcgcttttt atagggccgc cgccgccgcc gcctcgccat aaaaggaaac tttcggagcg
11221 cgccgctctg attggctgcc gccgcacctc tccgcctccc cccccccccc cccccccccc
11281 cccccccccc ccccggcgcg cgcccccccc ccccccccgc ccccatcgct gcacaaaata
11341 attaaaaaat aaataaatac aaaattgggg gtggggaggg gggggagatg gggagagtga
11401 agcagaacgt ggggctcacc tcgaccatgg taatagcgat gactaatacg tagatgtact
11461 gccaagtagg aaagtcccat aaggtcatgt actgggcata atgccaggcg ggccatttac
11521 cgtcattgac gtcaataggg ggcgtacttg gcatatgata cacttgatgt actgccaagt
11581 gggcagttta ccgtaaatac tccacccatt gacgtcaatg gaaagtccct attggcgtta
11641 ctatgggaac atacgtcatt attgacgtca atgggcgggg gtcgttgggc ggtcagccag
11701 gcgggccatt taccgtaagt tatgtaacgc ggaactccat atatgggcta tgaactaatg
11761 accccgtaat tgattactat taataactag tcaataatca atgtcgagac ccatagagcc
11821 caccgcatcc ccagcatgcc tgctattgtc ttcccaatcc tcccccttgc tgtcctgccc
11881 caccccaccc cccagaatag aatgacacct actcagacaa tgcgatgcaa tttcctcatt
11941 ttattaggaa aggacagtgg gagtggcacc ttccagggtc aaggaaggca cgggggaggg
12001 gcaaacaaca gatggctggc aactagaagg cacagtcgag gctgatcagc gggtttaaac
12061 tcagaagaac tcgtcaagaa ggcgatagaa ggcgatgcgc tgcgaatcgg gagcggcgat
12121 accgtaaagc acgaggaagc ggtcagccca ttcgccgcca agctcttcag caatatcacg
12181 ggtagccaac gctatgtcct gatagcggtc cgccacaccc agccggccac agtcgatgaa
12241 tccagaaaag cggccatttt ccaccatgat attcggcaag caggcatcgc catgggtcac
12301 gacgagatca tcgccgtcgg gcatgcgcgc cttgagcctg gcgaacagtt cggctggcgc
12361 gagcccctga tgctcttcgt ccagatcatc ctgatcgaca agaccggctt ccatccgagt
12421 acgtgctcgc tcgatgcgat gtttcgcttg gtggtcgaat gggcaggtag ccggatcaag
12481 cgtatgcagc cgccgcattg catcagccat gatggatact ttctcggcag gagcaaggtg
12541 agatgacagg agatcctgcc ccggcacttc gcccaatagc agccagtccc ttcccgcttc
12601 agtgacaacg tcgagcacag ctgcgcaagg aacgcccgtc gtggccagcc acgatagccg
12661 cgctgcctcg tcctgcagtt cattcagggc accggacagg tcggtcttga caaaaagaac
12721 cgggcgcccc tgcgctgaca gccggaacac ggcggcatca gagcagccga ttgtctgttg
12781 tgcccagtca tagccgaata gcctctccac ccaagcggcc ggagaacctg cgtgcaatcc
12841 atcttgttca atggccgatc ccatggtggc ggtaccctcg agcctagggc cgggattctc
12901 ctccacgtca ccgcatgtta gaagacttcc tctgccctct ccgctgccag atctctcgag
12961 gccctgtggg aggaagagaa gaggtcagaa gcttataact tcgtataatg tatgctatac
13021 gaagttattg ccccactgtg gggtggaggg gacagataaa agtacccaga accagagcca
13081 cattaaccgg ccctgggaat ataaggtggt cccagctcgg ggacacagga tccctggagg
13141 cagcaaacat gctgtcctga agtggacata ggggcccggg ttggaggaag aagactagct
13201 gagctctcgg acccctggaa gatgccatga cagggggctg gaagagctag cacagactag
13261 agaggtaagg ggggtagggg agctgcccaa atgaaaggag tgagaggtga cccgaatcca
13321 caggagaacg gggtgtccag gcaaagaaag caagaggatg gagaggtggc taaagccagg
13381 gagacggggt actttggggt tgtccagaaa aacggtgatg atgcaggcct acaagaaggg
13441 gaggcgggac gcaagggaga catccgtcgg agaaggccat cctaagaaac gagagatggc
13501 acaggcccca gaaggagaag gaaaagggaa cccagcgagt gaagacggca tggggttggg
13561 tgagggagga gagatgcccg gagaggaccc agacacgggg aggatccgct cagaggacat
13621 cacgtggtgc agcgccgaga aggaagtgct ccggaaagag catccttggg cagcaacaca
13681 gcagagagca aggggaagag ggagtggagg aagacggaac ctgaaggagg cggcagggaa
13741 ggatctgggc cagccgtaga ggtgacccag gccacaagct gcagacagaa agcggcacag
13801 gcccagggga gagaatgctg gtcagagaaa gca
//
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