| 质粒名称: | PB-TRE-dCas9-KRAB-MeCP2 |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 122267 |
| 存储实验室: | Andrea Califano |
|
载体骨架基本信息 |
|
| 载体名称: | PB-TRE |
| 完整质粒大小: | 13424 bp |
| 载体类型: | 哺乳细胞表达 |
| 筛选标记: | Hygromycin |
| 载体抗性: | 氨苄霉素 |
| 生长温度: | 30 ℃ |
| 宿主菌: | NEB Stable |
| 拷贝数: | 未知 |
|
插入基因信息 |
|
| 插入基因名称: | dCas9-KRAB-MeCP2 |
| 物种来源: | Synthetic |
| 插入大小: | 5346 bp |
| 标签/融合蛋白: | dCas9-KRAB-MeCP2 (C terminal on insert) |
| 克隆方法: | Gibson Cloning |
| 5’测序引物 | GCCCAAATTCTCGATTCACGC |
| 3’测序引物 | CTCGGTGGGGTATCGACAGA |
|
文献引用方法 |
|
| 材料和方法部分: | PB-TRE-dCas9-KRAB-MeCP2 was a gift from Andrea Califano (Addgene plasmid # 122267 ; http://n2t.net/addgene:122267 ; RRID:Addgene_122267) |
| 参考文献部分: | Interrogation of genome-wide, experimentally dissected gene regulatory networks reveals mechanisms underlying dynamic cellular state control. Tan X, Worley J, Turunen M, Wong K, Fernández EC, Paull E, Jones S, Wang J, Noh H, Salvatori B, Chavez A, Califano A. bioRxiv 2021.06.28.449297 10.1101/2021.06.28.449297 |
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 PB-TRE-dCas9-KRAB-MeCP2 13424 bp ds-DNA circular SYN 12-MAY-2021
DEFINITION SP-dCas9-KRAB-MeCP2 with doxycycline-inducible expression.
ACCESSION .
VERSION .
KEYWORDS PB-TRE-dCas9-KRAB-MeCP2
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 13424)
TITLE Validation of VIPER-inferred master regulators by pooled CRISPR
screening
JOURNAL Unpublished
REFERENCE 2 (bases 1 to 13424)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58
http://www.biofeng.com
FEATURES Location/Qualifiers
source 1..13424
/organism="synthetic DNA construct"
/mol_type="other DNA"
protein_bind 17..35
/gene="tetO"
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O2 for the tetR and tetA genes"
primer_bind 80..104
/label=LNCX
/note="Human CMV promoter, forward primer"
regulatory 198..207
/regulatory_class="other"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
CDS 204..4307
/codon_start=1
/product="catalytically dead mutant of the Cas9
endonuclease from the Streptococcus pyogenes Type II
CRISPR/Cas system"
/label=Cas9m4
/note="RNA-guided DNA-binding protein that lacks
endonuclease activity due to the mutations D10A, D839A,
H840A, and N863A (Mali et al., 2013)"
/translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
LYYLQNGRDMYVDQELDINRLSDYDVAAIVPQSFLKDDSIDNKVLTRSDKARGKSDNVP
SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
DATLIHQSITGLYETRIDLSQLGGD"
CDS 4311..4337
/codon_start=1
/product="HA (human influenza hemagglutinin) epitope tag"
/label=HA
/translation="YPYDVPDYA"
CDS 4356..4376
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 4383..4403
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 4443..4628
/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 4662..4682
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 4692..5549
/codon_start=1
/product="transcriptional repression domain from rat
methyl-CpG-binding protein 2 (Nan et al., 1997)"
/label=MeCP2
/translation="VQVKRVLEKSPGKLLVKMPFQASPGGKGEGGGATTSAQVMVIKRP
GRKRKAEADPQAIPKKRGRKPGSVVAAAAAEAKKKAVKESSIRSVQETVLPIKKRKTRE
TVSIEVKEVVKPLLVSTLGEKSGKGLKTCKSPGRKSKESSPKGRSSSASSPPKKEHHHH
HHHAESPKAPMPLLPPPPPPEPQSSEDPISPPEPQDLSSSICKEEKMPRAGSLESDGCP
KEPAKTQPMVAAAATTTTTTTTTVAEKYKHRGEGERKDIVSSSMPRPNREEPVDSRTPV
TERVS"
primer_bind complement(5604..5623)
/label=TK-pA-R
/note="Thymidine kinase polyA, reverse primer"
polyA_signal 5648..5696
/label=HSV TK poly(A) signal
/note="herpes simplex virus thymidine kinase
polyadenylation signal (Cole and Stacy, 1985)"
promoter 5927..6138
/label=EF-1-alpha core promoter
/note="core promoter for human elongation factor
EF-1-alpha"
LTR 6151..6419
/label=5' LTR (truncated)
/note="truncated 5' long terminal repeat (LTR) from human
T-cell leukemia virus (HTLV) type 1"
CDS 6453..7196
/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"
CDS 7197..7250
/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"
CDS 7272..8291
/codon_start=1
/gene="aph(4)-Ia"
/product="aminoglycoside phosphotransferase from E. coli"
/label=HygR
/note="confers resistance to hygromycin"
/translation="KPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGYV
LRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPET
ELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQTV
MDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFGD
SQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGNF
DDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE"
primer_bind complement(8348..8368)
/label=WPRE-R
/note="WPRE, reverse primer"
polyA_signal 8402..8523
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(8439..8458)
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
primer_bind 8493..8512
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
repeat_region 9286..9320
/label=piggyBac left (5') inverted repeat
/note="piggyBac transposon-specific inverted terminal
repeat sequence (ITR)"
primer_bind complement(9811..9827)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(9811..9827)
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind complement(9824..9846)
/label=M13/pUC Reverse
/note="In lacZ gene"
protein_bind 9835..9851
/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(9859..9889)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 9904..9925
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(10042..10059)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(10213..10801)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(10293..10312)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS complement(10972..11832)
/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 11595..11614
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
promoter complement(11833..11937)
/gene="bla"
/label=AmpR promoter
rep_origin complement(11963..12418)
/direction=LEFT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(12100..12121)
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind 12312..12331
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 12545..12567
/label=M13/pUC Forward
/note="In lacZ gene"
primer_bind 12559..12576
/label=M13 Forward
/note="In lacZ gene. Also called M13-F20 or M13 (-21)
Forward"
primer_bind 12560..12576
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
repeat_region 12706..12768
/label=piggyBac right (3') inverted repeat
/note="piggyBac transposon-specific inverted terminal
repeat sequence (ITR)"
promoter 13217..171
/label=TRE3G promoter
/note="3rd-generation Tet-responsive promoter that can be
activated by binding of Tet-On(R) 3G"
protein_bind 13225..13243
/gene="tetO"
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O2 for the tetR and tetA genes"
protein_bind 13261..13279
/gene="tetO"
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O2 for the tetR and tetA genes"
protein_bind 13297..13315
/gene="tetO"
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O2 for the tetR and tetA genes"
protein_bind 13333..13351
/gene="tetO"
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O2 for the tetR and tetA genes"
protein_bind 13369..13387
/gene="tetO"
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O2 for the tetR and tetA genes"
protein_bind 13405..13423
/gene="tetO"
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O2 for the tetR and tetA genes"
ORIGIN
1 cgtatatcca gtttactccc tatcagtgat agagaacgta taagctttag gcgtgtacgg
61 tgggcgccta taaaagcaga gctcgtttag tgaaccgtca gatcgcctgg agcaattcca
121 caacactttt gtcttatacc aactttccgt accacttcct accctcgtaa aggtctagag
181 ctagcgaatt cgaatttgcc accatggaca agaagtactc cattgggctc gctatcggca
241 caaacagcgt cggctgggcc gtcattacgg acgagtacaa ggtgccgagc aaaaaattca
301 aagttctggg caataccgat cgccacagca taaagaagaa cctcattggc gccctcctgt
361 tcgactccgg ggagacggcc gaagccacgc ggctcaaaag aacagcacgg cgcagatata
421 cccgcagaaa gaatcggatc tgctacctgc aggagatctt tagtaatgag atggctaagg
481 tggatgactc tttcttccat aggctggagg agtccttttt ggtggaggag gataaaaagc
541 acgagcgcca cccaatcttt ggcaatatcg tggacgaggt ggcgtaccat gaaaagtacc
601 caaccatata tcatctgagg aagaagcttg tagacagtac tgataaggct gacttgcggt
661 tgatctatct cgcgctggcg catatgatca aatttcgggg acacttcctc atcgaggggg
721 acctgaaccc agacaacagc gatgtcgaca aactctttat ccaactggtt cagacttaca
781 atcagctttt cgaagagaac ccgatcaacg catccggagt tgacgccaaa gcaatcctga
841 gcgctaggct gtccaaatcc cggcggctcg aaaacctcat cgcacagctc cctggggaga
901 agaagaacgg cctgtttggt aatcttatcg ccctgtcact cgggctgacc cccaacttta
961 aatctaactt cgacctggcc gaagatgcca agcttcaact gagcaaagac acctacgatg
1021 atgatctcga caatctgctg gcccagatcg gcgaccagta cgcagacctt tttttggcgg
1081 caaagaacct gtcagacgcc attctgctga gtgatattct gcgagtgaac acggagatca
1141 ccaaagctcc gctgagcgct agtatgatca agcgctatga tgagcaccac caagacttga
1201 ctttgctgaa ggcccttgtc agacagcaac tgcctgagaa gtacaaggaa attttcttcg
1261 atcagtctaa aaatggctac gccggataca ttgacggcgg agcaagccag gaggaatttt
1321 acaaatttat taagcccatc ttggaaaaaa tggacggcac cgaggagctg ctggtaaagc
1381 ttaacagaga agatctgttg cgcaaacagc gcactttcga caatggaagc atcccccacc
1441 agattcacct gggcgaactg cacgctatcc tcaggcggca agaggatttc tacccctttt
1501 tgaaagataa cagggaaaag attgagaaaa tcctcacatt tcggataccc tactatgtag
1561 gccccctcgc ccggggaaat tccagattcg cgtggatgac tcgcaaatca gaagagacca
1621 tcactccctg gaacttcgag gaagtcgtgg ataagggggc ctctgcccag tccttcatcg
1681 aaaggatgac taactttgat aaaaatctgc ctaacgaaaa ggtgcttcct aaacactctc
1741 tgctgtacga gtacttcaca gtttataacg agctcaccaa ggtcaaatac gtcacagaag
1801 ggatgagaaa gccagcattc ctgtctggag agcagaagaa agctatcgtg gacctcctct
1861 tcaagacgaa ccggaaagtt accgtgaaac agctcaaaga agactatttc aaaaagattg
1921 aatgtttcga ctctgttgaa atcagcggag tggaggatcg cttcaacgca tccctgggaa
1981 cgtatcacga tctcctgaaa atcattaaag acaaggactt cctggacaat gaggagaacg
2041 aggacattct tgaggacatt gtcctcaccc ttacgttgtt tgaagatagg gagatgattg
2101 aagaacgctt gaaaacttac gctcatctct tcgacgacaa agtcatgaaa cagctcaaga
2161 ggcgccgata tacaggatgg gggcggctgt caagaaaact gatcaatggg atccgagaca
2221 agcagagtgg aaagacaatc ctggattttc ttaagtccga tggatttgcc aaccggaact
2281 tcatgcagtt gatccatgat gactctctca cctttaagga ggacatccag aaagcacaag
2341 tttctggcca gggggacagt cttcacgagc acatcgctaa tcttgcaggt agcccagcta
2401 tcaaaaaggg aatactgcag accgttaagg tcgtggatga actcgtcaaa gtaatgggaa
2461 ggcataagcc cgagaatatc gttatcgaga tggcccgaga gaaccaaact acccagaagg
2521 gacagaagaa cagtagggaa aggatgaaga ggattgaaga gggtataaaa gaactggggt
2581 cccaaatcct taaggaacac ccagttgaaa acacccagct tcagaatgag aagctctacc
2641 tgtactacct gcagaacggc agggacatgt acgtggatca ggaactggac atcaatcggc
2701 tctccgacta cgacgtggct gctatcgtgc cccagtcttt tctcaaagat gattctattg
2761 ataataaagt gttgacaaga tccgataaag ctagagggaa gagtgataac gtcccctcag
2821 aagaagttgt caagaaaatg aaaaattatt ggcggcagct gctgaacgcc aaactgatca
2881 cacaacggaa gttcgataat ctgactaagg ctgaacgagg tggcctgtct gagttggata
2941 aagccggctt catcaaaagg cagcttgttg agacacgcca gatcaccaag cacgtggccc
3001 aaattctcga ttcacgcatg aacaccaagt acgatgaaaa tgacaaactg attcgagagg
3061 tgaaagttat tactctgaag tctaagctgg tctcagattt cagaaaggac tttcagtttt
3121 ataaggtgag agagatcaac aattaccacc atgcgcatga tgcctacctg aatgcagtgg
3181 taggcactgc acttatcaaa aaatatccca agcttgaatc tgaatttgtt tacggagact
3241 ataaagtgta cgatgttagg aaaatgatcg caaagtctga gcaggaaata ggcaaggcca
3301 ccgctaagta cttcttttac agcaatatta tgaatttttt caagaccgag attacactgg
3361 ccaatggaga gattcggaag cgaccactta tcgaaacaaa cggagaaaca ggagaaatcg
3421 tgtgggacaa gggtagggat ttcgcgacag tccggaaggt cctgtccatg ccgcaggtga
3481 acatcgttaa aaagaccgaa gtacagaccg gaggcttctc caaggaaagt atcctcccga
3541 aaaggaacag cgacaagctg atcgcacgca aaaaagattg ggaccccaag aaatacggcg
3601 gattcgattc tcctacagtc gcttacagtg tactggttgt ggccaaagtg gagaaaggga
3661 agtctaaaaa actcaaaagc gtcaaggaac tgctgggcat cacaatcatg gagcgatcaa
3721 gcttcgaaaa aaaccccatc gactttctcg aggcgaaagg atataaagag gtcaaaaaag
3781 acctcatcat taagcttccc aagtactctc tctttgagct tgaaaacggc cggaaacgaa
3841 tgctcgctag tgcgggcgag ctgcagaaag gtaacgagct ggcactgccc tctaaatacg
3901 ttaatttctt gtatctggcc agccactatg aaaagctcaa agggtctccc gaagataatg
3961 agcagaagca gctgttcgtg gaacaacaca aacactacct tgatgagatc atcgagcaaa
4021 taagcgaatt ctccaaaaga gtgatcctcg ccgacgctaa cctcgataag gtgctttctg
4081 cttacaataa gcacagggat aagcccatca gggagcaggc agaaaacatt atccacttgt
4141 ttactctgac caacttgggc gcgcctgcag ccttcaagta cttcgacacc accatagaca
4201 gaaagcggta cacctctaca aaggaggtcc tggacgccac actgattcat cagtcaatta
4261 cggggctcta tgaaacaaga atcgacctct ctcagctcgg tggagacgcc tatccctatg
4321 acgtgcccga ttatgccagc ctgggcagcg gctcccccaa gaaaaaacgc aaggtggaag
4381 atcctaagaa aaagcggaaa gtggacggca ttggtagtgg gagcaacggc agcagcggat
4441 cccggacact ggtgaccttc aaggatgtat ttgtggactt caccagggag gagtggaagc
4501 tgctggacac tgctcagcag atcgtgtaca gaaatgtgat gctggagaac tataagaacc
4561 tggtttcctt gggttatcag cttactaagc cagatgtgat cctccggttg gagaagggag
4621 aagagccctc gggaggtggt tcgggtggct ctggatcaag cccaaagaag aaacggaagg
4681 tggaagcctc agtgcaggtg aaaagggtgc tggaaaaatc ccccggcaaa ctcctcgtga
4741 agatgccctt ccaggcttcc cctggcggaa aaggtgaagg gggtggcgca accacatctg
4801 cccaggtcat ggtcatcaag cgacctggaa ggaaaagaaa ggccgaggct gaccctcagg
4861 ccattccaaa gaaacgggga cgcaagccag ggtccgtggt cgcagctgca gcagctgagg
4921 ctaagaaaaa ggcagtgaag gaaagctcca tccgcagtgt gcaggagact gtcctgccca
4981 tcaagaagag gaagactagg gagaccgtgt ccatcgaggt caaagaagtg gtcaagcccc
5041 tgctcgtgtc caccctgggc gaaaaatctg gaaaggggct caaaacatgc aagtcacctg
5101 gacggaaaag caaggagtct agtccaaagg ggcgctcaag ctccgcttct agtcccccta
5161 aaaaggaaca ccatcaccat caccatcacg ccgagtctcc taaggctcct atgccactgc
5221 tcccaccacc tccaccacct gagccacagt caagcgaaga ccccatcagc ccacccgagc
5281 ctcaggatct gtcctctagt atttgcaaag aggaaaagat gcccagagca ggcagcctgg
5341 agagtgatgg ctgtccaaaa gaacccgcca agacccagcc tatggtggca gccgctgcaa
5401 ctaccaccac aaccacaact accacagtgg ccgaaaaata caagcatcgc ggcgagggcg
5461 aacgaaagga cattgtgtca agctccatgc ccagacctaa ccgggaggaa ccagtcgata
5521 gtaggacacc cgtgactgag agagtctcat gaaagggttc gatccctacc ggttagtaat
5581 gagtttaaac gggggaggct aactgaaaca cggaaggaga caataccgga aggaacccgc
5641 gctatgacgg caataaaaag acagaataaa acgcacgggt gttgggtcgt ttgttcataa
5701 acgcggggtt cggtcccagg gctggcactc tgtcgatacc ccaccgagac cccattgggg
5761 ccaatacgcc cgcgtttctt ccttttcccc accccacccc ccaagttcgg gtgaaggccc
5821 agggctcgca gccaacgtcg gggcggcagg ccctgccata gcagatctgc gaaatcggat
5881 ccagctaaat ctaagtggat ctgcgatcgc tccggtgccc gtcagtgggc agagcgcaca
5941 tcgcccacag tccccgagaa gttgggggga ggggtcggca attgaacggg tgcctagaga
6001 aggtggcgcg gggtaaactg ggaaagtgat gtcgtgtact ggctccgcct ttttcccgag
6061 ggtgggggag aaccgtatat aagtgcagta gtcgccgtga acgttctttt tcgcaacggg
6121 tttgccgcca gaacacagct gaagcttcga ggggctcgca tctctccttc acgcgcccgc
6181 cgccctacct gaggccgcca tccacgccgg ttgagtcgcg ttctgccgcc tcccgcctgt
6241 ggtgcctcct gaactgcgtc cgccgtctag gtaagtttaa agctcaggtc gagaccgggc
6301 ctttgtccgg cgctcccttg gagcctacct agactcagcc ggctctccac gctttgcctg
6361 accctgcttg ctcaactcta cgtctttgtt tcgttttctg ttctgcgccg ttacagatct
6421 aagctgtgac cggcgcctac tctagagccg ccatgtctag actggacaag agcaaagtca
6481 taaactctgc tctggaatta ctcaatggag tcggtatcga aggcctgacg acaaggaaac
6541 tcgctcaaaa gctgggagtt gagcagccta ccctgtactg gcacgtgaag aacaagcggg
6601 ccctgctcga tgccctgcca atcgagatgc tggacaggca tcatacccac tcctgccccc
6661 tggaaggcga gtcatggcaa gactttctgc ggaacaacgc caagtcatac cgctgtgctc
6721 tcctctcaca tcgcgacggg gctaaagtgc atctcggcac ccgcccaaca gagaaacagt
6781 acgaaaccct ggaaaatcag ctcgcgttcc tgtgtcagca aggcttctcc ctggagaacg
6841 cactgtacgc tctgtccgcc gtgggccact ttacactggg ctgcgtattg gaggaacagg
6901 agcatcaagt agcaaaagag gaaagagaga cacctaccac cgattctatg cccccacttc
6961 tgaaacaagc aattgagctg ttcgaccggc agggagccga acctgccttc cttttcggcc
7021 tggaactaat catatgtggc ctggagaaac agctaaagtg cgaaagcggc gggccgaccg
7081 acgcccttga cgattttgac ttagacatgc tcccagccga tgcccttgac gactttgacc
7141 ttgatatgct gcctgctgac gctcttgacg attttgacct tgacatgctc cccggggagg
7201 gcagaggaag tcttctaaca tgcggtgacg tggaggagaa tcccggccct gaattcatgg
7261 atagatccgg aaagcctgaa ctcaccgcga cgtctgtcga gaagtttctg atcgaaaagt
7321 tcgacagcgt ctccgacctg atgcagctct cggagggcga agaatctcgt gctttcagct
7381 tcgatgtagg agggcgtgga tatgtcctgc gggtaaatag ctgcgccgat ggtttctaca
7441 aagatcgtta tgtttatcgg cactttgcat cggccgcgct cccgattccg gaagtgcttg
7501 acattgggga attcagcgag agcctgacct attgcatctc ccgccgtgca cagggtgtca
7561 cgttgcaaga cctgcctgaa accgaactgc ccgctgttct gcagccggtc gcggaggcca
7621 tggatgcgat cgctgcggcc gatcttagcc agacgagcgg gttcggccca ttcggaccgc
7681 aaggaatcgg tcaatacact acatggcgtg atttcatatg cgcgattgct gatccccatg
7741 tgtatcactg gcaaactgtg atggacgaca ccgtcagtgc gtccgtcgcg caggctctcg
7801 atgagctgat gctttgggcc gaggactgcc ccgaagtccg gcacctcgtg cacgcggatt
7861 tcggctccaa caatgtcctg acggacaatg gccgcataac agcggtcatt gactggagcg
7921 aggcgatgtt cggggattcc caatacgagg tcgccaacat cttcttctgg aggccgtggt
7981 tggcttgtat ggagcagcag acgcgctact tcgagcggag gcatccggag cttgcaggat
8041 cgccgcggct ccgggcgtat atgctccgca ttggtcttga ccaactctat cagagcttgg
8101 ttgacggcaa tttcgatgat gcagcttggg cgcagggtcg atgcgacgca atcgtccgat
8161 ccggagccgg gactgtcggg cgtacacaaa tcgcccgcag aagcgcggcc gtctggaccg
8221 atggctgtgt agaagtactc gccgatagtg gaaaccgacg ccccagcact cgtccgaggg
8281 caaaggaata agccaaggaa cctctggatt acaaaatttg tgaaagattg actggtattc
8341 ttaactatgt tgctcctttt acgctatgtg gatacgctgc tttaatgcct ttgtatcagt
8401 taacttgttt attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac
8461 aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc
8521 ttatcatgtc tggaattgac tcaaatgatg tcaattagtc tatcagaagc tcatctggtc
8581 tcccttccgg gggacaagac atccctgttt aatatttaaa cagcagtgtt cccaaactgg
8641 gttcttatat cccttgctct ggtcaaccag gttgcagggt ttcctgtcct cacaggaacg
8701 aagtccctaa agaaacagtg gcagccaggt ttagccccgg aattgactgg attccttttt
8761 tagggcccat tggtatggct ttttccccgt atccccccag gtgtctgcag gctcaaagag
8821 cagcgagaag cgttcagagg aaagcgatcc cgtgccacct tccccgtgcc cgggctgtcc
8881 ccgcacgctg ccggctcggg gatgcggggg gagcgccgga ccggagcgga gccccgggcg
8941 gctcgctgct gccccctagc gggggaggga cgtaattaca tccctggggg ctttgggggg
9001 gggctgtccc tgatatctat aacaagaaaa tatatatata ataagttatc acgtaagtag
9061 aacatgaaat aacaatataa ttatcgtatg agttaaatct taaaagtcac gtaaaagata
9121 atcatgcgtc attttgactc acgcggtcgt tatagttcaa aatcagtgac acttaccgca
9181 ttgacaagca cgcctcacgg gagctccaag cggcgactga gatgtcctaa atgcacagcg
9241 acggattcgc gctatttaga aagagagagc aatatttcaa gaatgcatgc gtcaatttta
9301 cgcagactat ctttctaggg ttaatctagc tgcatcagga tcatatcgtc gggtcttttt
9361 tccggctcag tcatcgccca agctggcgct atctgggcat cggggaggaa gaagcccgtg
9421 ccttttcccg cgaggttgaa gcggcatgga aagagtttgc cgaggatgac tgctgctgca
9481 ttgacgttga gcgaaaacgc acgtttacca tgatgattcg ggaaggtgtg gccatgcacg
9541 cctttaacgg tgaactgttc gttcaggcca cctgggatac cagttcgtcg cggcttttcc
9601 ggacacagtt ccggatggtc agcccgaagc gcatcagcaa cccgaacaat accggcgaca
9661 gccggaactg ccgtgccggt gtgcagatta atgacagcgg tgcggcgctg ggatattacg
9721 tcagcgagga cgggtatcct ggctggatgc cgcagaaatg gacatggata ccccgtgagt
9781 tacccggcgg gcgcgcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta
9841 tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc
9901 ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg
9961 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
10021 tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg
10081 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
10141 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
10201 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
10261 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
10321 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
10381 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
10441 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
10501 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
10561 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
10621 gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct
10681 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
10741 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
10801 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
10861 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
10921 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg
10981 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
11041 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc
11101 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc
11161 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
11221 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
11281 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
11341 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
11401 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
11461 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
11521 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
11581 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
11641 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
11701 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
11761 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
11821 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
11881 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
11941 atttccccga aaagtgccac ctaaattgta agcgttaata ttttgttaaa attcgcgtta
12001 aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat
12061 aaatcaaaag aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca
12121 ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc
12181 ccactacgtg aaccatcacc ctaatcaagt tttttggggt cgaggtgccg taaagcacta
12241 aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg
12301 gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg
12361 gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc
12421 cattcgccat tcaggctgcg caactgttgg gaagggcgat cggtgcgggc ctcttcgcta
12481 ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat taagttgggt aacgccaggg
12541 ttttcccagt cacgacgttg taaaacgacg gccagtgagc gcgcctcgtt cattcacgtt
12601 tttgaacccg tggaggacgg gcagactcgc ggtgcaaatg tgttttacag cgtgatggag
12661 cagatgaaga tgctcgacac gctgcagaac acgcagctag attaacccta gaaagataat
12721 catattgtga cgtacgttaa agataatcat gtgtaaaatt gacgcatgtg ttttatcggt
12781 ctgtatatcg aggtttattt attaatttga atagatatta agttttatta tatttacact
12841 tacatactaa taataaattc aacaaacaat ttatttatgt ttatttattt attaaaaaaa
12901 acaaaaactc aaaatttctt ctataaagta acaaaacttt tatgagggac agcccccccc
12961 caaagccccc agggatgtaa ttacgtccct cccccgctag ggggcagcag cgagccgccc
13021 ggggctccgc tccggtccgg cgctcccccc gcatccccga gccggcagcg tgcggggaca
13081 gcccgggcac ggggaaggtg gcacgggatc gctttcctct gaacgcttct cgctgctctt
13141 tgagcctgca gacacctggg gggatacggg gaaaaggcct ccacggccac tagtctttcg
13201 tcttcaagaa ttcctcgagt ttactcccta tcagtgatag agaacgtatg aagagtttac
13261 tccctatcag tgatagagaa cgtatgcaga ctttactccc tatcagtgat agagaacgta
13321 taaggagttt actccctatc agtgatagag aacgtatgac cagtttactc cctatcagtg
13381 atagagaacg tatctacagt ttactcccta tcagtgatag agaa
//
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