| 质粒名称: | pB-CAGGS-dCas9-KRAB-MeCP2 |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 110824 |
| 存储实验室: | Alejandro Chavez, George Church |
|
载体骨架基本信息 |
|
| 载体名称: | pB-CAGGS |
| 载体出品公司: | Clontech |
| 空载体大小: | 12882 bp |
| 完整质粒大小: | 12885 bp |
| 载体类型: | 哺乳细胞表达 |
| 筛选标记: | Blasticidin |
| 载体抗性: | 氨苄霉素 |
| 生长温度: | 30 ℃ |
| 宿主菌: | NEB Stable |
| 拷贝数: | 高拷贝 |
|
插入基因信息 |
|
| 插入基因名称: | dCas9-KRAB-MeCP2 / dCas9-KM |
| 物种来源: | Synthetic |
| 插入大小: | 5300 bp |
| 基因突变: | Cas9 mutations to make dCas9=D10A+D839A+H840A+N863A |
| 启动子: | CAGGS |
| 克隆方法: | Gibson Cloning |
| 5’测序引物 | TCAAGTACTTCGACACCACCA |
|
文献引用方法 |
|
| 材料和方法部分: | pB-CAGGS-dCas9-KRAB-MeCP2 was a gift from Alejandro Chavez & George Church (Addgene plasmid # 110824 ; http://n2t.net/addgene:110824 ; RRID:Addgene_110824) |
| 参考文献部分: | An enhanced CRISPR repressor for targeted mammalian gene regulation. Yeo NC, Chavez A, Lance-Byrne A, Chan Y, Menn D, Milanova D, Kuo CC, Guo X, Sharma S, Tung A, Cecchi RJ, Tuttle M, Pradhan S, Lim ET, Davidsohn N, Ebrahimkhani MR, Collins JJ, Lewis NE, Kiani S, Church GM. Nat Methods. 2018 Jul 16. pii: 10.1038/s41592-018-0048-5. doi: 10.1038/s41592-018-0048-5. 10.1038/s41592-018-0048-5 PubMed 30013045 |
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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-CAGGS-dCas9-KRAB-MeCP2 12885 bp ds-DNA circular SYN 12-MAY-2021
DEFINITION PiggyBac compatible plasmid expressing dCas9-KRAB-MeCP2.
ACCESSION .
VERSION .
KEYWORDS pB-CAGGS-dCas9-KRAB-MeCP2
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 12885)
AUTHORS Yeo NC, Chavez A, Lance-Byrne A, Chan Y, Menn D, Milanova D, Kuo CC,
Guo X, Sharma S, Tung A, Cecchi RJ, Tuttle M, Pradhan S, Lim ET,
Davidsohn N, Ebrahimkhani MR, Collins JJ, Lewis NE, Kiani S, Church
GM
TITLE An enhanced CRISPR repressor for targeted mammalian gene regulation.
JOURNAL Nat Methods. 2018 Jul 16. pii: 10.1038/s41592-018-0048-5. doi:
10.1038/s41592-018-0048-5.
PUBMED 30013045
REFERENCE 2 (bases 1 to 12885)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58
http://www.biofeng.com
FEATURES Location/Qualifiers
source 1..12885
/organism="synthetic DNA construct"
/mol_type="other DNA"
promoter complement(1..105)
/gene="bla"
/label=AmpR promoter
rep_origin complement(131..586)
/direction=LEFT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(268..289)
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind 480..499
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 713..735
/label=M13/pUC Forward
/note="In lacZ gene"
primer_bind 727..744
/label=M13 Forward
/note="In lacZ gene. Also called M13-F20 or M13 (-21)
Forward"
primer_bind 728..744
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
repeat_region 874..936
/label=piggyBac right (3') inverted repeat
/note="piggyBac transposon-specific inverted terminal
repeat sequence (ITR)"
enhancer 1357..1736
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
intron 2016..3032
/label=chimeric intron
/note="chimera between introns from chicken beta-actin and
rabbit beta-globin"
primer_bind 2956..2978
/label=pCAGGS-5
/note="Chimeric intron in CAG promoter, forward primer"
primer_bind 3040..3059
/label=pCAG-F
/note="Rabbit beta-globin intron, for pCAG plasmids,
forward primer"
regulatory 3129..3138
/regulatory_class="other"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
CDS 3135..7238
/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 7251..7271
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 7332..7526
/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
QLTKPDVILRLEKGEEPWLV"
CDS 7560..7580
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 7590..8447
/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(8502..8521)
/label=TK-pA-R
/note="Thymidine kinase polyA, reverse primer"
polyA_signal 8546..8594
/label=HSV TK poly(A) signal
/note="herpes simplex virus thymidine kinase
polyadenylation signal (Cole and Stacy, 1985)"
promoter 8826..9037
/label=EF-1-alpha core promoter
/note="core promoter for human elongation factor
EF-1-alpha"
regulatory 9047..9056
/regulatory_class="other"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
CDS 9053..9451
/codon_start=1
/gene="Aspergillus terreus BSD"
/product="blasticidin S deaminase"
/label=BSD
/note="confers resistance to blasticidin"
/translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
KAIVKDSDGQPTAVGIRELLPSGYVWEG"
polyA_signal 9455..9576
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(9492..9511)
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
primer_bind 9546..9565
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
repeat_region 10339..10373
/label=piggyBac left (5') inverted repeat
/note="piggyBac transposon-specific inverted terminal
repeat sequence (ITR)"
primer_bind complement(10864..10880)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(10864..10880)
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind complement(10877..10899)
/label=M13/pUC Reverse
/note="In lacZ gene"
protein_bind 10888..10904
/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(10912..10942)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 10957..10978
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(11095..11112)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(11266..11854)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(11346..11365)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS complement(12025..12885)
/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 12648..12667
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
ORIGIN
1 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
61 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
121 agtgccacct aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa
181 atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa
241 tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac
301 gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa
361 ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct
421 aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa
481 gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc
541 gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtccca ttcgccattc
601 aggctgcgca actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg
661 gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca
721 cgacgttgta aaacgacggc cagtgagcgc gcctcgttca ttcacgtttt tgaacccgtg
781 gaggacgggc agactcgcgg tgcaaatgtg ttttacagcg tgatggagca gatgaagatg
841 ctcgacacgc tgcagaacac gcagctagat taaccctaga aagataatca tattgtgacg
901 tacgttaaag ataatcatgt gtaaaattga cgcatgtgtt ttatcggtct gtatatcgag
961 gtttatttat taatttgaat agatattaag ttttattata tttacactta catactaata
1021 ataaattcaa caaacaattt atttatgttt atttatttat taaaaaaaac aaaaactcaa
1081 aatttcttct ataaagtaac aaaactttta tgagggacag ccccccccca aagcccccag
1141 ggatgtaatt acgtccctcc cccgctaggg ggcagcagcg agccgcccgg ggctccgctc
1201 cggtccggcg ctccccccgc atccccgagc cggcagcgtg cggggacagc ccgggcacgg
1261 ggaaggtggc acgggatcgc tttcctctga acgcttctcg ctgctctttg agcctgcaga
1321 cacctggggg gatacgggga aaaggcctcc acggccgaca ttgattattg actagttatt
1381 aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat
1441 aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa
1501 taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg
1561 agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc
1621 cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct
1681 tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt accatggtcg
1741 aggtgagccc cacgttctgc ttcactctcc ccatctcccc cccctcccca cccccaattt
1801 tgtatttatt tattttttaa ttattttgtg cagcgatggg ggcggggggg gggggggggs
1861 gcgcgccggg gggggggggg gggggggggg gggggggggg ggsgrggsgg rgaggtgcgg
1921 cggcagccaa tcagagcggc gcgctccgaa agtttccttt tatggcgagg cggcggcggc
1981 ggcggcccta taaaaagcga agcgcgcggc gggcgggagt cgctgcgcgc tgccttcgcc
2041 ccgtgccccg ctccgccgcc gcctcgcgcc gcccgccccg gctctgactg accgcgttac
2101 tcccacaggt gagcgggcgg gacggccctt ctcctccggg ctgtaattag cgcttggttt
2161 aatgacggct tgtttctttt ctgtggctgc gtgaaagcct tgaggggctc cgggagggcc
2221 ctttgtgcgg ggggagcggc tcggggggtg cgtgcgtgtg tgtgtgcgtg gggagcgccg
2281 cgtgcggctc cgcgctgccc ggcggctgtg agcgctgcgg gcgcggcgcg gggctttttg
2341 cgctccgcag tgtgcgcgag gggagcgcgg ccgggggcgg tgccccgcgg tgcggggggg
2401 gctgcgaggg gaacaaaggc tgcgtgcggg gtgtgtgcgt gggggggtga gcagggggtg
2461 tgggcgcgtc ggtcgggctg caaccccccc tgcacccccc tccccgagtt gctgagcacg
2521 gcccggcttc gggtgcgggg ctccgtacgg ggcgtggcgc ggggctcgcc gtgccgggcg
2581 gggggtggcg gcaggtgggg gtgccgggcg gggcggggcc gcctcgggcc ggggrgggct
2641 cgggggaggg gcgcggcggc ccccggagcg ccggcggctg tcgaggcgcg gcgagccgca
2701 gccattgcct tttatggtaa tcgtgcgaga gggcgcaggg acttcctttg tcccaaatct
2761 gtgcggagcc gaaatctggg aggcgccgcc gcaccccctc tagcgggcgc ggggcgaagc
2821 ggtgcggcgc cggcaggaag gaaatgggcg gggagggcct tcgtgcgtcg ccgcgccgcc
2881 gtccccttct ccctctccag cctcggggct gtccgcgggg ggacggctgc cttcgggggg
2941 gacggggcag ggcggggttc ggcttctggc gtgtgaccgg cggctctaga gcctctgcta
3001 accatgttca tgccttcttc tttttcctac agctcctggg caacgtgctg gttattgtgc
3061 tgtctcatca ttttggcaaa gaatctagat accctcgtaa aggtctagag ctagcggatc
3121 gaacccttgc caccatggac aagaagtact ccattgggct cgctatcggc acaaacagcg
3181 tcggctgggc cgtcattacg gacgagtaca aggtgccgag caaaaaattc aaagttctgg
3241 gcaataccga tcgccacagc ataaagaaga acctcattgg cgccctcctg ttcgactccg
3301 gggagacggc cgaagccacg cggctcaaaa gaacagcacg gcgcagatat acccgcagaa
3361 agaatcggat ctgctacctg caggagatct ttagtaatga gatggctaag gtggatgact
3421 ctttcttcca taggctggag gagtcctttt tggtggagga ggataaaaag cacgagcgcc
3481 acccaatctt tggcaatatc gtggacgagg tggcgtacca tgaaaagtac ccaaccatat
3541 atcatctgag gaagaagctt gtagacagta ctgataaggc tgacttgcgg ttgatctatc
3601 tcgcgctggc gcatatgatc aaatttcggg gacacttcct catcgagggg gacctgaacc
3661 cagacaacag cgatgtcgac aaactcttta tccaactggt tcagacttac aatcagcttt
3721 tcgaagagaa cccgatcaac gcatccggag ttgacgccaa agcaatcctg agcgctaggc
3781 tgtccaaatc ccggcggctc gaaaacctca tcgcacagct ccctggggag aagaagaacg
3841 gcctgtttgg taatcttatc gccctgtcac tcgggctgac ccccaacttt aaatctaact
3901 tcgacctggc cgaagatgcc aagcttcaac tgagcaaaga cacctacgat gatgatctcg
3961 acaatctgct ggcccagatc ggcgaccagt acgcagacct ttttttggcg gcaaagaacc
4021 tgtcagacgc cattctgctg agtgatattc tgcgagtgaa cacggagatc accaaagctc
4081 cgctgagcgc tagtatgatc aagcgctatg atgagcacca ccaagacttg actttgctga
4141 aggcccttgt cagacagcaa ctgcctgaga agtacaagga aattttcttc gatcagtcta
4201 aaaatggcta cgccggatac attgacggcg gagcaagcca ggaggaattt tacaaattta
4261 ttaagcccat cttggaaaaa atggacggca ccgaggagct gctggtaaag cttaacagag
4321 aagatctgtt gcgcaaacag cgcactttcg acaatggaag catcccccac cagattcacc
4381 tgggcgaact gcacgctatc ctcaggcggc aagaggattt ctaccccttt ttgaaagata
4441 acagggaaaa gattgagaaa atcctcacat ttcggatacc ctactatgta ggccccctcg
4501 cccggggaaa ttccagattc gcgtggatga ctcgcaaatc agaagagacc atcactccct
4561 ggaacttcga ggaagtcgtg gataaggggg cctctgccca gtccttcatc gaaaggatga
4621 ctaactttga taaaaatctg cctaacgaaa aggtgcttcc taaacactct ctgctgtacg
4681 agtacttcac agtttataac gagctcacca aggtcaaata cgtcacagaa gggatgagaa
4741 agccagcatt cctgtctgga gagcagaaga aagctatcgt ggacctcctc ttcaagacga
4801 accggaaagt taccgtgaaa cagctcaaag aagactattt caaaaagatt gaatgtttcg
4861 actctgttga aatcagcgga gtggaggatc gcttcaacgc atccctggga acgtatcacg
4921 atctcctgaa aatcattaaa gacaaggact tcctggacaa tgaggagaac gaggacattc
4981 ttgaggacat tgtcctcacc cttacgttgt ttgaagatag ggagatgatt gaagaacgct
5041 tgaaaactta cgctcatctc ttcgacgaca aagtcatgaa acagctcaag aggcgccgat
5101 atacaggatg ggggcggctg tcaagaaaac tgatcaatgg gatccgagac aagcagagtg
5161 gaaagacaat cctggatttt cttaagtccg atggatttgc caaccggaac ttcatgcagt
5221 tgatccatga tgactctctc acctttaagg aggacatcca gaaagcacaa gtttctggcc
5281 agggggacag tcttcacgag cacatcgcta atcttgcagg tagcccagct atcaaaaagg
5341 gaatactgca gaccgttaag gtcgtggatg aactcgtcaa agtaatggga aggcataagc
5401 ccgagaatat cgttatcgag atggcccgag agaaccaaac tacccagaag ggacagaaga
5461 acagtaggga aaggatgaag aggattgaag agggtataaa agaactgggg tcccaaatcc
5521 ttaaggaaca cccagttgaa aacacccagc ttcagaatga gaagctctac ctgtactacc
5581 tgcagaacgg cagggacatg tacgtggatc aggaactgga catcaatcgg ctctccgact
5641 acgacgtggc tgctatcgtg ccccagtctt ttctcaaaga tgattctatt gataataaag
5701 tgttgacaag atccgataaa gctagaggga agagtgataa cgtcccctca gaagaagttg
5761 tcaagaaaat gaaaaattat tggcggcagc tgctgaacgc caaactgatc acacaacgga
5821 agttcgataa tctgactaag gctgaacgag gtggcctgtc tgagttggat aaagccggct
5881 tcatcaaaag gcagcttgtt gagacacgcc agatcaccaa gcacgtggcc caaattctcg
5941 attcacgcat gaacaccaag tacgatgaaa atgacaaact gattcgagag gtgaaagtta
6001 ttactctgaa gtctaagctg gtctcagatt tcagaaagga ctttcagttt tataaggtga
6061 gagagatcaa caattaccac catgcgcatg atgcctacct gaatgcagtg gtaggcactg
6121 cacttatcaa aaaatatccc aagcttgaat ctgaatttgt ttacggagac tataaagtgt
6181 acgatgttag gaaaatgatc gcaaagtctg agcaggaaat aggcaaggcc accgctaagt
6241 acttctttta cagcaatatt atgaattttt tcaagaccga gattacactg gccaatggag
6301 agattcggaa gcgaccactt atcgaaacaa acggagaaac aggagaaatc gtgtgggaca
6361 agggtaggga tttcgcgaca gtccggaagg tcctgtccat gccgcaggtg aacatcgtta
6421 aaaagaccga agtacagacc ggaggcttct ccaaggaaag tatcctcccg aaaaggaaca
6481 gcgacaagct gatcgcacgc aaaaaagatt gggaccccaa gaaatacggc ggattcgatt
6541 ctcctacagt cgcttacagt gtactggttg tggccaaagt ggagaaaggg aagtctaaaa
6601 aactcaaaag cgtcaaggaa ctgctgggca tcacaatcat ggagcgatca agcttcgaaa
6661 aaaaccccat cgactttctc gaggcgaaag gatataaaga ggtcaaaaaa gacctcatca
6721 ttaagcttcc caagtactct ctctttgagc ttgaaaacgg ccggaaacga atgctcgcta
6781 gtgcgggcga gctgcagaaa ggtaacgagc tggcactgcc ctctaaatac gttaatttct
6841 tgtatctggc cagccactat gaaaagctca aagggtctcc cgaagataat gagcagaagc
6901 agctgttcgt ggaacaacac aaacactacc ttgatgagat catcgagcaa ataagcgaat
6961 tctccaaaag agtgatcctc gccgacgcta acctcgataa ggtgctttct gcttacaata
7021 agcacaggga taagcccatc agggagcagg cagaaaacat tatccacttg tttactctga
7081 ccaacttggg cgcgcctgca gccttcaagt acttcgacac caccatagac agaaagcggt
7141 acacctctac aaaggaggtc ctggacgcca cactgattca tcagtcaatt acggggctct
7201 atgaaacaag aatcgacctc tctcagctcg gtggagacag cagggctgac cccaagaaga
7261 agaggaaggt gagtggtgga ggaagtggcg ggtcagggtc gatggacgcg aaatcactta
7321 cggcatggtc gagaacactg gttacgttca aggacgtgtt tgtggacttt acacgtgagg
7381 agtggaaatt gctggatact gcgcaacaaa ttgtgtatcg aaatgtcatg cttgagaatt
7441 acaagaacct cgtcagtctc ggataccagt tgacgaaacc ggatgtgatc cttaggctcg
7501 aaaaggggga agaaccttgg ctggtatcgg gaggtggttc gggtggctct ggatcaagcc
7561 caaagaagaa acggaaggtg gaagcctcag tgcaggtgaa aagggtgctg gaaaaatccc
7621 ccggcaaact cctcgtgaag atgcccttcc aggcttcccc tggcggaaaa ggtgaagggg
7681 gtggcgcaac cacatctgcc caggtcatgg tcatcaagcg acctggaagg aaaagaaagg
7741 ccgaggctga ccctcaggcc attccaaaga aacggggacg caagccaggg tccgtggtcg
7801 cagctgcagc agctgaggct aagaaaaagg cagtgaagga aagctccatc cgcagtgtgc
7861 aggagactgt cctgcccatc aagaagagga agactaggga gaccgtgtcc atcgaggtca
7921 aagaagtggt caagcccctg ctcgtgtcca ccctgggcga aaaatctgga aaggggctca
7981 aaacatgcaa gtcacctgga cggaaaagca aggagtctag tccaaagggg cgctcaagct
8041 ccgcttctag tccccctaaa aaggaacacc atcaccatca ccatcacgcc gagtctccta
8101 aggctcctat gccactgctc ccaccacctc caccacctga gccacagtca agcgaagacc
8161 ccatcagccc acccgagcct caggatctgt cctctagtat ttgcaaagag gaaaagatgc
8221 ccagagcagg cagcctggag agtgatggct gtccaaaaga acccgccaag acccagccta
8281 tggtggcagc cgctgcaact accaccacaa ccacaactac cacagtggcc gaaaaataca
8341 agcatcgcgg cgagggcgaa cgaaaggaca ttgtgtcaag ctccatgccc agacctaacc
8401 gggaggaacc agtcgatagt aggacacccg tgactgagag agtctcatag aagaattcga
8461 tccctaccgg ttagtaatga gtttaaacgg gggaggctaa ctgaaacacg gaaggagaca
8521 ataccggaag gaacccgcgc tatgacggca ataaaaagac agaataaaac gcacgggtgt
8581 tgggtcgttt gttcataaac gcggggttcg gtcccagggc tggcactctg tcgatacccc
8641 accgagaccc cattggggcc aatacgcccg cgtttcttcc ttttccccac cccacccccc
8701 aagttcgggt gaaggcccag ggctcgcagc caacgtcggg gcggcaggcc ctgccatagc
8761 agatctgcga aatcggatcc gcggccgcac tgattctagc tgcagaggct ccggtgcccg
8821 tcagtgggca gagcgcacat cgcccacagt ccccgagaag ttggggggag gggtcggcaa
8881 ttgaaccggt gcctagagaa ggtggcgcgg ggtaaactgg gaaagtgatg tcgtgtactg
8941 gctccgcctt tttcccgagg gtgggggaga accgtatata agtgcagtag tcgccgtgaa
9001 cgttcttttt cgcaacgggt ttgccgccag aacacaggat cctcgagcca ccatggccaa
9061 gcctttgtct caagaagaat ccaccctcat tgaaagagca acggctacaa tcaacagcat
9121 ccccatctct gaagactaca gcgtcgccag cgcagctctc tctagcgacg gccgcatctt
9181 cactggtgtc aatgtatatc attttactgg gggaccttgt gcagaactcg tggtgctggg
9241 cactgctgct gctgcggcag ctggcaacct gacttgtatc gtcgcgatcg gaaatgagaa
9301 caggggcatc ttgagcccct gcggacggtg ccgacaggtg cttctcgatc tgcatcctgg
9361 gatcaaagcc atagtgaagg acagtgatgg acagccgacg gcagttggga ttcgtgaatt
9421 gctgccctct ggttatgtgt gggagggcta agttaacttg tttattgcag cttataatgg
9481 ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc
9541 tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctggaatt gactcaaatg
9601 atgtcaatta gtctatcaga agctcatctg gtctcccttc cgggggacaa gacatccctg
9661 tttaatattt aaacagcagt gttcccaaac tgggttctta tatcccttgc tctggtcaac
9721 caggttgcag ggtttcctgt cctcacagga acgaagtccc taaagaaaca gtggcagcca
9781 ggtttagccc cggaattgac tggattcctt ttttagggcc cattggtatg gctttttccc
9841 cgtatccccc caggtgtctg caggctcaaa gagcagcgag aagcgttcag aggaaagcga
9901 tcccgtgcca ccttccccgt gcccgggctg tccccgcacg ctgccggctc ggggatgcgg
9961 ggggagcgcc ggaccggagc ggagccccgg gcggctcgct gctgccccct agcgggggag
10021 ggacgtaatt acatccctgg gggctttggg ggggggctgt ccctgatatc tataacaaga
10081 aaatatatat ataataagtt atcacgtaag tagaacatga aataacaata taattatcgt
10141 atgagttaaa tcttaaaagt cacgtaaaag ataatcatgc gtcattttga ctcacgcggt
10201 cgttatagtt caaaatcagt gacacttacc gcattgacaa gcacgcctca cgggagctcc
10261 aagcggcgac tgagatgtcc taaatgcaca gcgacggatt cgcgctattt agaaagagag
10321 agcaatattt caagaatgca tgcgtcaatt ttacgcagac tatctttcta gggttaatct
10381 agctgcatca ggatcatatc gtcgggtctt ttttccggct cagtcatcgc ccaagctggc
10441 gctatctggg catcggggag gaagaagccc gtgccttttc ccgcgaggtt gaagcggcat
10501 ggaaagagtt tgccgaggat gactgctgct gcattgacgt tgagcgaaaa cgcacgttta
10561 ccatgatgat tcgggaaggt gtggccatgc acgcctttaa cggtgaactg ttcgttcagg
10621 ccacctggga taccagttcg tcgcggcttt tccggacaca gttccggatg gtcagcccga
10681 agcgcatcag caacccgaac aataccggcg acagccggaa ctgccgtgcc ggtgtgcaga
10741 ttaatgacag cggtgcggcg ctgggatatt acgtcagcga ggacgggtat cctggctgga
10801 tgccgcagaa atggacatgg ataccccgtg agttacccgg cgggcgcgct tggcgtaatc
10861 atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg
10921 agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat
10981 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
11041 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct
11101 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
11161 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
11221 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
11281 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
11341 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
11401 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
11461 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
11521 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
11581 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
11641 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
11701 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
11761 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
11821 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
11881 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
11941 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
12001 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
12061 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
12121 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc
12181 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
12241 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
12301 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg
12361 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
12421 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
12481 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
12541 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
12601 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
12661 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
12721 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
12781 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
12841 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcat
//
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