| 质粒名称: | pSLQ2817 pPB: CAG-PYL1-VPR-IRES-Puro-WPRE-SV40PA PGK-ABI-tagBFP-SpdCas9 |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 84239 |
| 存储实验室: | Stanley Qi |
|
载体骨架基本信息 |
|
| 载体名称: | PB |
| 载体出品公司: | System Biosciences |
| 载体抗性: | 氨苄青霉素 |
| 完整质粒大小: | 15416 bp |
| 载体类型: | 哺乳动物表达，CRISPR，合成生物学,PiggyBac |
| 筛选标记: | 嘌呤霉素 |
| 高拷贝/低拷贝: | 高拷贝 |
| 生长条件: | 37 ℃ |
|
插入基因信息 |
|
| 插入基因名称: | ABI-tagBFP-Sp dCas9 |
| 物种来源: | A. thaliana (mustard weed), Synthetic; S. pyogenes |
| 插入大小: | 5880 bp |
| 启动子 | PGK |
| 标签/融合蛋白: |
ABI（插入件上的N端子）
tagBFP（插入件上的N端子）
HA tag（插入C端子）
2xNLS（SV40）（插入件上的C端子）
|
| 克隆方法: | Ligation Independent克隆 |
| 5' 测序引物 | gaaggtcctccggaggcc |
| 3' 测序引物 | CGGTCTGTATATCGAGGTTTATTTATTAATTTG |
|
文献引用方法 |
|
| 材料和方法部分: | pSLQ2817 pPB: CAG-PYL1-VPR-IRES-Puro-WPRE-SV40PA PGK-ABI-tagBFP-SpdCas9 was a gift from Stanley Qi (Addgene plasmid # 84239 ; http://n2t.net/addgene:84239 ; RRID:Addgene_84239) |
| 参考文献部分: | Complex transcriptional modulation with orthogonal and inducible dCas9 regulators. Gao Y, Xiong X, Wong S, Charles EJ, Lim WA, Qi LS. Nat Methods. 2016 Oct 24. doi: 10.1038/nmeth.4042. 10.1038/nmeth.4042 PubMed 27776111 |
pSLQ2817 pPB: CAG-PYL1-VPR-IRES-Puro-WPRE-SV40PA PGK-ABI-tag
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 pSLQ2817 pPB: CAG-PYL1-VPR-IRES-Puro-WPRE-SV40PA
PGK-ABI-tagBF... 15416 bp ds-DNA circular SYN 06-AUG-2018
DEFINITION Expresses ABA-inducible VPR-Sp dCas9.
ACCESSION .
VERSION .
KEYWORDS pSLQ2817 pPB: CAG-PYL1-VPR-IRES-Puro-WPRE-SV40PA
PGK-ABI-tagBF...
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 15416)
AUTHORS Gao Y, Xiong X, Wong S, Charles EJ, Lim WA, Qi LS
TITLE Complex transcriptional modulation with orthogonal and inducible
dCas9 regulators.
JOURNAL Nat Methods. 2016 Oct 24. doi: 10.1038/nmeth.4042.
PUBMED 27776111
REFERENCE 2 (bases 1 to 15416)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Aug 6, 2018 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..15416
/organism="synthetic DNA construct"
/mol_type="other DNA"
promoter 7..284
/label=chicken beta-actin promoter
intron 286..1303
/label=chimeric intron
/note="chimera between introns from chicken beta-actin and
rabbit beta-globin"
primer_bind 1227..1249
/label=pCAGGS-5
/note="Chimeric intron in CAG promoter, forward primer"
primer_bind 1311..1330
/label=pCAG-F
/note="Rabbit beta-globin intron, for pCAG plasmids,
forward primer"
regulatory 1358..1367
/regulatory_class="other"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
CDS 1940..2089
/codon_start=1
/product="tetrameric repeat of the minimal activation
domain of herpes simplex virus VP16 (Beerli et al., 1998)"
/label=VP64
/translation="DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDF
DLDML"
CDS 2096..2116
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 2546..2917
/codon_start=1
/product="transcriptional activation domain of human RelA,
also known as p65 (O'Shea and Perkins, 2008)"
/label=RelA (p65) AD
/translation="PTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNS
EFQQLLNQGIPVAPHTTEPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGD
EDFSSIADMDFSALLSQISS"
CDS 2936..3505
/codon_start=1
/product="transcriptional activation domain from the human
herpesvirus 4 (Epstein-Barr virus) replication and
transcription activator Rta/BRLF1 (Hardwick et al., 1992;
Chavez et al., 2015)"
/label=Rta AD
/translation="RDSREGMFLPKPEAGSAISDVFEGREVCQPKRIRPFHPPGSPWAN
RPLPASLAPTPTGPVHEPVGSLTPAPVPQPLDPAPAVTPEASHLLEDPDEETSQAVKAL
REMADTVIPQKEEAAICGQMDLSHPPPRGHLDELTTTLESMTEDLNLDSPLTPELNEIL
DTFLNDECLLHAMHISTGLSIFDTSLF"
misc_feature 3523..4108
/label=IRES2
/note="internal ribosome entry site (IRES) of the
encephalomyocarditis virus (EMCV)"
primer_bind complement(3690..3707)
/label=IRES reverse
/note="IRES internal ribosome entry site, reverse primer.
Also called pCDH-rev"
primer_bind 3917..3936
/label=IRES-F
/note="IRES internal ribosome entry site, forward primer"
CDS 4130..4729
/codon_start=1
/gene="pac from Streptomyces alboniger"
/product="puromycin N-acetyltransferase"
/label=PuroR
/note="confers resistance to puromycin"
/translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
primer_bind complement(4130..4149)
/label=Puro-R
/note="Puromycin resistance gene, reverse primer. Also
called puro-variant-R"
primer_bind 4626..4646
/label=Puro-F
/note="Puromycin resistance gene, forward primer"
misc_feature 4767..5355
/label=WPRE
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
primer_bind complement(4820..4840)
/label=WPRE-R
/note="WPRE, reverse primer"
CDS complement(5238..5249)
/codon_start=1
/product="Factor Xa recognition and cleavage site"
/label=Factor Xa site
/translation="IEGR"
primer_bind complement(5394..5413)
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
primer_bind 5448..5467
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
promoter 5509..6008
/label=PGK promoter
/note="mouse phosphoglycerate kinase 1 promoter"
primer_bind complement(5544..5567)
/label=MSCV-rev
/note="Murine stem cell virus, reverse primer"
primer_bind 5941..5960
/label=mPGK-F
/note="Mouse PGK promoter, forward primer"
CDS 6968..7666
/codon_start=1
/product="monomeric blue fluorescent protein"
/label=TagBFP
/note="mammalian codon-optimized"
/translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
DLPSKLGHKLN"
regulatory 7682..7691
/regulatory_class="other"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
CDS 7688..11791
/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 11795..11821
/codon_start=1
/product="HA (human influenza hemagglutinin) epitope tag"
/label=HA
/translation="YPYDVPDYA"
CDS 11840..11860
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 11867..11887
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
repeat_region 12104..12166
/label=piggyBac right (3') inverted repeat
/note="piggyBac transposon-specific inverted terminal
repeat sequence (ITR)"
primer_bind complement(12217..12237)
/label=T3
/note="T3 promoter, forward primer"
promoter complement(12217..12235)
/label=T3 promoter
/note="promoter for bacteriophage T3 RNA polymerase"
primer_bind complement(12256..12272)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(12256..12272)
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind complement(12269..12291)
/label=M13/pUC Reverse
/note="In lacZ gene"
protein_bind 12280..12296
/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(12304..12334)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 12349..12370
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(12487..12504)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(12658..13246)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(12738..12757)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS complement(13417..14277)
/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 14040..14059
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
promoter complement(14278..14382)
/gene="bla"
/label=AmpR promoter
rep_origin complement(14408..14863)
/direction=LEFT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(14545..14566)
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind 14757..14776
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 14990..15012
/label=M13/pUC Forward
/note="In lacZ gene"
primer_bind 15004..15021
/label=M13 Forward
/note="In lacZ gene. Also called M13-F20 or M13 (-21)
Forward"
primer_bind 15005..15021
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
primer_bind 15031..15050
/label=T7
/note="T7 promoter, forward primer"
promoter 15031..15049
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
repeat_region 15079..15113
/label=piggyBac left (5') inverted repeat
/note="piggyBac transposon-specific inverted terminal
repeat sequence (ITR)"
ORIGIN
1 ccatggtcga ggtgagcccc acgttctgct tcactctccc catctccccc ccctccccac
61 ccccaatttt gtatttattt attttttaat tattttgtgc agcgatgggg gcgggggggg
121 ggggggggcg cgcgccaggc ggggcggggc ggggcgaggg gcggggcggg gcgaggcgga
181 gaggtgcggc ggcagccaat cagagcggcg cgctccgaaa gtttcctttt atggcgaggc
241 ggcggcggcg gcggccctat aaaaagcgaa gcgcgcggcg ggcggggagt cgctgcgacg
301 ctgccttcgc cccgtgcccc gctccgccgc cgcctcgcgc cgcccgcccc ggctctgact
361 gaccgcgtta ctcccacagg tgagcgggcg ggacggccct tctcctccgg gctgtaatta
421 gcgcttggtt taatgacggc ttgtttcttt tctgtggctg cgtgaaagcc ttgaggggct
481 ccgggagggc cctttgtgcg gggggagcgg ctcggggggt gcgtgcgtgt gtgtgtgcgt
541 ggggagcgcc gcgtgcggct ccgcgctgcc cggcggctgt gagcgctgcg ggcgcggcgc
601 ggggctttgt gcgctccgca gtgtgcgcga ggggagcgcg gccgggggcg gtgccccgcg
661 gtgcgggggg ggctgcgagg ggaacaaagg ctgcgtgcgg ggtgtgtgcg tgggggggtg
721 agcagggggt gtgggcgcgt cggtcgggct gcaacccccc ctgcaccccc ctccccgagt
781 tgctgagcac ggcccggctt cgggtgcggg gctccgtacg gggcgtggcg cggggctcgc
841 cgtgccgggc ggggggtggc ggcaggtggg ggtgccgggc ggggcggggc cgcctcgggc
901 cggggagggc tcgggggagg ggcgcggcgg cccccggagc gccggcggct gtcgaggcgc
961 ggcgagccgc agccattgcc ttttatggta atcgtgcgag agggcgcagg gacttccttt
1021 gtcccaaatc tgtgcggagc cgaaatctgg gaggcgccgc cgcaccccct ctagcgggcg
1081 cggggcgaag cggtgcggcg ccggcaggaa ggaaatgggc ggggagggcc ttcgtgcgtc
1141 gccgcgccgc cgtccccttc tccctctcca gcctcggggc tgtccgcggg gggacggctg
1201 ccttcggggg ggacggggca gggcggggtt cggcttctgg cgtgtgaccg gcggctctag
1261 agcctctgct aaccatgttc atgccttctt ctttttccta cagctcctgg gcaacgtgct
1321 ggttattgtg ctgtctcatc attttggcaa agaattcgcc accatgggtg ggggcgcgcc
1381 aactcaagac gaattcaccc aactctccca atcaatcgcc gagttccaca cgtaccaact
1441 cggtaacggc cgttgctcat ctctcctagc tcagcgaatc cacgcgccgc cggaaacagt
1501 atggtccgtg gtgagacgtt tcgataggcc acagatttac aaacacttca tcaaaagctg
1561 taacgtgagt gaagatttcg agatgcgagt gggatgcacg cgcgacgtga acgtgataag
1621 tggattaccg gcgaatacgt ctcgagagag attagatctg ttggacgatg atcggagagt
1681 gactgggttt agtataaccg gtggtgaaca taggctgagg aattataaat cggttacgac
1741 ggttcataga tttgagaaag aagaagaaga agaaaggatc tggaccgttg ttttggaatc
1801 ttatgttgtt gatgtaccgg aaggtaattc ggaggaagat acgagattgt ttgctgatac
1861 ggttattaga ttgaatcttc agaaacttgc ttcgatcact gaagctatga acgaattctc
1921 aagcgctgca ggtactagtg atgctttaga cgattttgac ttagatatgc ttggttcaga
1981 cgcgttagac gacttcgacc tagacatgtt aggctcagat gcattggacg acttcgattt
2041 agatatgttg ggctccgatg ccctagatga ctttgatcta gatatgctag gtagtcccaa
2101 aaagaagagg aaagtgggat cccagtatct gcccgacaca gatgatagac accgaatcga
2161 agagaaacgc aagcgaacgt atgaaacctt caaatcgatc atgaagaaat cgcccttctc
2221 gggtccgacc gatcccaggc ccccaccgag aaggattgcg gtcccgtccc gctcgtcggc
2281 cagcgtgccg aagcctgcgc cgcagcccta ccccttcacg tcgagcctga gcacaatcaa
2341 ttatgacgag ttcccgacga tggtgttccc ctcgggacaa atctcacaag cctcggcgct
2401 cgcaccagcg cctccccaag tccttccgca agcgcctgcc ccagcgcctg caccggcaat
2461 ggtgtccgcc ctcgcacagg cccctgcgcc cgtccccgtg ctcgcgcctg gaccgcccca
2521 ggcggtcgct ccaccggctc cgaagccgac gcaggccgga gagggaacac tctccgaagc
2581 acttcttcaa ctccagtttg atgacgagga tcttggagca ctccttggaa actcgacaga
2641 ccctgcggtg tttaccgacc tcgcgtcagt agataactcc gaatttcagc agcttttgaa
2701 ccagggtatc ccggtcgcgc cacatacaac ggagcccatg ttgatggaat accccgaagc
2761 aatcacgaga cttgtgacgg gagcgcagcg gcctcccgat cccgcacccg cacctttggg
2821 ggcacctggc ctccctaacg gacttttgag cggcgacgag gatttctcct ccatcgccga
2881 tatggatttc tcagccttgc tgtcacagat ttccagcggc tctggcagcg gcagccggga
2941 ttccagggaa gggatgtttt tgccgaagcc tgaggccggc tccgctatta gtgacgtgtt
3001 tgagggccgc gaggtgtgcc agccaaaacg aatccggcca tttcatcctc caggaagtcc
3061 atgggccaac cgcccactcc ccgccagcct cgcaccaaca ccaaccggtc cagtacatga
3121 gccagtcggg tcactgaccc cggcaccagt ccctcagcca ctggatccag cgcccgcagt
3181 gactcccgag gccagtcacc tgttggagga tcccgatgaa gagacgagcc aggctgtcaa
3241 agcccttcgg gagatggccg atactgtgat tccccagaag gaagaggctg caatctgtgg
3301 ccaaatggac ctttcccatc cgcccccaag gggccatctg gatgagctga caaccacact
3361 tgagtccatg accgaggatc tgaacctgga ctcacccctg accccggaat tgaacgagat
3421 tctggatacc ttcctgaacg acgagtgcct cttgcatgcc atgcatatca gcacaggact
3481 gtccatcttc gacacatctc tgttttgaat ttaaataatt cgcccctctc cctccccccc
3541 ccctaacgtt actggccgaa gccgcttgga ataaggccgg tgtgcgtttg tctatatgtt
3601 attttccacc atattgccgt cttttggcaa tgtgagggcc cggaaacctg gccctgtctt
3661 cttgacgagc attcctaggg gtctttcccc tctcgccaaa ggaatgcaag gtctgttgaa
3721 tgtcgtgaag gaagcagttc ctctggaagc ttcttgaaga caaacaacgt ctgtagcgac
3781 cctttgcagg cagcggaacc ccccacctgg cgacaggtgc ctctgcggcc aaaagccacg
3841 tgtataagat acacctgcaa aggcggcaca accccagtgc cacgttgtga gttggatagt
3901 tgtggaaaga gtcaaatggc tctcctcaag cgtattcaac aaggggctga aggatgccca
3961 gaaggtaccc cattgtatgg gatctgatct ggggcctcgg tgcacatgct ttacgtgtgt
4021 ttagtcgagg ttaaaaaacg tctaggcccc ccgaaccacg gggacgtggt tttcctttga
4081 aaaacacgat gataatatgg ccacaaccgg atccggagct accatgacca tgaccgagta
4141 caagcccacg gtgcgcctcg ccacccgcga cgacgtcccc agggccgtac gcaccctcgc
4201 cgccgcgttc gccgactacc ccgccacgcg ccacaccgtc gatccggacc gccacatcga
4261 gcgggtcacc gagctgcaag aactcttcct cacgcgcgtc gggctcgaca tcggcaaggt
4321 gtgggtcgcg gacgacggcg ccgcggtggc ggtctggacc acgccggaga gcgtcgaagc
4381 gggggcggtg ttcgccgaga tcggcccgcg catggccgag ttgagcggtt cccggctggc
4441 cgcgcagcaa cagatggaag gcctcctggc gccgcaccgg cccaaggagc ccgcgtggtt
4501 cctggccacc gtcggcgtct cgcccgacca ccagggcaag ggtctgggca gcgccgtcgt
4561 gctccccgga gtggaggcgg ccgagcgcgc cggggtgccc gccttcctgg agacctccgc
4621 gccccgcaac ctccccttct acgagcggct cggcttcacc gtcaccgccg acgtcgaggt
4681 gcccgaagga ccgcgcacct ggtgcatgac ccgcaagccc ggtgcctgag tttaaaccat
4741 gcaagcttga tatcaagctt atcgataatc aacctctgga ttacaaaatt tgtgaaagat
4801 tgactggtat tcttaactat gttgctcctt ttacgctatg tggatacgct gctttaatgc
4861 ctttgtatca tgctattgct tcccgtatgg ctttcatttt ctcctccttg tataaatcct
4921 ggttgctgtc tctttatgag gagttgtggc ccgttgtcag gcaacgtggc gtggtgtgca
4981 ctgtgtttgc tgacgcaacc cccactggtt ggggcattgc caccacctgt cagctccttt
5041 ccgggacttt cgctttcccc ctccctattg ccacggcgga actcatcgcc gcctgccttg
5101 cccgctgctg gacaggggct cggctgttgg gcactgacaa ttccgtggtg ttgtcgggga
5161 aatcatcgtc ctttccttgg ctgctcgcct gtgttgccac ctggattctg cgcgggacgt
5221 ccttctgcta cgtcccttcg gccctcaatc cagcggacct tccttcccgc ggcctgctgc
5281 cggctctgcg gcctcttccg cgtcttcgcc ttcgccctca gacgagtcgg atctcccttt
5341 gggccgcctc cccgcatcct tgtttattgc agcttataat ggttacaaat aaagcaatag
5401 catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa
5461 actcatcaat gtatcttatc atgtctggat cctgcaggta attctaccgg gtaggggagg
5521 cgcttttccc aaggcagtct ggagcatgcg ctttagcagc cccgctgggc acttggcgct
5581 acacaagtgg cctctggcct cgcacacatt ccacatccac cggtaggcgc caaccggctc
5641 cgttctttgg tggccccttc gcgccacctt ctactcctcc cctagtcagg aagttccccc
5701 ccgccccgca gctcgcgtcg tgcaggacgt gacaaatgga agtagcacgt ctcactaggc
5761 tcgtgcagat ggacagcacc gctgagcaat ggaagcgggt aggcctttgg ggcagcggcc
5821 aatagcagct ttgctccttc gctttctggg ctcagaggct gggaaggggt gggtccgggg
5881 gcgggctcag gggcgggctc aggggcgggg cgggcgcccg aaggtcctcc ggaggcccgg
5941 cattctgcac gcttcaaaag cgcacgtctg ccgcgctgtt ctcctcttcc tcatctccgg
6001 gcctttcgac ctgcagccca agcttgtcga cgccaccatg acgcgtgtgc ctttgtatgg
6061 ttttacttcg atttgtggaa gaagacctga gatggaagct gctgtttcga ctataccaag
6121 attccttcaa tcttcctctg gttcgatgtt agatggtcgg tttgatcctc aatccgccgc
6181 tcatttcttc ggtgtttacg acggccatgg cggttctcag gtagcgaact attgtagaga
6241 gaggatgcat ttggctttgg cggaggagat agctaaggag aaaccgatgc tctgcgatgg
6301 tgatacgtgg ctggagaagt ggaagaaagc tcttttcaac tcgttcctga gagttgactc
6361 ggagattgag tcagttgcgc cggagacggt tgggtcaacg tcggtggttg ccgttgtttt
6421 cccgtctcac atcttcgtcg ctaactgcgg tgactctaga gccgttcttt gccgcggcaa
6481 aactgcactt ccattatccg ttgaccataa accggataga gaagatgaag ctgcgaggat
6541 tgaagccgca ggagggaaag tgattcagtg gaatggagct cgtgttttcg gtgttctcgc
6601 catgtcgaga tccattggcg atagatactt gaaaccatcc atcattcctg atccggaagt
6661 gacggctgtg aagagagtaa aagaagatga ttgtctgatt ttggcgagtg acggggtttg
6721 ggatgtaatg acggatgaag aagcgtgtga gatggcaagg aagcggattc tcttgtggca
6781 caagaaaaac gcggtggctg gggatgcatc gttgctcgcg gatgagcgga gaaaggaagg
6841 gaaagatcct gcggcgatgt ccgcggctga gtatttgtca aagctggcga tacagagagg
6901 aagcaaagac aacataagtg tggtggtggt tgatttgaag catatgtcaa gcgctgcagg
6961 tgctaccatg agcgagctga ttaaggagaa catgcacatg aagctgtaca tggagggcac
7021 cgtggacaac catcacttca agtgcacatc cgagggcgaa ggcaagccct acgagggcac
7081 ccagaccatg agaatcaagg tggtcgaggg cggccctctc cccttcgcct tcgacatcct
7141 ggctactagc ttcctctacg gcagcaagac cttcatcaac cacacccagg gcatccccga
7201 cttcttcaag cagtccttcc ctgagggctt cacatgggag agagtcacca catacgaaga
7261 cgggggcgtg ctgaccgcta cccaggacac cagcctccag gacggctgcc tcatctacaa
7321 cgtcaagatc agaggggtga acttcacatc caacggccct gtgatgcaga agaaaacact
7381 cggctgggag gccttcaccg agacgctgta ccccgctgac ggcggcctgg aaggcagaaa
7441 cgacatggcc ctgaagctcg tgggcgggag ccatctgatc gcaaacatca agaccacata
7501 tagatccaag aaacccgcta agaacctcaa gatgcctggc gtctactatg tggactacag
7561 actggaaaga atcaaggagg ccaacaacga gacctacgtc gagcagcacg aggtggcagt
7621 ggccagatac tgcgacctcc ctagcaaact ggggcacaag cttaattcaa gcgctgcagg
7681 tgccaccatg gacaagaagt acagcatcgg cctggccatc ggcaccaact ctgtgggctg
7741 ggccgtgatc accgacgagt acaaggtgcc cagcaagaaa ttcaaggtgc tgggcaacac
7801 cgaccggcac agcatcaaga agaacctgat cggcgccctg ctgttcgaca gcggagaaac
7861 agccgaggcc acccggctga agagaaccgc cagaagaaga tacaccagac ggaagaaccg
7921 gatctgctat ctgcaagaga tcttcagcaa cgagatggcc aaggtggacg acagcttctt
7981 ccacagactg gaagagtcct tcctggtgga agaggataag aagcacgagc ggcaccccat
8041 cttcggcaac atcgtggacg aggtggccta ccacgagaag taccccacca tctaccacct
8101 gagaaagaaa ctggtggaca gcaccgacaa ggccgacctg cggctgatct atctggccct
8161 ggcccacatg atcaagttcc ggggccactt cctgatcgag ggcgacctga accccgacaa
8221 cagcgacgtg gacaagctgt tcatccagct ggtgcagacc tacaaccagc tgttcgagga
8281 aaaccccatc aacgccagcg gcgtggacgc caaggccatc ctgtctgcca gactgagcaa
8341 gagcagacgg ctggaaaatc tgatcgccca gctgcccggc gagaagaaga atggcctgtt
8401 cggcaacctg attgccctga gcctgggcct gacccccaac ttcaagagca acttcgacct
8461 ggccgaggat gccaaactgc agctgagcaa ggacacctac gacgacgacc tggacaacct
8521 gctggcccag atcggcgacc agtacgccga cctgtttctg gccgccaaga acctgtccga
8581 cgccatcctg ctgagcgaca tcctgagagt gaacaccgag atcaccaagg cccccctgag
8641 cgcctctatg atcaagagat acgacgagca ccaccaggac ctgaccctgc tgaaagctct
8701 cgtgcggcag cagctgcctg agaagtacaa agagattttc ttcgaccaga gcaagaacgg
8761 ctacgccggc tacatcgatg gcggagccag ccaggaagag ttctacaagt tcatcaagcc
8821 catcctggaa aagatggacg gcaccgagga actgctcgtg aagctgaaca gagaggacct
8881 gctgcggaag cagcggacct tcgacaacgg cagcatcccc caccagatcc acctgggaga
8941 gctgcacgcc attctgcggc ggcaggaaga tttttaccca ttcctgaagg acaaccggga
9001 aaagatcgag aagatcctga ccttccgcat cccctactac gtgggccctc tggccagggg
9061 aaacagcaga ttcgcctgga tgaccagaaa gagcgaggaa accatcaccc cctggaactt
9121 cgaggaagtg gtggacaagg gcgccagcgc ccagagcttc atcgagcgga tgaccaactt
9181 cgataagaac ctgcccaacg agaaggtgct gcccaagcac agcctgctgt acgagtactt
9241 caccgtgtac aacgagctga ccaaagtgaa atacgtgacc gagggaatga gaaagcccgc
9301 cttcctgagc ggcgagcaga aaaaagccat cgtggacctg ctgttcaaga ccaaccggaa
9361 agtgaccgtg aagcagctga aagaggacta cttcaagaaa atcgagtgct tcgactccgt
9421 ggaaatctcc ggcgtggaag atcggttcaa cgcctccctg ggcacatacc acgatctgct
9481 gaaaattatc aaggacaagg acttcctgga caatgaggaa aacgaggaca ttctggaaga
9541 tatcgtgctg accctgacac tgtttgagga cagagagatg atcgaggaac ggctgaaaac
9601 ctatgcccac ctgttcgacg acaaagtgat gaagcagctg aagcggcgga gatacaccgg
9661 ctggggcagg ctgagccgga agctgatcaa cggcatccgg gacaagcagt ccggcaagac
9721 aatcctggat ttcctgaagt ccgacggctt cgccaacaga aacttcatgc agctgatcca
9781 cgacgacagc ctgaccttta aagaggacat ccagaaagcc caggtgtccg gccagggcga
9841 tagcctgcac gagcacattg ccaatctggc cggcagcccc gccattaaga agggcatcct
9901 gcagacagtg aaggtggtgg acgagctcgt gaaagtgatg ggccggcaca agcccgagaa
9961 catcgtgatc gaaatggcca gagagaacca gaccacccag aagggacaga agaacagccg
10021 cgagagaatg aagcggatcg aagagggcat caaagagctg ggcagccaga tcctgaaaga
10081 acaccccgtg gaaaacaccc agctgcagaa cgagaagctg tacctgtact acctgcagaa
10141 tgggcgggat atgtacgtgg accaggaact ggacatcaac cggctgtccg actacgatgt
10201 ggacgctatc gtgcctcaga gctttctgaa ggacgactcc atcgataaca aagtgctgac
10261 tcggagcgac aagaaccggg gcaagagcga caacgtgccc tccgaagagg tcgtgaagaa
10321 gatgaagaac tactggcgcc agctgctgaa tgccaagctg attacccaga ggaagttcga
10381 caatctgacc aaggccgaga gaggcggcct gagcgaactg gataaggccg gcttcatcaa
10441 gagacagctg gtggaaaccc ggcagatcac aaagcacgtg gcacagatcc tggactcccg
10501 gatgaacact aagtacgacg agaacgacaa actgatccgg gaagtgaaag tgatcaccct
10561 gaagtccaag ctggtgtccg atttccggaa ggatttccag ttttacaaag tgcgcgagat
10621 caacaactac caccacgccc acgacgccta cctgaacgcc gtcgtgggaa ccgccctgat
10681 caaaaagtac cctaagctgg aaagcgagtt cgtgtacggc gactacaagg tgtacgacgt
10741 gcggaagatg atcgccaaga gcgagcagga aatcggcaag gctaccgcca agtacttctt
10801 ctacagcaac atcatgaact ttttcaagac cgagattacc ctggccaacg gcgagatccg
10861 gaagcggcct ctgatcgaga caaacggcga aacaggcgag atcgtgtggg ataagggccg
10921 ggactttgcc accgtgcgga aagtgctgtc tatgccccaa gtgaatatcg tgaaaaagac
10981 cgaggtgcag acaggcggct tcagcaaaga gtctatcctg cccaagagga acagcgacaa
11041 gctgatcgcc agaaagaagg actgggaccc taagaagtac ggcggcttcg acagccccac
11101 cgtggcctat tctgtgctgg tggtggccaa agtggaaaag ggcaagtcca agaaactgaa
11161 gagtgtgaaa gagctgctgg ggatcaccat catggaaaga agcagcttcg agaagaatcc
11221 catcgacttt ctggaagcca agggctacaa agaagtgaaa aaggacctga tcatcaagct
11281 gcctaagtac tccctgttcg agctggaaaa cggccggaag agaatgctgg cctctgccgg
11341 cgaactgcag aagggaaacg aactggccct gccctccaaa tatgtgaact tcctgtacct
11401 ggccagccac tatgagaagc tgaagggctc ccccgaggat aatgagcaga aacagctgtt
11461 tgtggaacag cacaaacact acctggacga gatcatcgag cagatcagcg agttctccaa
11521 gagagtgatc ctggccgacg ctaatctgga caaggtgctg agcgcctaca acaagcacag
11581 agacaagcct atcagagagc aggccgagaa tatcatccac ctgtttaccc tgaccaatct
11641 gggagcccct gccgccttca agtactttga caccaccatc gaccggaaga ggtacaccag
11701 caccaaagag gtgctggacg ccaccctgat ccaccagagc atcaccggcc tgtacgagac
11761 acggatcgac ctgtctcagc tgggaggcga cgcctatccc tatgacgtgc ccgattatgc
11821 cagcctgggc agcggctccc ccaagaaaaa acgcaaggtg gaagatccta agaaaaagcg
11881 gaaagtggac ggcattggta gtgggagcaa cggatgactc gagctagtta aaagttttgt
11941 tactttatag aagaaatttt gagtttttgt ttttttttaa taaataaata aacataaata
12001 aattgtttgt tgaatttatt attagtatgt aagtgtaaat ataataaaac ttaatatcta
12061 ttcaaattaa taaataaacc tcgatataca gaccgataaa acacatgcgt caattttacg
12121 catgattatc tttaacgtac gtcacaatat gattatcttt ctagggttaa tctagtatac
12181 gcgtatgcgg ccgcttaatt aatccagctt ttgttccctt tagtgagggt taattgcgcg
12241 cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc
12301 acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta
12361 actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
12421 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc
12481 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
12541 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
12601 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
12661 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
12721 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
12781 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
12841 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
12901 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
12961 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
13021 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
13081 ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt
13141 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
13201 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
13261 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
13321 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
13381 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
13441 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
13501 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga
13561 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
13621 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
13681 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat
13741 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
13801 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
13861 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
13921 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
13981 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga
14041 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
14101 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
14161 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
14221 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact
14281 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat
14341 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt
14401 gccacctaaa ttgtaagcgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc
14461 agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag
14521 accgagatag ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg
14581 gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca
14641 tcaccctaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa
14701 gggagccccc gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg
14761 aagaaagcga aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta
14821 accaccacac ccgccgcgct taatgcgccg ctacagggcg cgtcccattc gccattcagg
14881 ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt cgctattacg ccagctggcg
14941 aaagggggat gtgctgcaag gcgattaagt tgggtaacgc cagggttttc ccagtcacga
15001 cgttgtaaaa cgacggccag tgagcgcgcg taatacgact cactataggg cgaattgggg
15061 cgcgccattc tagattaacc ctagaaagat agtctgcgta aaattgacgc atgcattctt
15121 gaaatattgc tctctctttc taaatagcgc gaatccgtcg ctgtgcattt aggacatctc
15181 agtcgccgct tggagctccc gtgaggcgtg cttgtcaatg cggtaagtgt cactgatttt
15241 gaactataac gaccgcgtga gtcaaaatga cgcatgatta tcttttacgt gacttttaag
15301 atttaactca tacgataatt atattgttat ttcatgttct acttacgtga taacttatta
15361 tatatatatt ttcttgttat agatatcaac tagaatgcta gcatgggccc agatct
//
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