| 质粒名称: | pLV hU6-sgRNA hUbC-dCas9-KRAB-T2a-Puro |
|---|---|
| 出品公司: | 爱迪基因 |
| 目录编号: | 71236 |
| 存储实验室: | Charles Gersbach |
|
载体骨架基本信息 |
|
| 载体名称: | FUGW |
| 载体出品公司: | -- |
| 载体抗性: | 氨苄青霉素 |
| 空载体大小: | -- |
| 完整质粒大小: | 15000 bp |
| 载体修饰: | -- |
| 载体类型: | 慢病毒,CRISPR |
| 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: | humanized dCas9-KRAB T2A Puro |
| 物种来源: | S. Pyogenes |
| 插入大小: | -- |
| 基因突变: | D10A and H840A |
| GenBank编号 | -- |
| 启动子 | |
| 标签/融合蛋白: | Flag（插入N端子） |
| 克隆方法: | -- |
| 5' 克隆位点: | -- |
| 3' 克隆位点: | -- |
| 5' 测序引物 | -- |
| 3' 测序引物 | -- |
|
文献引用方法 |
|
| 材料和方法部分: | pLV hU6-sgRNA hUbC-dCas9-KRAB-T2a-Puro was a gift from Charles Gersbach (Addgene plasmid # 71236 ; http://n2t.net/addgene:71236 ; RRID:Addgene_71236) |
| 参考文献部分: | Highly specific epigenome editing by CRISPR-Cas9 repressors for silencing of distal regulatory elements. Thakore PI, D'Ippolito AM, Song L, Safi A, Shivakumar NK, Kabadi AM, Reddy TE, Crawford GE, Gersbach CA. Nat Methods. 2015 Dec;12(12):1143-9. doi: 10.1038/nmeth.3630. Epub 2015 Oct 26. 10.1038/nmeth.3630 PubMed 26501517 |
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 pLV hU6-sgRNA hUbC-dCas9-KRAB-T2a-Puro 14900 bp ds-DNA circular SYN 02-MAY-2019
DEFINITION Express sgRNA and dCas9-KRAB from lentiviral vector.
ACCESSION .
VERSION .
KEYWORDS pLV hU6-sgRNA hUbC-dCas9-KRAB-T2a-Puro
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 14900)
AUTHORS Thakore PI, D'Ippolito AM, Song L, Safi A, Shivakumar NK, Kabadi AM,
Reddy TE, Crawford GE, Gersbach CA
TITLE Highly specific epigenome editing by CRISPR-Cas9 repressors for
silencing of distal regulatory elements.
JOURNAL Nat Methods. 2015 Dec;12(12):1143-9. doi: 10.1038/nmeth.3630. Epub
2015 Oct 26.
PUBMED 26501517
REFERENCE 2 (bases 1 to 14900)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 2, 2019 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..14900
/organism="synthetic DNA construct"
/mol_type="other DNA"
primer_bind 262..281
/label=hUBCpro-F
/note="Human Ubiquitin C (UbC) promoter, forward primer"
regulatory 454..463
/regulatory_class="other"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
CDS 463..528
/codon_start=1
/product="three tandem FLAG(R) epitope tags, followed by an
enterokinase cleavage site"
/label=3xFLAG
/translation="DYKDHDGDYKDHDIDYKDDDDK"
CDS 535..555
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 565..4668
/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 4681..4701
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 4735..4929
/codon_start=1
/gene="ZNF10"
/gene_synonym="KOX1"
/product="Kruppel-associated box (KRAB) transcriptional
repression domain from the human zinc finger protein ZNF10
(Margolin et al., 1994)"
/label=KRAB
/translation="RTLVTFKDVFVDFTREEWKLLDTAQQILYRNVMLENYKNLVSLGY
QLTKPDVILRLEKGEEPWLV"
CDS 4996..5016
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 5023..5076
/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 5077..5676
/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(5077..5096)
/label=Puro-R
/note="Puromycin resistance gene, reverse primer. Also
called puro-variant-R"
primer_bind 5573..5593
/label=Puro-F
/note="Puromycin resistance gene, forward primer"
protein_bind 5730..5763
/label=loxP
/bound_moiety="Cre recombinase"
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (GCATACAT)."
misc_feature 5788..6376
/label=WPRE
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
primer_bind complement(5841..5861)
/label=WPRE-R
/note="WPRE, reverse primer"
CDS complement(6259..6270)
/codon_start=1
/product="Factor Xa recognition and cleavage site"
/label=Factor Xa site
/translation="IEGR"
primer_bind complement(6379..6395)
/label=KS primer
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(6380..6396)
/label=pBluescriptKS
/note="For pBluescript vector"
LTR 6901..7081
/label=5' LTR (truncated)
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
primer_bind complement(7107..7124)
/label=BGH-rev
/note="Bovine growth hormone terminator, reverse primer.
Also called BGH reverse"
polyA_signal 7113..7337
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
rep_origin 7383..7811
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(7470..7489)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 7680..7701
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind complement(7820..7840)
/label=pBABE 3'
/note="SV40 enhancer, reverse primer for pBABE vectors"
promoter 7825..8154
/label=SV40 promoter
/note="SV40 enhancer and early promoter"
rep_origin 8005..8140
/label=SV40 ori
/note="SV40 origin of replication"
primer_bind 8067..8086
/label=SV40pro-F
/note="SV40 promoter/origin, forward primer"
promoter 8202..8249
/label=EM7 promoter
/note="synthetic bacterial promoter "
CDS 8268..8642
/codon_start=1
/gene="Sh ble from Streptoalloteichus hindustanus"
/product="antibiotic-binding protein"
/label=BleoR
/note="confers resistance to bleomycin, phleomycin, and
Zeocin(TM)"
/translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
EFALRDPAGNCVHFVAEEQD"
polyA_signal 8772..8893
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(8809..8828)
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
primer_bind 8863..8882
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
primer_bind complement(8942..8958)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(8942..8958)
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind complement(8955..8977)
/label=M13/pUC Reverse
/note="In lacZ gene"
protein_bind 8966..8982
/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(8990..9020)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 9035..9056
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(9173..9190)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(9344..9932)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(9424..9443)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS complement(10103..10963)
/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 10726..10745
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
promoter complement(10964..11068)
/gene="bla"
/label=AmpR promoter
primer_bind complement(11143..11162)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
enhancer 11334..11713
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 11715..11913
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
primer_bind 11868..11888
/label=CMV-F
/note="Human CMV immediate early promoter, forward primer"
LTR 11931..12111
/label=5' LTR (truncated)
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
misc_feature 12158..12283
/label=HIV-1 Psi
/note="packaging signal of human immunodeficiency virus
type 1"
misc_feature 12776..13009
/label=RRE
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
misc_feature 13536..13653
/label=cPPT/CTS
/note="central polypurine tract and central termination
sequence of HIV-1"
protein_bind 13697..13730
/label=loxP
/bound_moiety="Cre recombinase"
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (GCATACAT)."
misc_RNA complement(13741..13816)
/label=gRNA scaffold
/note="guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system"
promoter complement(13841..14081)
/label=U6 promoter
/note="RNA polymerase III promoter for human U6 snRNA"
primer_bind complement(13891..13910)
/label=LKO.1 5'
/note="Human U6 promoter, forward primer"
primer_bind complement(14061..14081)
/label=hU6-F
/note="Human U6 promoter, forward primer"
promoter 14114..425
/label=UbC promoter
/note="human ubiquitin C promoter"
ORIGIN
1 gtgccgttgg gcagtgcacc cgtacctttg ggagcgcgcg ccctcgtcgt gtcgtgacgt
61 cacccgttct gttggcttat aatgcagggt ggggccacct gccggtaggt gtgcggtagg
121 cttttctccg tcgcaggacg cagggttcgg gcctagggta ggctctcctg aatcgacagg
181 cgccggacct ctggtgaggg gagggataag tgaggcgtca gtttctttgg tcggttttat
241 gtacctatct tcttaagtag ctgaagctcc ggttttgaac tatgcgctcg gggttggcga
301 gtgtgttttg tgaagttttt taggcacctt ttgaaatgta atcatttggg tcaatatgta
361 attttcagtg ttagactagt aaattgtccg ctaaattctg gccgtttttg gcttttttgt
421 tagacgaagc ttgggctgca ggtcgactct agagccacca tggactacaa agaccatgac
481 ggtgattata aagatcatga catcgattac aaggatgacg atgacaagat ggcccccaag
541 aagaagagga aggtgggccg cggaatggac aagaagtact ccattgggct cgccatcggc
601 acaaacagcg tcggctgggc cgtcattacg gacgagtaca aggtgccgag caaaaaattc
661 aaagttctgg gcaataccga tcgccacagc ataaagaaga acctcattgg cgccctcctg
721 ttcgactccg gggaaaccgc cgaagccacg cggctcaaaa gaacagcacg gcgcagatat
781 acccgcagaa agaatcggat ctgctacctg caggagatct ttagtaatga gatggctaag
841 gtggatgact ctttcttcca taggctggag gagtcctttt tggtggagga ggataaaaag
901 cacgagcgcc acccaatctt tggcaatatc gtggacgagg tggcgtacca tgaaaagtac
961 ccaaccatat atcatctgag gaagaagctt gtagacagta ctgataaggc tgacttgcgg
1021 ttgatctatc tcgcgctggc gcatatgatc aaatttcggg gacacttcct catcgagggg
1081 gacctgaacc cagacaacag cgatgtcgac aaactcttta tccaactggt tcagacttac
1141 aatcagcttt tcgaagagaa cccgatcaac gcatccggag ttgacgccaa agcaatcctg
1201 agcgctaggc tgtccaaatc ccggcggctc gaaaacctca tcgcacagct ccctggggag
1261 aagaagaacg gcctgtttgg taatcttatc gccctgtcac tcgggctgac ccccaacttt
1321 aaatctaact tcgacctggc cgaagatgcc aagcttcaac tgagcaaaga cacctacgat
1381 gatgatctcg acaatctgct ggcccagatc ggcgaccagt acgcagacct ttttttggcg
1441 gcaaagaacc tgtcagacgc cattctgctg agtgatattc tgcgagtgaa cacggagatc
1501 accaaagctc cgctgagcgc tagtatgatc aagcgctatg atgagcacca ccaagacttg
1561 actttgctga aggcccttgt cagacagcaa ctgcctgaga agtacaagga aattttcttc
1621 gatcagtcta aaaatggcta cgccggatac attgacggcg gagcaagcca ggaggaattt
1681 tacaaattta ttaagcccat cttggaaaaa atggacggca ccgaggagct gctggtaaag
1741 cttaacagag aagatctgtt gcgcaaacag cgcactttcg acaatggaag catcccccac
1801 cagattcacc tgggcgaact gcacgctatc ctcaggcggc aagaggattt ctaccccttt
1861 ttgaaagata acagggaaaa gattgagaaa atcctcacat ttcggatacc ctactatgta
1921 ggccccctcg cccggggaaa ttccagattc gcgtggatga ctcgcaaatc agaagagacc
1981 atcactccct ggaacttcga ggaagtcgtg gataaggggg cctctgccca gtccttcatc
2041 gaaaggatga ctaactttga taaaaatctg cctaacgaaa aggtgcttcc taaacactct
2101 ctgctgtacg agtacttcac agtttataac gagctcacca aggtcaaata cgtcacagaa
2161 gggatgagaa agccagcatt cctgtctgga gagcagaaga aagctatcgt ggacctcctc
2221 ttcaagacga accggaaagt taccgtgaaa cagctcaaag aagactattt caaaaagatt
2281 gaatgtttcg actctgttga aatcagcgga gtggaggatc gcttcaacgc atccctggga
2341 acgtatcacg atctcctgaa aatcattaaa gacaaggact tcctggacaa tgaggagaac
2401 gaggacattc ttgaggacat tgtcctcacc cttacgttgt ttgaagatag ggagatgatt
2461 gaagaacgct tgaaaactta cgctcatctc ttcgacgaca aagtcatgaa acagctcaag
2521 aggcgccgat atacaggatg ggggcggctg tcaagaaaac tgatcaatgg gatccgagac
2581 aagcagagtg gaaagacaat cctggatttt cttaagtccg atggatttgc caaccggaac
2641 ttcatgcagt tgatccatga tgactctctc acctttaagg aggacatcca gaaagcacaa
2701 gtttctggcc agggggacag tcttcacgag cacatcgcta atcttgcagg tagcccagct
2761 atcaaaaagg gaatactgca gaccgttaag gtcgtggatg aactcgtcaa agtaatggga
2821 aggcataagc ccgagaatat cgttatcgag atggcccgag agaaccaaac tacccagaag
2881 ggacagaaga acagtaggga aaggatgaag aggattgaag agggtataaa agaactgggg
2941 tcccaaatcc ttaaggaaca cccagttgaa aacacccagc ttcagaatga gaagctctac
3001 ctgtactacc tgcagaacgg cagggacatg tacgtggatc aggaactgga catcaatcgg
3061 ctctccgact acgacgtgga tgccatcgtg ccccagtctt ttctcaaaga tgattctatt
3121 gataataaag tgttgacaag atccgataaa aatagaggga agagtgataa cgtcccctca
3181 gaagaagttg tcaagaaaat gaaaaattat tggcggcagc tgctgaacgc caaactgatc
3241 acacaacgga agttcgataa tctgactaag gctgaacgag gtggcctgtc tgagttggat
3301 aaagccggct tcatcaaaag gcagcttgtt gagacacgcc agatcaccaa gcacgtggcc
3361 caaattctcg attcacgcat gaacaccaag tacgatgaaa atgacaaact gattcgagag
3421 gtgaaagtta ttactctgaa gtctaagctg gtctcagatt tcagaaagga ctttcagttt
3481 tataaggtga gagagatcaa caattaccac catgcgcatg atgcctacct gaatgcagtg
3541 gtaggcactg cacttatcaa aaaatatccc aagcttgaat ctgaatttgt ttacggagac
3601 tataaagtgt acgatgttag gaaaatgatc gcaaagtctg agcaggaaat aggcaaggcc
3661 accgctaagt acttctttta cagcaatatt atgaattttt tcaagaccga gattacactg
3721 gccaatggag agattcggaa gcgaccactt atcgaaacaa acggagaaac aggagaaatc
3781 gtgtgggaca agggtaggga tttcgcgaca gtccggaagg tcctgtccat gccgcaggtg
3841 aacatcgtta aaaagaccga agtacagacc ggaggcttct ccaaggaaag tatcctcccg
3901 aaaaggaaca gcgacaagct gatcgcacgc aaaaaagatt gggaccccaa gaaatacggc
3961 ggattcgatt ctcctacagt cgcttacagt gtactggttg tggccaaagt ggagaaaggg
4021 aagtctaaaa aactcaaaag cgtcaaggaa ctgctgggca tcacaatcat ggagcgatca
4081 agcttcgaaa aaaaccccat cgactttctc gaggcgaaag gatataaaga ggtcaaaaaa
4141 gacctcatca ttaagcttcc caagtactct ctctttgagc ttgaaaacgg ccggaaacga
4201 atgctcgcta gtgcgggcga gctgcagaaa ggtaacgagc tggcactgcc ctctaaatac
4261 gttaatttct tgtatctggc cagccactat gaaaagctca aagggtctcc cgaagataat
4321 gagcagaagc agctgttcgt ggaacaacac aaacactacc ttgatgagat catcgagcaa
4381 ataagcgaat tctccaaaag agtgatcctc gccgacgcta acctcgataa ggtgctttct
4441 gcttacaata agcacaggga taagcccatc agggagcagg cagaaaacat tatccacttg
4501 tttactctga ccaacttggg cgcgcctgca gccttcaagt acttcgacac caccatagac
4561 agaaagcggt acacctctac aaaggaggtc ctggacgcca cactgattca tcagtcaatt
4621 acggggctct atgaaacaag aatcgacctc tctcagctcg gtggagacag cagggctgac
4681 cccaagaaga agaggaaggt ggctagcgat gctaagtcac tgactgcctg gtcccggaca
4741 ctggtgacct tcaaggatgt gtttgtggac ttcaccaggg aggagtggaa gctgctggac
4801 actgctcagc agatcctgta cagaaatgtg atgctggaga actataagaa cctggtttcc
4861 ttgggttatc agcttactaa gccagatgtg atcctccggt tggagaaggg agaagagccc
4921 tggctggtgg agagagaaat tcaccaagag acccatcctg attcagagac tgcatttgaa
4981 atcaaatcat cagttccgaa aaagaaacgc aaagttgcta gcgagggcag aggaagtctt
5041 ctaacatgcg gtgacgtgga ggagaatccc ggccctatga ccgagtacaa gcccacggtg
5101 cgcctcgcca cccgcgacga cgtccccagg gccgtacgca ccctcgccgc cgcgttcgcc
5161 gactaccccg ccacgcgcca caccgtcgat ccggaccgcc acatcgagcg ggtcaccgag
5221 ctgcaagaac tcttcctcac gcgcgtcggg ctcgacatcg gcaaggtgtg ggtcgcggac
5281 gacggcgccg cggtggcggt ctggaccacg ccggagagcg tcgaagcggg ggcggtgttc
5341 gccgagatcg gcccgcgcat ggccgagttg agcggttccc ggctggccgc gcagcaacag
5401 atggaaggcc tcctggcgcc gcaccggccc aaggagcccg cgtggttcct ggccaccgtc
5461 ggcgtgtcgc ccgaccacca gggcaagggt ctgggcagcg ccgtcgtgct ccccggagtg
5521 gaggcggccg agcgcgccgg ggtgcccgcc ttcctggaga cctccgcgcc ccgcaacctc
5581 cccttctacg agcggctcgg cttcaccgtc accgccgacg tcgaggtgcc cgaaggaccg
5641 cgcacctggt gcatgacccg caagcccggt gcctgaccag cacactggcg gccgttacta
5701 gcttctgcag cacgaccggt tgataataga taacttcgta tagcatacat tatacgaagt
5761 tatgaattcg atatcaagct tatcgataat caacctctgg attacaaaat ttgtgaaaga
5821 ttgactggta ttcttaacta tgttgctcct tttacgctat gtggatacgc tgctttaatg
5881 cctttgtatc atgctattgc ttcccgtatg gctttcattt tctcctcctt gtataaatcc
5941 tggttgctgt ctctttatga ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc
6001 actgtgtttg ctgacgcaac ccccactggt tggggcattg ccaccacctg tcagctcctt
6061 tccgggactt tcgctttccc cctccctatt gccacggcgg aactcatcgc cgcctgcctt
6121 gcccgctgct ggacaggggc tcggctgttg ggcactgaca attccgtggt gttgtcgggg
6181 aaatcatcgt cctttccttg gctgctcgcc tgtgttgcca cctggattct gcgcgggacg
6241 tccttctgct acgtcccttc ggccctcaat ccagcggacc ttccttcccg cggcctgctg
6301 ccggctctgc ggcctcttcc gcgtcttcgc cttcgccctc agacgagtcg gatctccctt
6361 tgggccgcct ccccgcatcg ataccgtcga cctcgagacc tagaaaaaca tggagcaatc
6421 acaagtagca atacagcagc taccaatgct gattgtgcct ggctagaagc acaagaggag
6481 gaggaggtgg gttttccagt cacacctcag gtacctttaa gaccaatgac ttacaaggca
6541 gctgtagatc ttagccactt tttaaaagaa aaggggggac tggaagggct aattcactcc
6601 caacgaagac aagatatcct tgatctgtgg atctaccaca cacaaggcta cttccctgat
6661 tggcagaact acacaccagg gccagggatc agatatccac tgacctttgg atggtgctac
6721 aagctagtac cagttgagca agagaaggta gaagaagcca atgaaggaga gaacacccgc
6781 ttgttacacc ctgtgagcct gcatgggatg gatgacccgg agagagaagt attagagtgg
6841 aggtttgaca gccgcctagc atttcatcac atggcccgag agctgcatcc ggactgtact
6901 gggtctctct ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca
6961 ctgcttaagc ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg
7021 tgtgactctg gtaactagag atccctcaga cccttttagt cagtgtggaa aatctctagc
7081 agggcccgtt taaacccgct gatcagcctc gactgtgcct tctagttgcc agccatctgt
7141 tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc
7201 ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg
7261 tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga
7321 tgcggtgggc tctatggctt ctgaggcgga aagaaccagc tggggctcta gggggtatcc
7381 ccacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
7441 cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
7501 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
7561 tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg
7621 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag
7681 tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt
7741 ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt
7801 taacgcgaat taattctgtg gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc
7861 ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag gtgtggaaag
7921 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
7981 atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct
8041 ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc ctctgcctct
8101 gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagctc
8161 ccgggagctt gtatatccat tttcggatct gatcagcacg tgttgacaat taatcatcgg
8221 catagtatat cggcatagta taatacgaca aggtgaggaa ctaaaccatg gccaagttga
8281 ccagtgccgt tccggtgctc accgcgcgcg acgtcgccgg agcggtcgag ttctggaccg
8341 accggctcgg gttctcccgg gacttcgtgg aggacgactt cgccggtgtg gtccgggacg
8401 acgtgaccct gttcatcagc gcggtccagg accaggtggt gccggacaac accctggcct
8461 gggtgtgggt gcgcggcctg gacgagctgt acgccgagtg gtcggaggtc gtgtccacga
8521 acttccggga cgcctccggg ccggccatga ccgagatcgg cgagcagccg tgggggcggg
8581 agttcgccct gcgcgacccg gccggcaact gcgtgcactt cgtggccgag gagcaggact
8641 gacacgtgct acgagatttc gattccaccg ccgccttcta tgaaaggttg ggcttcggaa
8701 tcgttttccg ggacgccggc tggatgatcc tccagcgcgg ggatctcatg ctggagttct
8761 tcgcccaccc caacttgttt attgcagctt ataatggtta caaataaagc aatagcatca
8821 caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca
8881 tcaatgtatc ttatcatgtc tgtataccgt cgacctctag ctagagcttg gcgtaatcat
8941 ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag
9001 ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg
9061 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
9121 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca
9181 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
9241 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
9301 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
9361 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
9421 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
9481 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
9541 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
9601 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
9661 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
9721 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
9781 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
9841 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
9901 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
9961 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
10021 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
10081 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
10141 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
10201 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
10261 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
10321 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
10381 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
10441 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
10501 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
10561 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
10621 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
10681 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
10741 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
10801 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
10861 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
10921 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
10981 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
11041 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtcg
11101 acggatcggg agatctcccg atcccctatg gtgcactctc agtacaatct gctctgatgc
11161 cgcatagtta agccagtatc tgctccctgc ttgtgtgttg gaggtcgctg agtagtgcgc
11221 gagcaaaatt taagctacaa caaggcaagg cttgaccgac aattgcatga agaatctgct
11281 tagggttagg cgttttgcgc tgcttcgcga tgtacgggcc agatatacgc gttgacattg
11341 attattgact agttattaat agtaatcaat tacggggtca ttagttcata gcccatatat
11401 ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc
11461 ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca
11521 ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta
11581 tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta
11641 tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat
11701 cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat agcggtttga
11761 ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt tttggcacca
11821 aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg
11881 taggcgtgta cggtgggagg tctatataag cagcgcgttt tgcctgtact gggtctctct
11941 ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca ctgcttaagc
12001 ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg
12061 gtaactagag atccctcaga cccttttagt cagtgtggaa aatctctagc agtggcgccc
12121 gaacagggac ttgaaagcga aagggaaacc agaggagctc tctcgacgca ggactcggct
12181 tgctgaagcg cgcacggcaa gaggcgaggg gcggcgactg gtgagtacgc caaaaatttt
12241 gactagcgga ggctagaagg agagagatgg gtgcgagagc gtcagtatta agcgggggag
12301 aattagatcg cgatgggaaa aaattcggtt aaggccaggg ggaaagaaaa aatataaatt
12361 aaaacatata gtatgggcaa gcagggagct agaacgattc gcagttaatc ctggcctgtt
12421 agaaacatca gaaggctgta gacaaatact gggacagcta caaccatccc ttcagacagg
12481 atcagaagaa cttagatcat tatataatac agtagcaacc ctctattgtg tgcatcaaag
12541 gatagagata aaagacacca aggaagcttt agacaagata gaggaagagc aaaacaaaag
12601 taagaccacc gcacagcaag cggccgctga tcttcagacc tggaggagga gatatgaggg
12661 acaattggag aagtgaatta tataaatata aagtagtaaa aattgaacca ttaggagtag
12721 cacccaccaa ggcaaagaga agagtggtgc agagagaaaa aagagcagtg ggaataggag
12781 ctttgttcct tgggttcttg ggagcagcag gaagcactat gggcgcagcg tcaatgacgc
12841 tgacggtaca ggccagacaa ttattgtctg gtatagtgca gcagcagaac aatttgctga
12901 gggctattga ggcgcaacag catctgttgc aactcacagt ctggggcatc aagcagctcc
12961 aggcaagaat cctggctgtg gaaagatacc taaaggatca acagctcctg gggatttggg
13021 gttgctctgg aaaactcatt tgcaccactg ctgtgccttg gaatgctagt tggagtaata
13081 aatctctgga acagatttgg aatcacacga cctggatgga gtgggacaga gaaattaaca
13141 attacacaag cttaatacac tccttaattg aagaatcgca aaaccagcaa gaaaagaatg
13201 aacaagaatt attggaatta gataaatggg caagtttgtg gaattggttt aacataacaa
13261 attggctgtg gtatataaaa ttattcataa tgatagtagg aggcttggta ggtttaagaa
13321 tagtttttgc tgtactttct atagtgaata gagttaggca gggatattca ccattatcgt
13381 ttcagaccca cctcccaacc ccgaggggac ccgacaggcc cgaaggaata gaagaagaag
13441 gtggagagag agacagagac agatccattc gattagtgaa cggatcggca ctgcgtgcgc
13501 caattctgca gacaaatggc agtattcatc cacaatttta aaagaaaagg ggggattggg
13561 gggtacagtg caggggaaag aatagtagac ataatagcaa cagacataca aactaaagaa
13621 ttacaaaaac aaattacaaa aattcaaaat tttcgggttt attacaggga cagcagagat
13681 ccagtttggt taattaataa cttcgtatag catacattat acgaagttat taaaaaaaaa
13741 gcaccgactc ggtgccactt tttcaagttg ataacggact agccttattt taacttgcta
13801 tttctagctc taaaactgag acgtccgtct ccggtgtttc gtcctttcca caagatatat
13861 aaagccaaga aatcgaaata ctttcaagtt acggtaagca tatgatagtc cattttaaaa
13921 cataatttta aaactgcaaa ctacccaaga aattattact ttctacgtca cgtattttgt
13981 actaatatct ttgtgtttac agtcaaatta attccaatta tctctctaac agccttgtat
14041 cgtatatgca aatatgaagg aatcatggga aataggccct cttaattaat taacccgtgt
14101 cggctccaga tctggcctcc gcgccgggtt ttggcgcctc ccgcgggcgc ccccctcctc
14161 acggcgagcg ctgccacgtc agacgaaggg cgcagcgagc gtcctgatcc ttccgcccgg
14221 acgctcagga cagcggcccg ctgctcataa gactcggcct tagaacccca gtatcagcag
14281 aaggacattt taggacggga cttgggtgac tctagggcac tggttttctt tccagagagc
14341 ggaacaggcg aggaaaagta gtcccttctc ggcgattctg cggagggatc tccgtggggc
14401 ggtgaacgcc gatgattata taaggacgcg ccgggtgtgg cacagctagt tccgtcgcag
14461 ccgggatttg ggtcgcggtt cttgtttgtg gatcgctgtg atcgtcactt ggtgagtagc
14521 gggctgctgg gctggccggg gctttcgtgg ccgccgggcc gctcggtggg acggaagcgt
14581 gtggagagac cgccaagggc tgtagtctgg gtccgcgagc aaggttgccc tgaactgggg
14641 gttgggggga gcgcagcaaa atggcggctg ttcccgagtc ttgaatggaa gacgcttgtg
14701 aggcgggctg tgaggtcgtt gaaacaaggt ggggggcatg gtgggcggca agaacccaag
14761 gtcttgaggc cttcgctaat gcgggaaagc tcttattcgg gtgagatggg ctggggcacc
14821 atctggggac cctgacgtga agtttgtcac tgactggaga actcggtttg tcgtctgttg
14881 cgggggcggc agttatggcg
//
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