| 质粒名称: | pLV hU6-sgRNA hUbC-dCas9-KRAB-T2a-GFP |
|---|---|
| 出品公司: | 爱迪基因 |
| 目录编号: | 71237 |
| 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: | -- |
| 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 GFP |
| 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: | 标志（插入时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-GFP was a gift from Charles Gersbach (Addgene plasmid # 71237 ; http://n2t.net/addgene:71237 ; RRID:Addgene_71237) |
| 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-GFP 14982 bp ds-DNA circular SYN 06-AUG-2018
DEFINITION Express sgRNA and dCas9-KRAB from 3rd generation lentiviral vector.
ACCESSION .
VERSION .
KEYWORDS pLV hU6-sgRNA hUbC-dCas9-KRAB-T2a-GFP
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 14982)
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 14982)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Aug 6, 2018 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..14982
/organism="synthetic DNA construct"
/mol_type="other DNA"
enhancer 1..380
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 382..580
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
primer_bind 535..555
/label=CMV-F
/note="Human CMV immediate early promoter, forward primer"
LTR 598..778
/label=5' LTR (truncated)
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
misc_feature 825..950
/label=HIV-1 Psi
/note="packaging signal of human immunodeficiency virus
type 1"
misc_feature 1443..1676
/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 2203..2320
/label=cPPT/CTS
/note="central polypurine tract and central termination
sequence of HIV-1"
protein_bind 2364..2397
/label=loxP
/bound_moiety="Cre recombinase"
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (GCATACAT)."
misc_RNA complement(2408..2483)
/label=gRNA scaffold
/note="guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system"
promoter complement(2508..2748)
/label=U6 promoter
/note="RNA polymerase III promoter for human U6 snRNA"
primer_bind complement(2558..2577)
/label=LKO.1 5'
/note="Human U6 promoter, forward primer"
primer_bind complement(2728..2748)
/label=hU6-F
/note="Human U6 promoter, forward primer"
promoter 2781..3992
/label=UbC promoter
/note="human ubiquitin C promoter"
primer_bind 3829..3848
/label=hUBCpro-F
/note="Human Ubiquitin C (UbC) promoter, forward primer"
regulatory 4021..4030
/regulatory_class="other"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
CDS 4030..4095
/codon_start=1
/product="three tandem FLAG(R) epitope tags, followed by an
enterokinase cleavage site"
/label=3xFLAG
/translation="DYKDHDGDYKDHDIDYKDDDDK"
CDS 4102..4122
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 4132..8235
/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 8248..8268
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 8302..8496
/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 8563..8583
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 8590..8643
/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 8650..9363
/codon_start=1
/product="enhanced GFP"
/label=EGFP
/note="mammalian codon-optimized"
/translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
FVTAAGITLGMDELYK"
primer_bind complement(8692..8713)
/label=EGFP-N
/note="EGFP, reverse primer"
primer_bind complement(8953..8972)
/label=EXFP-R
/note="For distinguishing EGFP variants, reverse primer"
primer_bind 9300..9321
/label=EGFP-C
/note="EGFP, forward primer"
protein_bind 9379..9412
/label=loxP
/bound_moiety="Cre recombinase"
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (GCATACAT)."
misc_feature 9437..10025
/label=WPRE
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
primer_bind complement(9490..9510)
/label=WPRE-R
/note="WPRE, reverse primer"
CDS complement(9908..9919)
/codon_start=1
/product="Factor Xa recognition and cleavage site"
/label=Factor Xa site
/translation="IEGR"
primer_bind complement(10028..10044)
/label=KS primer
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(10029..10045)
/label=pBluescriptKS
/note="For pBluescript vector"
LTR 10550..10730
/label=5' LTR (truncated)
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
primer_bind complement(10756..10773)
/label=BGH-rev
/note="Bovine growth hormone terminator, reverse primer.
Also called BGH reverse"
polyA_signal 10762..10986
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
rep_origin 11032..11460
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(11119..11138)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 11329..11350
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind complement(11469..11489)
/label=pBABE 3'
/note="SV40 enhancer, reverse primer for pBABE vectors"
promoter 11474..11803
/label=SV40 promoter
/note="SV40 enhancer and early promoter"
rep_origin 11654..11789
/label=SV40 ori
/note="SV40 origin of replication"
primer_bind 11716..11735
/label=SV40pro-F
/note="SV40 promoter/origin, forward primer"
promoter 11851..11898
/label=EM7 promoter
/note="synthetic bacterial promoter "
CDS 11917..12291
/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 12421..12542
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(12458..12477)
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
primer_bind 12512..12531
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
primer_bind complement(12591..12607)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(12591..12607)
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind complement(12604..12626)
/label=M13/pUC Reverse
/note="In lacZ gene"
protein_bind 12615..12631
/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(12639..12669)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 12684..12705
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(12822..12839)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(12993..13581)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(13073..13092)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS complement(13752..14612)
/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 14375..14394
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
promoter complement(14613..14717)
/gene="bla"
/label=AmpR promoter
primer_bind complement(14792..14811)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
ORIGIN
1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag cgcgttttgc ctgtactggg
601 tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg
661 cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt
721 gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt
781 ggcgcccgaa cagggacttg aaagcgaaag ggaaaccaga ggagctctct cgacgcagga
841 ctcggcttgc tgaagcgcgc acggcaagag gcgaggggcg gcgactggtg agtacgccaa
901 aaattttgac tagcggaggc tagaaggaga gagatgggtg cgagagcgtc agtattaagc
961 gggggagaat tagatcgcga tgggaaaaaa ttcggttaag gccaggggga aagaaaaaat
1021 ataaattaaa acatatagta tgggcaagca gggagctaga acgattcgca gttaatcctg
1081 gcctgttaga aacatcagaa ggctgtagac aaatactggg acagctacaa ccatcccttc
1141 agacaggatc agaagaactt agatcattat ataatacagt agcaaccctc tattgtgtgc
1201 atcaaaggat agagataaaa gacaccaagg aagctttaga caagatagag gaagagcaaa
1261 acaaaagtaa gaccaccgca cagcaagcgg ccgctgatct tcagacctgg aggaggagat
1321 atgagggaca attggagaag tgaattatat aaatataaag tagtaaaaat tgaaccatta
1381 ggagtagcac ccaccaaggc aaagagaaga gtggtgcaga gagaaaaaag agcagtggga
1441 ataggagctt tgttccttgg gttcttggga gcagcaggaa gcactatggg cgcagcgtca
1501 atgacgctga cggtacaggc cagacaatta ttgtctggta tagtgcagca gcagaacaat
1561 ttgctgaggg ctattgaggc gcaacagcat ctgttgcaac tcacagtctg gggcatcaag
1621 cagctccagg caagaatcct ggctgtggaa agatacctaa aggatcaaca gctcctgggg
1681 atttggggtt gctctggaaa actcatttgc accactgctg tgccttggaa tgctagttgg
1741 agtaataaat ctctggaaca gatttggaat cacacgacct ggatggagtg ggacagagaa
1801 attaacaatt acacaagctt aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa
1861 aagaatgaac aagaattatt ggaattagat aaatgggcaa gtttgtggaa ttggtttaac
1921 ataacaaatt ggctgtggta tataaaatta ttcataatga tagtaggagg cttggtaggt
1981 ttaagaatag tttttgctgt actttctata gtgaatagag ttaggcaggg atattcacca
2041 ttatcgtttc agacccacct cccaaccccg aggggacccg acaggcccga aggaatagaa
2101 gaagaaggtg gagagagaga cagagacaga tccattcgat tagtgaacgg atcggcactg
2161 cgtgcgccaa ttctgcagac aaatggcagt attcatccac aattttaaaa gaaaaggggg
2221 gattgggggg tacagtgcag gggaaagaat agtagacata atagcaacag acatacaaac
2281 taaagaatta caaaaacaaa ttacaaaaat tcaaaatttt cgggtttatt acagggacag
2341 cagagatcca gtttggttaa ttaataactt cgtatagcat acattatacg aagttattaa
2401 aaaaaaagca ccgactcggt gccacttttt caagttgata acggactagc cttattttaa
2461 cttgctattt ctagctctaa aactgagacg tccgtctccg gtgtttcgtc ctttccacaa
2521 gatatataaa gccaagaaat cgaaatactt tcaagttacg gtaagcatat gatagtccat
2581 tttaaaacat aattttaaaa ctgcaaacta cccaagaaat tattactttc tacgtcacgt
2641 attttgtact aatatctttg tgtttacagt caaattaatt ccaattatct ctctaacagc
2701 cttgtatcgt atatgcaaat atgaaggaat catgggaaat aggccctctt aattaattaa
2761 cccgtgtcgg ctccagatct ggcctccgcg ccgggttttg gcgcctcccg cgggcgcccc
2821 cctcctcacg gcgagcgctg ccacgtcaga cgaagggcgc agcgagcgtc ctgatccttc
2881 cgcccggacg ctcaggacag cggcccgctg ctcataagac tcggccttag aaccccagta
2941 tcagcagaag gacattttag gacgggactt gggtgactct agggcactgg ttttctttcc
3001 agagagcgga acaggcgagg aaaagtagtc ccttctcggc gattctgcgg agggatctcc
3061 gtggggcggt gaacgccgat gattatataa ggacgcgccg ggtgtggcac agctagttcc
3121 gtcgcagccg ggatttgggt cgcggttctt gtttgtggat cgctgtgatc gtcacttggt
3181 gagtagcggg ctgctgggct ggccggggct ttcgtggccg ccgggccgct cggtgggacg
3241 gaagcgtgtg gagagaccgc caagggctgt agtctgggtc cgcgagcaag gttgccctga
3301 actgggggtt ggggggagcg cagcaaaatg gcggctgttc ccgagtcttg aatggaagac
3361 gcttgtgagg cgggctgtga ggtcgttgaa acaaggtggg gggcatggtg ggcggcaaga
3421 acccaaggtc ttgaggcctt cgctaatgcg ggaaagctct tattcgggtg agatgggctg
3481 gggcaccatc tggggaccct gacgtgaagt ttgtcactga ctggagaact cggtttgtcg
3541 tctgttgcgg gggcggcagt tatggcggtg ccgttgggca gtgcacccgt acctttggga
3601 gcgcgcgccc tcgtcgtgtc gtgacgtcac ccgttctgtt ggcttataat gcagggtggg
3661 gccacctgcc ggtaggtgtg cggtaggctt ttctccgtcg caggacgcag ggttcgggcc
3721 tagggtaggc tctcctgaat cgacaggcgc cggacctctg gtgaggggag ggataagtga
3781 ggcgtcagtt tctttggtcg gttttatgta cctatcttct taagtagctg aagctccggt
3841 tttgaactat gcgctcgggg ttggcgagtg tgttttgtga agttttttag gcaccttttg
3901 aaatgtaatc atttgggtca atatgtaatt ttcagtgtta gactagtaaa ttgtccgcta
3961 aattctggcc gtttttggct tttttgttag acgaagcttg ggctgcaggt cgactctaga
4021 gccaccatgg actacaaaga ccatgacggt gattataaag atcatgacat cgattacaag
4081 gatgacgatg acaagatggc ccccaagaag aagaggaagg tgggccgcgg aatggacaag
4141 aagtactcca ttgggctcgc catcggcaca aacagcgtcg gctgggccgt cattacggac
4201 gagtacaagg tgccgagcaa aaaattcaaa gttctgggca ataccgatcg ccacagcata
4261 aagaagaacc tcattggcgc cctcctgttc gactccgggg aaaccgccga agccacgcgg
4321 ctcaaaagaa cagcacggcg cagatatacc cgcagaaaga atcggatctg ctacctgcag
4381 gagatcttta gtaatgagat ggctaaggtg gatgactctt tcttccatag gctggaggag
4441 tcctttttgg tggaggagga taaaaagcac gagcgccacc caatctttgg caatatcgtg
4501 gacgaggtgg cgtaccatga aaagtaccca accatatatc atctgaggaa gaagcttgta
4561 gacagtactg ataaggctga cttgcggttg atctatctcg cgctggcgca tatgatcaaa
4621 tttcggggac acttcctcat cgagggggac ctgaacccag acaacagcga tgtcgacaaa
4681 ctctttatcc aactggttca gacttacaat cagcttttcg aagagaaccc gatcaacgca
4741 tccggagttg acgccaaagc aatcctgagc gctaggctgt ccaaatcccg gcggctcgaa
4801 aacctcatcg cacagctccc tggggagaag aagaacggcc tgtttggtaa tcttatcgcc
4861 ctgtcactcg ggctgacccc caactttaaa tctaacttcg acctggccga agatgccaag
4921 cttcaactga gcaaagacac ctacgatgat gatctcgaca atctgctggc ccagatcggc
4981 gaccagtacg cagacctttt tttggcggca aagaacctgt cagacgccat tctgctgagt
5041 gatattctgc gagtgaacac ggagatcacc aaagctccgc tgagcgctag tatgatcaag
5101 cgctatgatg agcaccacca agacttgact ttgctgaagg cccttgtcag acagcaactg
5161 cctgagaagt acaaggaaat tttcttcgat cagtctaaaa atggctacgc cggatacatt
5221 gacggcggag caagccagga ggaattttac aaatttatta agcccatctt ggaaaaaatg
5281 gacggcaccg aggagctgct ggtaaagctt aacagagaag atctgttgcg caaacagcgc
5341 actttcgaca atggaagcat cccccaccag attcacctgg gcgaactgca cgctatcctc
5401 aggcggcaag aggatttcta cccctttttg aaagataaca gggaaaagat tgagaaaatc
5461 ctcacatttc ggatacccta ctatgtaggc cccctcgccc ggggaaattc cagattcgcg
5521 tggatgactc gcaaatcaga agagaccatc actccctgga acttcgagga agtcgtggat
5581 aagggggcct ctgcccagtc cttcatcgaa aggatgacta actttgataa aaatctgcct
5641 aacgaaaagg tgcttcctaa acactctctg ctgtacgagt acttcacagt ttataacgag
5701 ctcaccaagg tcaaatacgt cacagaaggg atgagaaagc cagcattcct gtctggagag
5761 cagaagaaag ctatcgtgga cctcctcttc aagacgaacc ggaaagttac cgtgaaacag
5821 ctcaaagaag actatttcaa aaagattgaa tgtttcgact ctgttgaaat cagcggagtg
5881 gaggatcgct tcaacgcatc cctgggaacg tatcacgatc tcctgaaaat cattaaagac
5941 aaggacttcc tggacaatga ggagaacgag gacattcttg aggacattgt cctcaccctt
6001 acgttgtttg aagataggga gatgattgaa gaacgcttga aaacttacgc tcatctcttc
6061 gacgacaaag tcatgaaaca gctcaagagg cgccgatata caggatgggg gcggctgtca
6121 agaaaactga tcaatgggat ccgagacaag cagagtggaa agacaatcct ggattttctt
6181 aagtccgatg gatttgccaa ccggaacttc atgcagttga tccatgatga ctctctcacc
6241 tttaaggagg acatccagaa agcacaagtt tctggccagg gggacagtct tcacgagcac
6301 atcgctaatc ttgcaggtag cccagctatc aaaaagggaa tactgcagac cgttaaggtc
6361 gtggatgaac tcgtcaaagt aatgggaagg cataagcccg agaatatcgt tatcgagatg
6421 gcccgagaga accaaactac ccagaaggga cagaagaaca gtagggaaag gatgaagagg
6481 attgaagagg gtataaaaga actggggtcc caaatcctta aggaacaccc agttgaaaac
6541 acccagcttc agaatgagaa gctctacctg tactacctgc agaacggcag ggacatgtac
6601 gtggatcagg aactggacat caatcggctc tccgactacg acgtggatgc catcgtgccc
6661 cagtcttttc tcaaagatga ttctattgat aataaagtgt tgacaagatc cgataaaaat
6721 agagggaaga gtgataacgt cccctcagaa gaagttgtca agaaaatgaa aaattattgg
6781 cggcagctgc tgaacgccaa actgatcaca caacggaagt tcgataatct gactaaggct
6841 gaacgaggtg gcctgtctga gttggataaa gccggcttca tcaaaaggca gcttgttgag
6901 acacgccaga tcaccaagca cgtggcccaa attctcgatt cacgcatgaa caccaagtac
6961 gatgaaaatg acaaactgat tcgagaggtg aaagttatta ctctgaagtc taagctggtc
7021 tcagatttca gaaaggactt tcagttttat aaggtgagag agatcaacaa ttaccaccat
7081 gcgcatgatg cctacctgaa tgcagtggta ggcactgcac ttatcaaaaa atatcccaag
7141 cttgaatctg aatttgttta cggagactat aaagtgtacg atgttaggaa aatgatcgca
7201 aagtctgagc aggaaatagg caaggccacc gctaagtact tcttttacag caatattatg
7261 aattttttca agaccgagat tacactggcc aatggagaga ttcggaagcg accacttatc
7321 gaaacaaacg gagaaacagg agaaatcgtg tgggacaagg gtagggattt cgcgacagtc
7381 cggaaggtcc tgtccatgcc gcaggtgaac atcgttaaaa agaccgaagt acagaccgga
7441 ggcttctcca aggaaagtat cctcccgaaa aggaacagcg acaagctgat cgcacgcaaa
7501 aaagattggg accccaagaa atacggcgga ttcgattctc ctacagtcgc ttacagtgta
7561 ctggttgtgg ccaaagtgga gaaagggaag tctaaaaaac tcaaaagcgt caaggaactg
7621 ctgggcatca caatcatgga gcgatcaagc ttcgaaaaaa accccatcga ctttctcgag
7681 gcgaaaggat ataaagaggt caaaaaagac ctcatcatta agcttcccaa gtactctctc
7741 tttgagcttg aaaacggccg gaaacgaatg ctcgctagtg cgggcgagct gcagaaaggt
7801 aacgagctgg cactgccctc taaatacgtt aatttcttgt atctggccag ccactatgaa
7861 aagctcaaag ggtctcccga agataatgag cagaagcagc tgttcgtgga acaacacaaa
7921 cactaccttg atgagatcat cgagcaaata agcgaattct ccaaaagagt gatcctcgcc
7981 gacgctaacc tcgataaggt gctttctgct tacaataagc acagggataa gcccatcagg
8041 gagcaggcag aaaacattat ccacttgttt actctgacca acttgggcgc gcctgcagcc
8101 ttcaagtact tcgacaccac catagacaga aagcggtaca cctctacaaa ggaggtcctg
8161 gacgccacac tgattcatca gtcaattacg gggctctatg aaacaagaat cgacctctct
8221 cagctcggtg gagacagcag ggctgacccc aagaagaaga ggaaggtggc tagcgatgct
8281 aagtcactga ctgcctggtc ccggacactg gtgaccttca aggatgtgtt tgtggacttc
8341 accagggagg agtggaagct gctggacact gctcagcaga tcctgtacag aaatgtgatg
8401 ctggagaact ataagaacct ggtttccttg ggttatcagc ttactaagcc agatgtgatc
8461 ctccggttgg agaagggaga agagccctgg ctggtggaga gagaaattca ccaagagacc
8521 catcctgatt cagagactgc atttgaaatc aaatcatcag ttccgaaaaa gaaacgcaaa
8581 gttgctagcg agggcagagg aagtcttcta acatgcggtg acgtggagga gaatcccggc
8641 cctggtaccg tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag
8701 ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc
8761 acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg
8821 cccaccctcg tgaccaccct gacctacggc gtgcagtgct tcagccgcta ccccgaccac
8881 atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc
8941 atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac
9001 accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg
9061 gggcacaagc tggagtacaa ctacaacagc cacaacgtct atatcatggc cgacaagcag
9121 aagaacggca tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag
9181 ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac
9241 aaccactacc tgagcaccca gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac
9301 atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctgtac
9361 aagaccggtt gataatagat aacttcgtat agcatacatt atacgaagtt atgaattcga
9421 tatcaagctt atcgataatc aacctctgga ttacaaaatt tgtgaaagat tgactggtat
9481 tcttaactat gttgctcctt ttacgctatg tggatacgct gctttaatgc ctttgtatca
9541 tgctattgct tcccgtatgg ctttcatttt ctcctccttg tataaatcct ggttgctgtc
9601 tctttatgag gagttgtggc ccgttgtcag gcaacgtggc gtggtgtgca ctgtgtttgc
9661 tgacgcaacc cccactggtt ggggcattgc caccacctgt cagctccttt ccgggacttt
9721 cgctttcccc ctccctattg ccacggcgga actcatcgcc gcctgccttg cccgctgctg
9781 gacaggggct cggctgttgg gcactgacaa ttccgtggtg ttgtcgggga aatcatcgtc
9841 ctttccttgg ctgctcgcct gtgttgccac ctggattctg cgcgggacgt ccttctgcta
9901 cgtcccttcg gccctcaatc cagcggacct tccttcccgc ggcctgctgc cggctctgcg
9961 gcctcttccg cgtcttcgcc ttcgccctca gacgagtcgg atctcccttt gggccgcctc
10021 cccgcatcga taccgtcgac ctcgagacct agaaaaacat ggagcaatca caagtagcaa
10081 tacagcagct accaatgctg attgtgcctg gctagaagca caagaggagg aggaggtggg
10141 ttttccagtc acacctcagg tacctttaag accaatgact tacaaggcag ctgtagatct
10201 tagccacttt ttaaaagaaa aggggggact ggaagggcta attcactccc aacgaagaca
10261 agatatcctt gatctgtgga tctaccacac acaaggctac ttccctgatt ggcagaacta
10321 cacaccaggg ccagggatca gatatccact gacctttgga tggtgctaca agctagtacc
10381 agttgagcaa gagaaggtag aagaagccaa tgaaggagag aacacccgct tgttacaccc
10441 tgtgagcctg catgggatgg atgacccgga gagagaagta ttagagtgga ggtttgacag
10501 ccgcctagca tttcatcaca tggcccgaga gctgcatccg gactgtactg ggtctctctg
10561 gttagaccag atctgagcct gggagctctc tggctaacta gggaacccac tgcttaagcc
10621 tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg
10681 taactagaga tccctcagac ccttttagtc agtgtggaaa atctctagca gggcccgttt
10741 aaacccgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct
10801 cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg
10861 aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc
10921 aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct
10981 ctatggcttc tgaggcggaa agaaccagct ggggctctag ggggtatccc cacgcgccct
11041 gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg
11101 ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg
11161 gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
11221 ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct
11281 gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt
11341 tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta taagggattt
11401 tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt
11461 aattctgtgg aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag
11521 aagtatgcaa agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc
11581 cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc
11641 cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg
11701 ctgactaatt ttttttattt atgcagaggc cgaggccgcc tctgcctctg agctattcca
11761 gaagtagtga ggaggctttt ttggaggcct aggcttttgc aaaaagctcc cgggagcttg
11821 tatatccatt ttcggatctg atcagcacgt gttgacaatt aatcatcggc atagtatatc
11881 ggcatagtat aatacgacaa ggtgaggaac taaaccatgg ccaagttgac cagtgccgtt
11941 ccggtgctca ccgcgcgcga cgtcgccgga gcggtcgagt tctggaccga ccggctcggg
12001 ttctcccggg acttcgtgga ggacgacttc gccggtgtgg tccgggacga cgtgaccctg
12061 ttcatcagcg cggtccagga ccaggtggtg ccggacaaca ccctggcctg ggtgtgggtg
12121 cgcggcctgg acgagctgta cgccgagtgg tcggaggtcg tgtccacgaa cttccgggac
12181 gcctccgggc cggccatgac cgagatcggc gagcagccgt gggggcggga gttcgccctg
12241 cgcgacccgg ccggcaactg cgtgcacttc gtggccgagg agcaggactg acacgtgcta
12301 cgagatttcg attccaccgc cgccttctat gaaaggttgg gcttcggaat cgttttccgg
12361 gacgccggct ggatgatcct ccagcgcggg gatctcatgc tggagttctt cgcccacccc
12421 aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca
12481 aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct
12541 tatcatgtct gtataccgtc gacctctagc tagagcttgg cgtaatcatg gtcatagctg
12601 tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata
12661 aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca
12721 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
12781 gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg
12841 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta
12901 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc
12961 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
13021 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
13081 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
13141 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt
13201 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
13261 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
13321 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
13381 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta
13441 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
13501 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
13561 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag
13621 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc
13681 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact
13741 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt
13801 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta
13861 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta
13921 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc
13981 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat
14041 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt
14101 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg
14161 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca
14221 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta
14281 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg
14341 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact
14401 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg
14461 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt
14521 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga
14581 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc
14641 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa
14701 caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcga cggatcggga
14761 gatctcccga tcccctatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa
14821 gccagtatct gctccctgct tgtgtgttgg aggtcgctga gtagtgcgcg agcaaaattt
14881 aagctacaac aaggcaaggc ttgaccgaca attgcatgaa gaatctgctt agggttaggc
14941 gttttgcgct gcttcgcgat gtacgggcca gatatacgcg tt
//
