| 质粒名称: | pLV hUbC-VP64 dCas9 VP64-T2A-GFP |
|---|---|
| 出品公司: | 爱迪基因 |
| 目录编号: | 59791 |
| 存储实验室: | Charles Gersbach |
|
载体骨架基本信息 |
|
| 载体名称: | FUGW |
| 载体抗性: | 氨苄霉素 |
| 空载体大小: | 10000 bp |
| 完整质粒大小: | 15000 bp |
| 载体类型: | 哺乳细胞表达，Lentiviral, CRISPR |
| 高拷贝/低拷贝: | 未知 |
| 生长条件: | 37 ℃ |
|
插入基因信息 |
|
| 插入基因名称: | humanized VP64 dead Cas9 VP64 T2A GFP |
| 物种来源: | S. Pyogenes |
| 基因突变: | D10A and H840A |
| 标签/融合蛋白: |
Flag (N terminal on insert)
|
|
文献引用方法 |
|
| 材料和方法部分: | pLV hUbC-VP64 dCas9 VP64-T2A-GFP was a gift from Charles Gersbach (爱迪基因 plasmid # 59791) |
| 参考文献部分: | Multiplex CRISPR/Cas9-based genome engineering from a single lentiviral vector. Kabadi AM, Ousterout DG, Hilton IB, Gersbach CA. Nucleic Acids Res. 2014 Aug 13. pii: gku749. 10.1093/nar/gku749 PubMed 25122746 |
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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 pLV hUbC-VP64 dCas9 VP64-T2A-GFP 15099 bp ds-DNA circular SYN 10-JAN-2018
DEFINITION Co-expresses human optimized S. pyogenes dCas9 fused to two copies
of VP64 and GFP.
ACCESSION .
VERSION .
KEYWORDS pLV hUbC-VP64 dCas9 VP64-T2A-GFP
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 15099)
AUTHORS Kabadi AM, Ousterout DG, Hilton IB, Gersbach CA
TITLE Multiplex CRISPR/Cas9-based genome engineering from a single
lentiviral vector.
JOURNAL Nucleic Acids Res. 2014 Aug 13. pii: gku749.
PUBMED 25122746
COMMENT This file is created by Vector NTI
http://www.biofeng.com/
COMMENT ORIGDB|GenBank
COMMENT VNTAUTHORNAME|biofeng.com|
COMMENT VNTNAME|pLV hUbC-VP64 dCas9 VP64-T2A-GFP|
FEATURES Location/Qualifiers
source 1..15099
/organism="synthetic DNA construct"
/mol_type="other DNA"
primer_bind complement(47..66)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
enhancer 238..617
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 619..817
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
primer_bind 772..792
/label=CMV-F
/note="Human CMV immediate early promoter, forward primer"
LTR 835..1015
/label=5' LTR (truncated)
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
misc_feature 1062..1187
/label=HIV-1 Psi
/note="packaging signal of human immunodeficiency virus
type 1"
misc_feature 1680..1913
/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 2440..2557
/label=cPPT/CTS
/note="central polypurine tract and central termination
sequence of HIV-1"
protein_bind 2602..2635
/label=loxP
/bound_moiety="Cre recombinase"
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (GCATACAT)."
CDS complement(2622..2948)
/codon_start=1
/gene="lacZ fragment"
/product="LacZ-alpha fragment of beta-galactosidase"
/label=lacZ-alpha
/translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWDAPCSGATTVSY
HNFV"
primer_bind 2798..2820
/label=M13/pUC Forward
/note="In lacZ gene"
primer_bind 2812..2829
/label=M13 Forward
/note="In lacZ gene. Also called M13-F20 or M13 (-21)
Forward"
primer_bind 2813..2829
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
primer_bind 2839..2858
/label=T7
/note="T7 promoter, forward primer"
promoter 2839..2857
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
primer_bind complement(2905..2925)
/label=T3
/note="T3 promoter, forward primer"
promoter complement(2905..2923)
/label=T3 promoter
/note="promoter for bacteriophage T3 RNA polymerase"
primer_bind complement(2944..2960)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
protein_bind 2968..2984
/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(2992..3022)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 3037..3058
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 3124..4334
/label=UbC promoter
/note="human ubiquitin C promoter"
primer_bind 4171..4190
/label=hUBCpro-F
/note="Human Ubiquitin C (UbC) promoter, forward primer"
regulatory 4363..4372
/regulatory_class="other"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
CDS 4372..4437
/codon_start=1
/product="three tandem FLAG(R) epitope tags, followed by an
enterokinase cleavage site"
/label=3xFLAG
/translation="DYKDHDGDYKDHDIDYKDDDDK"
CDS 4453..4599
/codon_start=1
/product="tetrameric repeat of the minimal activation
domain of herpes simplex virus VP16 (Beerli et al., 1998)"
/label=VP64
/translation="DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDF
DLDM"
CDS 4606..4626
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 4636..8739
/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 8752..8772
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 8785..8934
/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 8944..8997
/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 9004..9717
/codon_start=1
/product="enhanced GFP"
/label=EGFP
/note="mammalian codon-optimized"
/translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
FVTAAGITLGMDELYK"
primer_bind complement(9046..9067)
/label=EGFP-N
/note="EGFP, reverse primer"
primer_bind complement(9307..9326)
/label=EXFP-R
/note="For distinguishing EGFP variants, reverse primer"
primer_bind 9654..9675
/label=EGFP-C
/note="EGFP, forward primer"
protein_bind 9733..9766
/label=loxP
/bound_moiety="Cre recombinase"
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (GCATACAT)."
misc_feature 9791..10379
/label=WPRE
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
primer_bind complement(9844..9864)
/label=WPRE-R
/note="WPRE, reverse primer"
CDS complement(10262..10273)
/codon_start=1
/product="Factor Xa recognition and cleavage site"
/label=Factor Xa site
/translation="IEGR"
primer_bind complement(10382..10398)
/label=KS primer
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(10383..10399)
/label=pBluescriptKS
/note="For pBluescript vector"
LTR 10904..11084
/label=5' LTR (truncated)
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
primer_bind complement(11110..11127)
/label=BGH-rev
/note="Bovine growth hormone terminator, reverse primer.
Also called BGH reverse"
polyA_signal 11116..11340
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
rep_origin 11386..11814
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(11473..11492)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 11683..11704
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind complement(11823..11843)
/label=pBABE 3'
/note="SV40 enhancer, reverse primer for pBABE vectors"
promoter 11828..12157
/label=SV40 promoter
/note="SV40 enhancer and early promoter"
rep_origin 12008..12143
/label=SV40 ori
/note="SV40 origin of replication"
primer_bind 12070..12089
/label=SV40pro-F
/note="SV40 promoter/origin, forward primer"
promoter 12205..12252
/label=EM7 promoter
/note="synthetic bacterial promoter "
CDS 12271..12645
/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 12775..12896
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(12812..12831)
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
primer_bind 12866..12885
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
primer_bind complement(12945..12961)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
protein_bind 12969..12985
/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(12993..13023)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 13038..13059
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(13176..13193)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(13347..13935)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(13427..13446)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS complement(14106..14966)
/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 14729..14748
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
promoter complement(14967..15071)
/gene="bla"
/label=AmpR promoter
ORIGIN
1 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg
61 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt
121 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc
181 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac
241 attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat
301 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg
361 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt
421 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
481 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
541 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag
601 tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt
661 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc
721 accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg
781 gcggtaggcg tgtacggtgg gaggtctata taagcagcgc gttttgcctg tactgggtct
841 ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt
901 aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac
961 tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtggc
1021 gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc
1081 ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa
1141 ttttgactag cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg
1201 ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata
1261 aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc
1321 tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga
1381 caggatcaga agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc
1441 aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca
1501 aaagtaagac caccgcacag caagcggccg ctgatcttca gacctggagg aggagatatg
1561 agggacaatt ggagaagtga attatataaa tataaagtag taaaaattga accattagga
1621 gtagcaccca ccaaggcaaa gagaagagtg gtgcagagag aaaaaagagc agtgggaata
1681 ggagctttgt tccttgggtt cttgggagca gcaggaagca ctatgggcgc agcgtcaatg
1741 acgctgacgg tacaggccag acaattattg tctggtatag tgcagcagca gaacaatttg
1801 ctgagggcta ttgaggcgca acagcatctg ttgcaactca cagtctgggg catcaagcag
1861 ctccaggcaa gaatcctggc tgtggaaaga tacctaaagg atcaacagct cctggggatt
1921 tggggttgct ctggaaaact catttgcacc actgctgtgc cttggaatgc tagttggagt
1981 aataaatctc tggaacagat ttggaatcac acgacctgga tggagtggga cagagaaatt
2041 aacaattaca caagcttaat acactcctta attgaagaat cgcaaaacca gcaagaaaag
2101 aatgaacaag aattattgga attagataaa tgggcaagtt tgtggaattg gtttaacata
2161 acaaattggc tgtggtatat aaaattattc ataatgatag taggaggctt ggtaggttta
2221 agaatagttt ttgctgtact ttctatagtg aatagagtta ggcagggata ttcaccatta
2281 tcgtttcaga cccacctccc aaccccgagg ggacccgaca ggcccgaagg aatagaagaa
2341 gaaggtggag agagagacag agacagatcc attcgattag tgaacggatc ggcactgcgt
2401 gcgccaattc tgcagacaaa tggcagtatt catccacaat tttaaaagaa aaggggggat
2461 tggggggtac agtgcagggg aaagaatagt agacataata gcaacagaca tacaaactaa
2521 agaattacaa aaacaaatta caaaaattca aaattttcgg gtttattaca gggacagcag
2581 agatccagtt tggttaatta aataacttcg tatagcatac attatacgaa gttatgataa
2641 gagacggtgg tggcgccgct acagggcgcg tcccattcgc cattcaggct gcgcaactgt
2701 tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt
2761 gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg
2821 acggccagtg agcgcgcgta atacgactca ctatagggcg aattgggtac cgggcccccc
2881 ctcgaggtcc tccagctttt gttcccttta gtgagggtta attgcgcgct tggcgtaatc
2941 atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg
3001 agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat
3061 tgcgttgcgc tcactgcccg ctttccactg catgacgtct ccacaattaa ttaagggtgc
3121 agcggcctcc gcgccgggtt ttggcgcctc ccgcgggcgc ccccctcctc acggcgagcg
3181 ctgccacgtc agacgaaggg cgcaggagcg ttcctgatcc ttccgcccgg acgctcagga
3241 cagcggcccg ctgctcataa gactcggcct tagaacccca gtatcagcag aaggacattt
3301 taggacggga cttgggtgac tctagggcac tggttttctt tccagagagc ggaacaggcg
3361 aggaaaagta gtcccttctc ggcgattctg cggagggatc tccgtggggc ggtgaacgcc
3421 gatgattata taaggacgcg ccgggtgtgg cacagctagt tccgtcgcag ccgggatttg
3481 ggtcgcggtt cttgtttgtg gatcgctgtg atcgtcactt ggtgagttgc gggctgctgg
3541 gctggccggg gctttcgtgg ccgccgggcc gctcggtggg acggaagcgt gtggagagac
3601 cgccaagggc tgtagtctgg gtccgcgagc aaggttgccc tgaactgggg gttgggggga
3661 gcgcacaaaa tggcggctgt tcccgagtct tgaatggaag acgcttgtaa ggcgggctgt
3721 gaggtcgttg aaacaaggtg gggggcatgg tgggcggcaa gaacccaagg tcttgaggcc
3781 ttcgctaatg cgggaaagct cttattcggg tgagatgggc tggggcacca tctggggacc
3841 ctgacgtgaa gtttgtcact gactggagaa ctcgggtttg tcgtctggtt gcgggggcgg
3901 cagttatgcg gtgccgttgg gcagtgcacc cgtacctttg ggagcgcgcg cctcgtcgtg
3961 tcgtgacgtc acccgttctg ttggcttata atgcagggtg gggccacctg ccggtaggtg
4021 tgcggtaggc ttttctccgt cgcaggacgc agggttcggg cctagggtag gctctcctga
4081 atcgacaggc gccggacctc tggtgagggg agggataagt gaggcgtcag tttctttggt
4141 cggttttatg tacctatctt cttaagtagc tgaagctccg gttttgaact atgcgctcgg
4201 ggttggcgag tgtgttttgt gaagtttttt aggcaccttt tgaaatgtaa tcatttgggt
4261 caatatgtaa ttttcagtgt tagactagta aattgtccgc taaattctgg ccgtttttgg
4321 cttttttgtt agacgaagct tgggctgcag gtcgactcta gagccaccat ggactacaaa
4381 gaccatgacg gtgattataa agatcatgac atcgattaca aggatgacga tgacaagcac
4441 ggtccgcggg ctgacgcatt ggacgatttt gatctggata tgctgggaag tgacgccctc
4501 gatgattttg accttgacat gcttggttcg gatgcccttg atgactttga cctcgacatg
4561 ctcggcagtg acgcccttga tgatttcgac ctggacatgg ttaaccccaa gaagaagagg
4621 aaggtgggcc gcggaatgga caagaagtac tccattgggc tcgccatcgg cacaaacagc
4681 gtcggctggg ccgtcattac ggacgagtac aaggtgccga gcaaaaaatt caaagttctg
4741 ggcaataccg atcgccacag cataaagaag aacctcattg gcgccctcct gttcgactcc
4801 ggggaaaccg ccgaagccac gcggctcaaa agaacagcac ggcgcagata tacccgcaga
4861 aagaatcgga tctgctacct gcaggagatc tttagtaatg agatggctaa ggtggatgac
4921 tctttcttcc ataggctgga ggagtccttt ttggtggagg aggataaaaa gcacgagcgc
4981 cacccaatct ttggcaatat cgtggacgag gtggcgtacc atgaaaagta cccaaccata
5041 tatcatctga ggaagaagct tgtagacagt actgataagg ctgacttgcg gttgatctat
5101 ctcgcgctgg cgcatatgat caaatttcgg ggacacttcc tcatcgaggg ggacctgaac
5161 ccagacaaca gcgatgtcga caaactcttt atccaactgg ttcagactta caatcagctt
5221 ttcgaagaga acccgatcaa cgcatccgga gttgacgcca aagcaatcct gagcgctagg
5281 ctgtccaaat cccggcggct cgaaaacctc atcgcacagc tccctgggga gaagaagaac
5341 ggcctgtttg gtaatcttat cgccctgtca ctcgggctga cccccaactt taaatctaac
5401 ttcgacctgg ccgaagatgc caagcttcaa ctgagcaaag acacctacga tgatgatctc
5461 gacaatctgc tggcccagat cggcgaccag tacgcagacc tttttttggc ggcaaagaac
5521 ctgtcagacg ccattctgct gagtgatatt ctgcgagtga acacggagat caccaaagct
5581 ccgctgagcg ctagtatgat caagcgctat gatgagcacc accaagactt gactttgctg
5641 aaggcccttg tcagacagca actgcctgag aagtacaagg aaattttctt cgatcagtct
5701 aaaaatggct acgccggata cattgacggc ggagcaagcc aggaggaatt ttacaaattt
5761 attaagccca tcttggaaaa aatggacggc accgaggagc tgctggtaaa gcttaacaga
5821 gaagatctgt tgcgcaaaca gcgcactttc gacaatggaa gcatccccca ccagattcac
5881 ctgggcgaac tgcacgctat cctcaggcgg caagaggatt tctacccctt tttgaaagat
5941 aacagggaaa agattgagaa aatcctcaca tttcggatac cctactatgt aggccccctc
6001 gcccggggaa attccagatt cgcgtggatg actcgcaaat cagaagagac catcactccc
6061 tggaacttcg aggaagtcgt ggataagggg gcctctgccc agtccttcat cgaaaggatg
6121 actaactttg ataaaaatct gcctaacgaa aaggtgcttc ctaaacactc tctgctgtac
6181 gagtacttca cagtttataa cgagctcacc aaggtcaaat acgtcacaga agggatgaga
6241 aagccagcat tcctgtctgg agagcagaag aaagctatcg tggacctcct cttcaagacg
6301 aaccggaaag ttaccgtgaa acagctcaaa gaagactatt tcaaaaagat tgaatgtttc
6361 gactctgttg aaatcagcgg agtggaggat cgcttcaacg catccctggg aacgtatcac
6421 gatctcctga aaatcattaa agacaaggac ttcctggaca atgaggagaa cgaggacatt
6481 cttgaggaca ttgtcctcac ccttacgttg tttgaagata gggagatgat tgaagaacgc
6541 ttgaaaactt acgctcatct cttcgacgac aaagtcatga aacagctcaa gaggcgccga
6601 tatacaggat gggggcggct gtcaagaaaa ctgatcaatg ggatccgaga caagcagagt
6661 ggaaagacaa tcctggattt tcttaagtcc gatggatttg ccaaccggaa cttcatgcag
6721 ttgatccatg atgactctct cacctttaag gaggacatcc agaaagcaca agtttctggc
6781 cagggggaca gtcttcacga gcacatcgct aatcttgcag gtagcccagc tatcaaaaag
6841 ggaatactgc agaccgttaa ggtcgtggat gaactcgtca aagtaatggg aaggcataag
6901 cccgagaata tcgttatcga gatggcccga gagaaccaaa ctacccagaa gggacagaag
6961 aacagtaggg aaaggatgaa gaggattgaa gagggtataa aagaactggg gtcccaaatc
7021 cttaaggaac acccagttga aaacacccag cttcagaatg agaagctcta cctgtactac
7081 ctgcagaacg gcagggacat gtacgtggat caggaactgg acatcaatcg gctctccgac
7141 tacgacgtgg atgccatcgt gccccagtct tttctcaaag atgattctat tgataataaa
7201 gtgttgacaa gatccgataa aaatagaggg aagagtgata acgtcccctc agaagaagtt
7261 gtcaagaaaa tgaaaaatta ttggcggcag ctgctgaacg ccaaactgat cacacaacgg
7321 aagttcgata atctgactaa ggctgaacga ggtggcctgt ctgagttgga taaagccggc
7381 ttcatcaaaa ggcagcttgt tgagacacgc cagatcacca agcacgtggc ccaaattctc
7441 gattcacgca tgaacaccaa gtacgatgaa aatgacaaac tgattcgaga ggtgaaagtt
7501 attactctga agtctaagct ggtctcagat ttcagaaagg actttcagtt ttataaggtg
7561 agagagatca acaattacca ccatgcgcat gatgcctacc tgaatgcagt ggtaggcact
7621 gcacttatca aaaaatatcc caagcttgaa tctgaatttg tttacggaga ctataaagtg
7681 tacgatgtta ggaaaatgat cgcaaagtct gagcaggaaa taggcaaggc caccgctaag
7741 tacttctttt acagcaatat tatgaatttt ttcaagaccg agattacact ggccaatgga
7801 gagattcgga agcgaccact tatcgaaaca aacggagaaa caggagaaat cgtgtgggac
7861 aagggtaggg atttcgcgac agtccggaag gtcctgtcca tgccgcaggt gaacatcgtt
7921 aaaaagaccg aagtacagac cggaggcttc tccaaggaaa gtatcctccc gaaaaggaac
7981 agcgacaagc tgatcgcacg caaaaaagat tgggacccca agaaatacgg cggattcgat
8041 tctcctacag tcgcttacag tgtactggtt gtggccaaag tggagaaagg gaagtctaaa
8101 aaactcaaaa gcgtcaagga actgctgggc atcacaatca tggagcgatc aagcttcgaa
8161 aaaaacccca tcgactttct cgaggcgaaa ggatataaag aggtcaaaaa agacctcatc
8221 attaagcttc ccaagtactc tctctttgag cttgaaaacg gccggaaacg aatgctcgct
8281 agtgcgggcg agctgcagaa aggtaacgag ctggcactgc cctctaaata cgttaatttc
8341 ttgtatctgg ccagccacta tgaaaagctc aaagggtctc ccgaagataa tgagcagaag
8401 cagctgttcg tggaacaaca caaacactac cttgatgaga tcatcgagca aataagcgaa
8461 ttctccaaaa gagtgatcct cgccgacgct aacctcgata aggtgctttc tgcttacaat
8521 aagcacaggg ataagcccat cagggagcag gcagaaaaca ttatccactt gtttactctg
8581 accaacttgg gcgcgcctgc agccttcaag tacttcgaca ccaccataga cagaaagcgg
8641 tacacctcta caaaggaggt cctggacgcc acactgattc atcagtcaat tacggggctc
8701 tatgaaacaa gaatcgacct ctctcagctc ggtggagaca gcagggctga ccccaagaag
8761 aagaggaagg tggctagccg cgccgacgcg ctggacgatt tcgatctcga catgctgggt
8821 tctgatgccc tcgatgactt tgacctggat atgttgggaa gcgacgcatt ggatgacttt
8881 gatctggaca tgctcggctc cgatgctctg gacgatttcg atctcgatat gttaatcgct
8941 agcgagggca gaggaagtct tctaacatgc ggtgacgtgg aggagaatcc cggccctggt
9001 accgtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac
9061 ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac
9121 ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc
9181 ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag
9241 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc
9301 ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg
9361 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac
9421 aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac
9481 ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc
9541 gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac
9601 tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc
9661 ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagacc
9721 ggttgataat agataacttc gtatagcata cattatacga agttatgaat tcgatatcaa
9781 gcttatcgat aatcaacctc tggattacaa aatttgtgaa agattgactg gtattcttaa
9841 ctatgttgct ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgctat
9901 tgcttcccgt atggctttca ttttctcctc cttgtataaa tcctggttgc tgtctcttta
9961 tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc
10021 aacccccact ggttggggca ttgccaccac ctgtcagctc ctttccggga ctttcgcttt
10081 ccccctccct attgccacgg cggaactcat cgccgcctgc cttgcccgct gctggacagg
10141 ggctcggctg ttgggcactg acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc
10201 ttggctgctc gcctgtgttg ccacctggat tctgcgcggg acgtccttct gctacgtccc
10261 ttcggccctc aatccagcgg accttccttc ccgcggcctg ctgccggctc tgcggcctct
10321 tccgcgtctt cgccttcgcc ctcagacgag tcggatctcc ctttgggccg cctccccgca
10381 tcgataccgt cgacctcgag acctagaaaa acatggagca atcacaagta gcaatacagc
10441 agctaccaat gctgattgtg cctggctaga agcacaagag gaggaggagg tgggttttcc
10501 agtcacacct caggtacctt taagaccaat gacttacaag gcagctgtag atcttagcca
10561 ctttttaaaa gaaaaggggg gactggaagg gctaattcac tcccaacgaa gacaagatat
10621 ccttgatctg tggatctacc acacacaagg ctacttccct gattggcaga actacacacc
10681 agggccaggg atcagatatc cactgacctt tggatggtgc tacaagctag taccagttga
10741 gcaagagaag gtagaagaag ccaatgaagg agagaacacc cgcttgttac accctgtgag
10801 cctgcatggg atggatgacc cggagagaga agtattagag tggaggtttg acagccgcct
10861 agcatttcat cacatggccc gagagctgca tccggactgt actgggtctc tctggttaga
10921 ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata
10981 aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta
11041 gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagggccc gtttaaaccc
11101 gctgatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg
11161 tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa
11221 ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca
11281 gcaaggggga ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg
11341 cttctgaggc ggaaagaacc agctggggct ctagggggta tccccacgcg ccctgtagcg
11401 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg
11461 ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc
11521 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc
11581 tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga
11641 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa
11701 ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg attttgccga
11761 tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattaattct
11821 gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat
11881 gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc
11941 aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac
12001 tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact
12061 aatttttttt atttatgcag aggccgaggc cgcctctgcc tctgagctat tccagaagta
12121 gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag ctcccgggag cttgtatatc
12181 cattttcgga tctgatcagc acgtgttgac aattaatcat cggcatagta tatcggcata
12241 gtataatacg acaaggtgag gaactaaacc atggccaagt tgaccagtgc cgttccggtg
12301 ctcaccgcgc gcgacgtcgc cggagcggtc gagttctgga ccgaccggct cgggttctcc
12361 cgggacttcg tggaggacga cttcgccggt gtggtccggg acgacgtgac cctgttcatc
12421 agcgcggtcc aggaccaggt ggtgccggac aacaccctgg cctgggtgtg ggtgcgcggc
12481 ctggacgagc tgtacgccga gtggtcggag gtcgtgtcca cgaacttccg ggacgcctcc
12541 gggccggcca tgaccgagat cggcgagcag ccgtgggggc gggagttcgc cctgcgcgac
12601 ccggccggca actgcgtgca cttcgtggcc gaggagcagg actgacacgt gctacgagat
12661 ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc
12721 ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaacttg
12781 tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa
12841 gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat
12901 gtctgtatac cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct
12961 gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt
13021 aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc
13081 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
13141 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
13201 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
13261 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
13321 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
13381 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
13441 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
13501 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
13561 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
13621 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
13681 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
13741 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
13801 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
13861 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
13921 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
13981 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
14041 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
14101 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
14161 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
14221 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
14281 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
14341 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
14401 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
14461 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
14521 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
14581 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
14641 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
14701 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
14761 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
14821 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
14881 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
14941 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
15001 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
15061 ggggttccgc gcacatttcc ccgaaaagtg ccacctgac
//
