| 质粒名称: | lenti dCAS-VP64_Blast |
|---|---|
| 出品公司: | 爱迪基因 |
| 目录编号: | 61425 |
| 存储实验室: | Feng Zhang |
|
载体骨架基本信息 |
|
| 载体名称: | plenti |
| 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: | -- |
| 载体修饰: | 人细胞色素c氧化酶亚基VIII（human cytochrome C oxidase subunit VIII） |
| 载体类型: | 哺乳细胞表达 |
| 筛选标记: | 新霉素（G418） |
| 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: | dCAS9(D10A, N863A)-VP64_2A_Blast |
| 物种来源: | -- |
| 插入大小: | -- |
| 基因突变: | -- |
| GenBank编号 | -- |
| 启动子 | -- |
| 标签/融合蛋白: | 人细胞色素c氧化酶亚基VIII（N端） |
| 克隆方法: | 限制性内切酶 |
| 5' 克隆位点: | AgeI(未破坏) |
| 3' 克隆位点: | NotI (未破坏) |
| 5' 测序引物 | CMV-F |
| 3' 测序引物 | SV40pA-R |
|
文献引用方法 |
|
| 材料和方法部分: |
lenti dCAS-VP64_Blast was a gift from Feng Zhang (Addgene plasmid # 61425 ; http://n2t.net/addgene:61425 ; RRID:Addgene_61425)
For viral preps, please replace (Addgene plasmid # 61425) in the above sentence with: (Addgene viral prep # 61425-LVC)
|
| 参考文献部分: | Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex. Konermann S, Brigham MD, Trevino AE, Joung J, Abudayyeh OO, Barcena C, Hsu PD, Habib N, Gootenberg JS, Nishimasu H, Nureki O, Zhang F. Nature. 2014 Dec 10. doi: 10.1038/nature14136. 10.1038/nature14136 PubMed 25494202 |
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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 lenti dCAS-VP64_Blast 14085 bp ds-DNA circular SYN 16-MAY-2019
DEFINITION 3rd generation lenti vector encoding dCAS9-VP64 with 2A Blast
resistance marker (EF1a-NLS-dCas9(N863)-VP64-2A-Blast-WPRE).
ACCESSION .
VERSION .
KEYWORDS lenti dCAS-VP64_Blast
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 14085)
AUTHORS Konermann S, Brigham MD, Trevino AE, Joung J, Abudayyeh OO, Barcena
C, Hsu PD, Habib N, Gootenberg JS, Nishimasu H, Nureki O, Zhang F
TITLE Genome-scale transcriptional activation by an engineered CRISPR-Cas9
complex.
JOURNAL Nature. 2014 Dec 10. doi: 10.1038/nature14136.
PUBMED 25494202
REFERENCE 2 (bases 1 to 14085)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 16, 2019 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..14085
/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"
promoter 2687..3865
/label=EF-1-alpha promoter
/note="strong constitutive promoter for human elongation
factor EF-1-alpha"
intron 2918..3856
/label=EF-1-alpha intron A
/note="intron upstream of the start codon of human
EF-1-alpha"
primer_bind 3813..3833
/label=EF1a-F
/note="Human elongation factor-1a promoter, forward primer"
CDS 3881..3928
/codon_start=1
/product="bipartite nuclear localization signal from
nucleoplasmin"
/label=nucleoplasmin NLS
/translation="KRPAATKKAGQAKKKK"
CDS 3929..8029
/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 N863A mutation in the HNH
catalytic domain"
/translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKARGKSDNVPS
EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
ATLIHQSITGLYETRIDLSQLGGD"
CDS 8084..8104
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 8129..8278
/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 8300..8353
/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 8354..8752
/codon_start=1
/gene="Aspergillus terreus BSD"
/product="blasticidin S deaminase"
/label=BSD
/note="confers resistance to blasticidin"
/translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
KAIVKDSDGQPTAVGIRELLPSGYVWEG"
misc_feature 8777..9365
/label=WPRE
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
primer_bind complement(8830..8850)
/label=WPRE-R
/note="WPRE, reverse primer"
CDS complement(9248..9259)
/codon_start=1
/product="Factor Xa recognition and cleavage site"
/label=Factor Xa site
/translation="IEGR"
primer_bind complement(9368..9384)
/label=KS primer
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(9369..9385)
/label=pBluescriptKS
/note="For pBluescript vector"
LTR 9890..10070
/label=5' LTR (truncated)
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
primer_bind complement(10096..10113)
/label=BGH-rev
/note="Bovine growth hormone terminator, reverse primer.
Also called BGH reverse"
polyA_signal 10102..10326
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
rep_origin 10372..10800
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(10459..10478)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 10669..10690
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind complement(10809..10829)
/label=pBABE 3'
/note="SV40 enhancer, reverse primer for pBABE vectors"
promoter 10814..11143
/label=SV40 promoter
/note="SV40 enhancer and early promoter"
rep_origin 10994..11129
/label=SV40 ori
/note="SV40 origin of replication"
primer_bind 11056..11075
/label=SV40pro-F
/note="SV40 promoter/origin, forward primer"
promoter 11191..11238
/label=EM7 promoter
/note="synthetic bacterial promoter "
CDS 11257..11631
/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 11761..11882
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(11798..11817)
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
primer_bind 11852..11871
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
primer_bind complement(11931..11947)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(11931..11947)
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind complement(11944..11966)
/label=M13/pUC Reverse
/note="In lacZ gene"
protein_bind 11955..11971
/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(11979..12009)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 12024..12045
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(12162..12179)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(12333..12921)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(12413..12432)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS complement(13092..13952)
/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 13715..13734
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
promoter complement(13953..14057)
/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 gctagctgca aagatggata aagttttaaa cagagaggaa
2641 tctttgcagc taatggacct tctaggtctt gaaaggagtg ggaattggct ccggtgcccg
2701 tcagtgggca gagcgcacat cgcccacagt ccccgagaag ttggggggag gggtcggcaa
2761 ttgaaccggt gcctagagaa ggtggcgcgg ggtaaactgg gaaagtgatg tcgtgtactg
2821 gctccgcctt tttcccgagg gtgggggaga accgtatata agtgcagtag tcgccgtgaa
2881 cgttcttttt cgcaacgggt ttgccgccag aacacaggta agtgccgtgt gtggttcccg
2941 cgggcctggc ctctttacgg gttatggccc ttgcgtgcct tgaattactt ccacctggct
3001 gcagtacgtg attcttgatc ccgagcttcg ggttggaagt gggtgggaga gttcgaggcc
3061 ttgcgcttaa ggagcccctt cgcctcgtgc ttgagttgag gcctggcctg ggcgctgggg
3121 ccgccgcgtg cgaatctggt ggcaccttcg cgcctgtctc gctgctttcg ataagtctct
3181 agccatttaa aatttttgat gacctgctgc gacgcttttt ttctggcaag atagtcttgt
3241 aaatgcgggc caagatctgc acactggtat ttcggttttt ggggccgcgg gcggcgacgg
3301 ggcccgtgcg tcccagcgca catgttcggc gaggcggggc ctgcgagcgc ggccaccgag
3361 aatcggacgg gggtagtctc aagctggccg gcctgctctg gtgcctggcc tcgcgccgcc
3421 gtgtatcgcc ccgccctggg cggcaaggct ggcccggtcg gcaccagttg cgtgagcgga
3481 aagatggccg cttcccggcc ctgctgcagg gagctcaaaa tggaggacgc ggcgctcggg
3541 agagcgggcg ggtgagtcac ccacacaaag gaaaagggcc tttccgtcct cagccgtcgc
3601 ttcatgtgac tccacggagt accgggcgcc gtccaggcac ctcgattagt tctcgagctt
3661 ttggagtacg tcgtctttag gttgggggga ggggttttat gcgatggagt ttccccacac
3721 tgagtgggtg gagactgaag ttaggccagc ttggcacttg atgtaattct ccttggaatt
3781 tgcccttttt gagtttggat cttggttcat tctcaagcct cagacagtgg ttcaaagttt
3841 ttttcttcca tttcaggtgt cgtgacgtac ggccaccatg aaaaggccgg cggccacgaa
3901 aaaggccggc caggcaaaaa agaaaaagga caagaagtac agcatcggcc tggccatcgg
3961 caccaactct gtgggctggg ccgtgatcac cgacgagtac aaggtgccca gcaagaaatt
4021 caaggtgctg ggcaacaccg accggcacag catcaagaag aacctgatcg gagccctgct
4081 gttcgacagc ggcgaaacag ccgaggccac ccggctgaag agaaccgcca gaagaagata
4141 caccagacgg aagaaccgga tctgctatct gcaagagatc ttcagcaacg agatggccaa
4201 ggtggacgac agcttcttcc acagactgga agagtccttc ctggtggaag aggataagaa
4261 gcacgagcgg caccccatct tcggcaacat cgtggacgag gtggcctacc acgagaagta
4321 ccccaccatc taccacctga gaaagaaact ggtggacagc accgacaagg ccgacctgcg
4381 gctgatctat ctggccctgg cccacatgat caagttccgg ggccacttcc tgatcgaggg
4441 cgacctgaac cccgacaaca gcgacgtgga caagctgttc atccagctgg tgcagaccta
4501 caaccagctg ttcgaggaaa accccatcaa cgccagcggc gtggacgcca aggccatcct
4561 gtctgccaga ctgagcaaga gcagacggct ggaaaatctg atcgcccagc tgcccggcga
4621 gaagaagaat ggcctgttcg gcaacctgat tgccctgagc ctgggcctga cccccaactt
4681 caagagcaac ttcgacctgg ccgaggatgc caaactgcag ctgagcaagg acacctacga
4741 cgacgacctg gacaacctgc tggcccagat cggcgaccag tacgccgacc tgtttctggc
4801 cgccaagaac ctgtccgacg ccatcctgct gagcgacatc ctgagagtga acaccgagat
4861 caccaaggcc cccctgagcg cctctatgat caagagatac gacgagcacc accaggacct
4921 gaccctgctg aaagctctcg tgcggcagca gctgcctgag aagtacaaag agattttctt
4981 cgaccagagc aagaacggct acgccggcta cattgacggc ggagccagcc aggaagagtt
5041 ctacaagttc atcaagccca tcctggaaaa gatggacggc accgaggaac tgctcgtgaa
5101 gctgaacaga gaggacctgc tgcggaagca gcggaccttc gacaacggca gcatccccca
5161 ccagatccac ctgggagagc tgcacgccat tctgcggcgg caggaagatt tttacccatt
5221 cctgaaggac aaccgggaaa agatcgagaa gatcctgacc ttccgcatcc cctactacgt
5281 gggccctctg gccaggggaa acagcagatt cgcctggatg accagaaaga gcgaggaaac
5341 catcaccccc tggaacttcg aggaagtggt ggacaagggc gcttccgccc agagcttcat
5401 cgagcggatg accaacttcg ataagaacct gcccaacgag aaggtgctgc ccaagcacag
5461 cctgctgtac gagtacttca ccgtgtataa cgagctgacc aaagtgaaat acgtgaccga
5521 gggaatgaga aagcccgcct tcctgagcgg cgagcagaaa aaggccatcg tggacctgct
5581 gttcaagacc aaccggaaag tgaccgtgaa gcagctgaaa gaggactact tcaagaaaat
5641 cgagtgcttc gactccgtgg aaatctccgg cgtggaagat cggttcaacg cctccctggg
5701 cacataccac gatctgctga aaattatcaa ggacaaggac ttcctggaca atgaggaaaa
5761 cgaggacatt ctggaagata tcgtgctgac cctgacactg tttgaggaca gagagatgat
5821 cgaggaacgg ctgaaaacct atgcccacct gttcgacgac aaagtgatga agcagctgaa
5881 gcggcggaga tacaccggct ggggcaggct gagccggaag ctgatcaacg gcatccggga
5941 caagcagtcc ggcaagacaa tcctggattt cctgaagtcc gacggcttcg ccaacagaaa
6001 cttcatgcag ctgatccacg acgacagcct gacctttaaa gaggacatcc agaaagccca
6061 ggtgtccggc cagggcgata gcctgcacga gcacattgcc aatctggccg gcagccccgc
6121 cattaagaag ggcatcctgc agacagtgaa ggtggtggac gagctcgtga aagtgatggg
6181 ccggcacaag cccgagaaca tcgtgatcga aatggccaga gagaaccaga ccacccagaa
6241 gggacagaag aacagccgcg agagaatgaa gcggatcgaa gagggcatca aagagctggg
6301 cagccagatc ctgaaagaac accccgtgga aaacacccag ctgcagaacg agaagctgta
6361 cctgtactac ctgcagaatg ggcgggatat gtacgtggac caggaactgg acatcaaccg
6421 gctgtccgac tacgatgtgg accacatcgt gcctcagagc tttctgaagg acgactccat
6481 cgacaacaag gtgctgacca gaagcgacaa ggcccggggc aagagcgaca acgtgccctc
6541 cgaagaggtc gtgaagaaga tgaagaacta ctggcggcag ctgctgaacg ccaagctgat
6601 tacccagaga aagttcgaca atctgaccaa ggccgagaga ggcggcctga gcgaactgga
6661 taaggccggc ttcatcaaga gacagctggt ggaaacccgg cagatcacaa agcacgtggc
6721 acagatcctg gactcccgga tgaacactaa gtacgacgag aatgacaagc tgatccggga
6781 agtgaaagtg atcaccctga agtccaagct ggtgtccgat ttccggaagg atttccagtt
6841 ttacaaagtg cgcgagatca acaactacca ccacgcccac gacgcctacc tgaacgccgt
6901 cgtgggaacc gccctgatca aaaagtaccc taagctggaa agcgagttcg tgtacggcga
6961 ctacaaggtg tacgacgtgc ggaagatgat cgccaagagc gagcaggaaa tcggcaaggc
7021 taccgccaag tacttcttct acagcaacat catgaacttt ttcaagaccg agattaccct
7081 ggccaacggc gagatccgga agcggcctct gatcgagaca aacggcgaaa ccggggagat
7141 cgtgtgggat aagggccggg attttgccac cgtgcggaaa gtgctgagca tgccccaagt
7201 gaatatcgtg aaaaagaccg aggtgcagac aggcggcttc agcaaagagt ctatcctgcc
7261 caagaggaac agcgataagc tgatcgccag aaagaaggac tgggacccta agaagtacgg
7321 cggcttcgac agccccaccg tggcctattc tgtgctggtg gtggccaaag tggaaaaggg
7381 caagtccaag aaactgaaga gtgtgaaaga gctgctgggg atcaccatca tggaaagaag
7441 cagcttcgag aagaatccca tcgactttct ggaagccaag ggctacaaag aagtgaaaaa
7501 ggacctgatc atcaagctgc ctaagtactc cctgttcgag ctggaaaacg gccggaagag
7561 aatgctggcc tctgccggcg aactgcagaa gggaaacgaa ctggccctgc cctccaaata
7621 tgtgaacttc ctgtacctgg ccagccacta tgagaagctg aagggctccc ccgaggataa
7681 tgagcagaaa cagctgtttg tggaacagca caagcactac ctggacgaga tcatcgagca
7741 gatcagcgag ttctccaaga gagtgatcct ggccgacgct aatctggaca aagtgctgtc
7801 cgcctacaac aagcaccggg ataagcccat cagagagcag gccgagaata tcatccacct
7861 gtttaccctg accaatctgg gagcccctgc cgccttcaag tactttgaca ccaccatcga
7921 ccggaagagg tacaccagca ccaaagaggt gctggacgcc accctgatcc accagagcat
7981 caccggcctg tacgagacac ggatcgacct gtctcagctg ggaggcgaca gcgctggagg
8041 aggtggaagc ggaggaggag gaagcggagg aggaggtagc ggacctaaga aaaagaggaa
8101 ggtggcggcc gctggatccg gacgggctga cgcattggac gattttgatc tggatatgct
8161 gggaagtgac gccctcgatg attttgacct tgacatgctt ggttcggatg cccttgatga
8221 ctttgacctc gacatgctcg gcagtgacgc ccttgatgat ttcgacctgg acatgctgat
8281 taactgtaca ggcagtggag agggcagagg aagtctgcta acatgcggtg acgtcgagga
8341 gaatcctggc ccaatggcca agcctttgtc tcaagaagaa tccaccctca ttgaaagagc
8401 aacggctaca atcaacagca tccccatctc tgaagactac agcgtcgcca gcgcagctct
8461 ctctagcgac ggccgcatct tcactggtgt caatgtatat cattttactg ggggaccttg
8521 tgcagaactc gtggtgctgg gcactgctgc tgctgcggca gctggcaacc tgacttgtat
8581 cgtcgcgatc ggaaatgaga acaggggcat cttgagcccc tgcggacggt gccgacaggt
8641 gcttctcgat ctgcatcctg ggatcaaagc catagtgaag gacagtgatg gacagccgac
8701 ggcagttggg attcgtgaat tgctgccctc tggttatgtg tgggagggct aagaattcga
8761 tatcaagctt atcggtaatc aacctctgga ttacaaaatt tgtgaaagat tgactggtat
8821 tcttaactat gttgctcctt ttacgctatg tggatacgct gctttaatgc ctttgtatca
8881 tgctattgct tcccgtatgg ctttcatttt ctcctccttg tataaatcct ggttgctgtc
8941 tctttatgag gagttgtggc ccgttgtcag gcaacgtggc gtggtgtgca ctgtgtttgc
9001 tgacgcaacc cccactggtt ggggcattgc caccacctgt cagctccttt ccgggacttt
9061 cgctttcccc ctccctattg ccacggcgga actcatcgcc gcctgccttg cccgctgctg
9121 gacaggggct cggctgttgg gcactgacaa ttccgtggtg ttgtcgggga aatcatcgtc
9181 ctttccttgg ctgctcgcct gtgttgccac ctggattctg cgcgggacgt ccttctgcta
9241 cgtcccttcg gccctcaatc cagcggacct tccttcccgc ggcctgctgc cggctctgcg
9301 gcctcttccg cgtcttcgcc ttcgccctca gacgagtcgg atctcccttt gggccgcctc
9361 cccgcatcga taccgtcgac ctcgagacct agaaaaacat ggagcaatca caagtagcaa
9421 tacagcagct accaatgctg attgtgcctg gctagaagca caagaggagg aggaggtggg
9481 ttttccagtc acacctcagg tacctttaag accaatgact tacaaggcag ctgtagatct
9541 tagccacttt ttaaaagaaa aggggggact ggaagggcta attcactccc aacgaagaca
9601 agatatcctt gatctgtgga tctaccacac acaaggctac ttccctgatt ggcagaacta
9661 cacaccaggg ccagggatca gatatccact gacctttgga tggtgctaca agctagtacc
9721 agttgagcaa gagaaggtag aagaagccaa tgaaggagag aacacccgct tgttacaccc
9781 tgtgagcctg catgggatgg atgacccgga gagagaagta ttagagtgga ggtttgacag
9841 ccgcctagca tttcatcaca tggcccgaga gctgcatccg gactgtactg ggtctctctg
9901 gttagaccag atctgagcct gggagctctc tggctaacta gggaacccac tgcttaagcc
9961 tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg
10021 taactagaga tccctcagac ccttttagtc agtgtggaaa atctctagca gggcccgttt
10081 aaacccgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct
10141 cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg
10201 aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc
10261 aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct
10321 ctatggcttc tgaggcggaa agaaccagct ggggctctag ggggtatccc cacgcgccct
10381 gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg
10441 ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg
10501 gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
10561 ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct
10621 gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt
10681 tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta taagggattt
10741 tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt
10801 aattctgtgg aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag
10861 aagtatgcaa agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc
10921 cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc
10981 cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg
11041 ctgactaatt ttttttattt atgcagaggc cgaggccgcc tctgcctctg agctattcca
11101 gaagtagtga ggaggctttt ttggaggcct aggcttttgc aaaaagctcc cgggagcttg
11161 tatatccatt ttcggatctg atcagcacgt gttgacaatt aatcatcggc atagtatatc
11221 ggcatagtat aatacgacaa ggtgaggaac taaaccatgg ccaagttgac cagtgccgtt
11281 ccggtgctca ccgcgcgcga cgtcgccgga gcggtcgagt tctggaccga ccggctcggg
11341 ttctcccggg acttcgtgga ggacgacttc gccggtgtgg tccgggacga cgtgaccctg
11401 ttcatcagcg cggtccagga ccaggtggtg ccggacaaca ccctggcctg ggtgtgggtg
11461 cgcggcctgg acgagctgta cgccgagtgg tcggaggtcg tgtccacgaa cttccgggac
11521 gcctccgggc cggccatgac cgagatcggc gagcagccgt gggggcggga gttcgccctg
11581 cgcgacccgg ccggcaactg cgtgcacttc gtggccgagg agcaggactg acacgtgcta
11641 cgagatttcg attccaccgc cgccttctat gaaaggttgg gcttcggaat cgttttccgg
11701 gacgccggct ggatgatcct ccagcgcggg gatctcatgc tggagttctt cgcccacccc
11761 aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca
11821 aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct
11881 tatcatgtct gtataccgtc gacctctagc tagagcttgg cgtaatcatg gtcatagctg
11941 tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata
12001 aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca
12061 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
12121 gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg
12181 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta
12241 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc
12301 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
12361 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
12421 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
12481 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt
12541 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
12601 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
12661 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
12721 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta
12781 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
12841 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
12901 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag
12961 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc
13021 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact
13081 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt
13141 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta
13201 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta
13261 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc
13321 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat
13381 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt
13441 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg
13501 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca
13561 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta
13621 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg
13681 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact
13741 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg
13801 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt
13861 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga
13921 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc
13981 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa
14041 caaatagggg ttccgcgcac atttccccga aaagtgccac ctgac
//
