| 质粒名称: | dCAS9-VP64_GFP |
|---|---|
| 出品公司: | 爱迪基因 |
| 目录编号: | 61422 |
| 存储实验室: | Feng Zhang |
|
载体骨架基本信息 |
|
| 载体名称: | lenti(AMP) |
| 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: | -- |
| 载体类型: | 哺乳细胞表达 |
| 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: | dCAS9(D10A,H840A)-VP64_2A_GFP |
| 物种来源: | -- |
| 插入大小: | -- |
| 基因突变: | -- |
| GenBank编号 | -- |
| 启动子 | -- |
| 标签/融合蛋白: | -- |
| 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: |
dCAS9-VP64_GFP was a gift from Feng Zhang (Addgene plasmid # 61422 ; http://n2t.net/addgene:61422 ; RRID:Addgene_61422)
For viral preps, please replace (Addgene plasmid # 61422) in the above sentence with: (Addgene viral prep # 61422-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 |
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 dCAS9-VP64_GFP 14547 bp ds-DNA circular SYN 06-AUG-2018
DEFINITION Expresses dCAS9-VP64 activator with 2A GFP.
ACCESSION .
VERSION .
KEYWORDS dCAS9-VP64_GFP
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 14547)
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 14547)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Aug 6, 2018 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..14547
/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 2682..3860
/label=EF-1-alpha promoter
/note="strong constitutive promoter for human elongation
factor EF-1-alpha"
intron 2913..3851
/label=EF-1-alpha intron A
/note="intron upstream of the start codon of human
EF-1-alpha"
primer_bind 3808..3828
/label=EF1a-F
/note="Human elongation factor-1a promoter, forward primer"
CDS 3879..3899
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 3909..8009
/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="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
ATLIHQSITGLYETRIDLSQLGGD"
CDS 8064..8084
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 8109..8258
/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 8280..8333
/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 8334..9050
/codon_start=1
/product="enhanced GFP"
/label=EGFP
/note="mammalian codon-optimized"
/translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
FVTAAGITLGMDELYK"
primer_bind complement(8376..8397)
/label=EGFP-N
/note="EGFP, reverse primer"
primer_bind complement(8637..8656)
/label=EXFP-R
/note="For distinguishing EGFP variants, reverse primer"
primer_bind 8984..9005
/label=EGFP-C
/note="EGFP, forward primer"
misc_feature 9075..9663
/label=WPRE
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
primer_bind complement(9128..9148)
/label=WPRE-R
/note="WPRE, reverse primer"
CDS complement(9546..9557)
/codon_start=1
/product="Factor Xa recognition and cleavage site"
/label=Factor Xa site
/translation="IEGR"
primer_bind complement(9666..9682)
/label=KS primer
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(9667..9683)
/label=pBluescriptKS
/note="For pBluescript vector"
LTR 10188..10368
/label=5' LTR (truncated)
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
primer_bind complement(10394..10411)
/label=BGH-rev
/note="Bovine growth hormone terminator, reverse primer.
Also called BGH reverse"
polyA_signal 10400..10624
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
rep_origin 10834..11262
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(10921..10940)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 11131..11152
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind complement(11271..11291)
/label=pBABE 3'
/note="SV40 enhancer, reverse primer for pBABE vectors"
promoter 11276..11605
/label=SV40 promoter
/note="SV40 enhancer and early promoter"
rep_origin 11456..11591
/label=SV40 ori
/note="SV40 origin of replication"
primer_bind 11518..11537
/label=SV40pro-F
/note="SV40 promoter/origin, forward primer"
promoter 11653..11700
/label=EM7 promoter
/note="synthetic bacterial promoter "
CDS 11719..12093
/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 12223..12344
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(12260..12279)
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
primer_bind 12314..12333
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
primer_bind complement(12393..12409)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(12393..12409)
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind complement(12406..12428)
/label=M13/pUC Reverse
/note="In lacZ gene"
protein_bind 12417..12433
/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(12441..12471)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 12486..12507
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(12624..12641)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(12795..13383)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(12875..12894)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS complement(13554..14414)
/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 14177..14196
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
promoter complement(14415..14519)
/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 atgcaaagat ggataaagtt ttaaacagag aggaatcttt
2641 gcagctaatg gaccttctag gtcttgaaag gagtgggaat tggctccggt gcccgtcagt
2701 gggcagagcg cacatcgccc acagtccccg agaagttggg gggaggggtc ggcaattgaa
2761 ccggtgccta gagaaggtgg cgcggggtaa actgggaaag tgatgtcgtg tactggctcc
2821 gcctttttcc cgagggtggg ggagaaccgt atataagtgc agtagtcgcc gtgaacgttc
2881 tttttcgcaa cgggtttgcc gccagaacac aggtaagtgc cgtgtgtggt tcccgcgggc
2941 ctggcctctt tacgggttat ggcccttgcg tgccttgaat tacttccacc tggctgcagt
3001 acgtgattct tgatcccgag cttcgggttg gaagtgggtg ggagagttcg aggccttgcg
3061 cttaaggagc cccttcgcct cgtgcttgag ttgaggcctg gcctgggcgc tggggccgcc
3121 gcgtgcgaat ctggtggcac cttcgcgcct gtctcgctgc tttcgataag tctctagcca
3181 tttaaaattt ttgatgacct gctgcgacgc tttttttctg gcaagatagt cttgtaaatg
3241 cgggccaaga tctgcacact ggtatttcgg tttttggggc cgcgggcggc gacggggccc
3301 gtgcgtccca gcgcacatgt tcggcgaggc ggggcctgcg agcgcggcca ccgagaatcg
3361 gacgggggta gtctcaagct ggccggcctg ctctggtgcc tggcctcgcg ccgccgtgta
3421 tcgccccgcc ctgggcggca aggctggccc ggtcggcacc agttgcgtga gcggaaagat
3481 ggccgcttcc cggccctgct gcagggagct caaaatggag gacgcggcgc tcgggagagc
3541 gggcgggtga gtcacccaca caaaggaaaa gggcctttcc gtcctcagcc gtcgcttcat
3601 gtgactccac ggagtaccgg gcgccgtcca ggcacctcga ttagttctcg agcttttgga
3661 gtacgtcgtc tttaggttgg ggggaggggt tttatgcgat ggagtttccc cacactgagt
3721 gggtggagac tgaagttagg ccagcttggc acttgatgta attctccttg gaatttgccc
3781 tttttgagtt tggatcttgg ttcattctca agcctcagac agtggttcaa agtttttttc
3841 ttccatttca ggtgtcgtga cgtacggcca ccatgagccc caagaagaag agaaaggtgg
3901 aggccagcga caagaagtac agcatcggcc tggccatcgg caccaactct gtgggctggg
3961 ccgtgatcac cgacgagtac aaggtgccca gcaagaaatt caaggtgctg ggcaacaccg
4021 accggcacag catcaagaag aacctgatcg gagccctgct gttcgacagc ggcgaaacag
4081 ccgaggccac ccggctgaag agaaccgcca gaagaagata caccagacgg aagaaccgga
4141 tctgctatct gcaagagatc ttcagcaacg agatggccaa ggtggacgac agcttcttcc
4201 acagactgga agagtccttc ctggtggaag aggataagaa gcacgagcgg caccccatct
4261 tcggcaacat cgtggacgag gtggcctacc acgagaagta ccccaccatc taccacctga
4321 gaaagaaact ggtggacagc accgacaagg ccgacctgcg gctgatctat ctggccctgg
4381 cccacatgat caagttccgg ggccacttcc tgatcgaggg cgacctgaac cccgacaaca
4441 gcgacgtgga caagctgttc atccagctgg tgcagaccta caaccagctg ttcgaggaaa
4501 accccatcaa cgccagcggc gtggacgcca aggccatcct gtctgccaga ctgagcaaga
4561 gcagacggct ggaaaatctg atcgcccagc tgcccggcga gaagaagaat ggcctgttcg
4621 gcaacctgat tgccctgagc ctgggcctga cccccaactt caagagcaac ttcgacctgg
4681 ccgaggatgc caaactgcag ctgagcaagg acacctacga cgacgacctg gacaacctgc
4741 tggcccagat cggcgaccag tacgccgacc tgtttctggc cgccaagaac ctgtccgacg
4801 ccatcctgct gagcgacatc ctgagagtga acaccgagat caccaaggcc cccctgagcg
4861 cctctatgat caagagatac gacgagcacc accaggacct gaccctgctg aaagctctcg
4921 tgcggcagca gctgcctgag aagtacaaag agattttctt cgaccagagc aagaacggct
4981 acgccggcta cattgacggc ggagccagcc aggaagagtt ctacaagttc atcaagccca
5041 tcctggaaaa gatggacggc accgaggaac tgctcgtgaa gctgaacaga gaggacctgc
5101 tgcggaagca gcggaccttc gacaacggca gcatccccca ccagatccac ctgggagagc
5161 tgcacgccat tctgcggcgg caggaagatt tttacccatt cctgaaggac aaccgggaaa
5221 agatcgagaa gatcctgacc ttccgcatcc cctactacgt gggccctctg gccaggggaa
5281 acagcagatt cgcctggatg accagaaaga gcgaggaaac catcaccccc tggaacttcg
5341 aggaagtggt ggacaagggc gcttccgccc agagcttcat cgagcggatg accaacttcg
5401 ataagaacct gcccaacgag aaggtgctgc ccaagcacag cctgctgtac gagtacttca
5461 ccgtgtataa cgagctgacc aaagtgaaat acgtgaccga gggaatgaga aagcccgcct
5521 tcctgagcgg cgagcagaaa aaggccatcg tggacctgct gttcaagacc aaccggaaag
5581 tgaccgtgaa gcagctgaaa gaggactact tcaagaaaat cgagtgcttc gactccgtgg
5641 aaatctccgg cgtggaagat cggttcaacg cctccctggg cacataccac gatctgctga
5701 aaattatcaa ggacaaggac ttcctggaca atgaggaaaa cgaggacatt ctggaagata
5761 tcgtgctgac cctgacactg tttgaggaca gagagatgat cgaggaacgg ctgaaaacct
5821 atgcccacct gttcgacgac aaagtgatga agcagctgaa gcggcggaga tacaccggct
5881 ggggcaggct gagccggaag ctgatcaacg gcatccggga caagcagtcc ggcaagacaa
5941 tcctggattt cctgaagtcc gacggcttcg ccaacagaaa cttcatgcag ctgatccacg
6001 acgacagcct gacctttaaa gaggacatcc agaaagccca ggtgtccggc cagggcgata
6061 gcctgcacga gcacattgcc aatctggccg gcagccccgc cattaagaag ggcatcctgc
6121 agacagtgaa ggtggtggac gagctcgtga aagtgatggg ccggcacaag cccgagaaca
6181 tcgtgatcga aatggccaga gagaaccaga ccacccagaa gggacagaag aacagccgcg
6241 agagaatgaa gcggatcgaa gagggcatca aagagctggg cagccagatc ctgaaagaac
6301 accccgtgga aaacacccag ctgcagaacg agaagctgta cctgtactac ctgcagaatg
6361 ggcgggatat gtacgtggac caggaactgg acatcaaccg gctgtccgac tacgatgtgg
6421 acgctatcgt gcctcagagc tttctgaagg acgactccat cgacaacaag gtgctgacca
6481 gaagcgacaa gaaccggggc aagagcgaca acgtgccctc cgaagaggtc gtgaagaaga
6541 tgaagaacta ctggcggcag ctgctgaacg ccaagctgat tacccagaga aagttcgaca
6601 atctgaccaa ggccgagaga ggcggcctga gcgaactgga taaggccggc ttcatcaaga
6661 gacagctggt ggaaacccgg cagatcacaa agcacgtggc acagatcctg gactcccgga
6721 tgaacactaa gtacgacgag aatgacaagc tgatccggga agtgaaagtg atcaccctga
6781 agtccaagct ggtgtccgat ttccggaagg atttccagtt ttacaaagtg cgcgagatca
6841 acaactacca ccacgcccac gacgcctacc tgaacgccgt cgtgggaacc gccctgatca
6901 aaaagtaccc taagctggaa agcgagttcg tgtacggcga ctacaaggtg tacgacgtgc
6961 ggaagatgat cgccaagagc gagcaggaaa tcggcaaggc taccgccaag tacttcttct
7021 acagcaacat catgaacttt ttcaagaccg agattaccct ggccaacggc gagatccgga
7081 agcggcctct gatcgagaca aacggcgaaa ccggggagat cgtgtgggat aagggccggg
7141 attttgccac cgtgcggaaa gtgctgagca tgccccaagt gaatatcgtg aaaaagaccg
7201 aggtgcagac aggcggcttc agcaaagagt ctatcctgcc caagaggaac agcgataagc
7261 tgatcgccag aaagaaggac tgggacccta agaagtacgg cggcttcgac agccccaccg
7321 tggcctattc tgtgctggtg gtggccaaag tggaaaaggg caagtccaag aaactgaaga
7381 gtgtgaaaga gctgctgggg atcaccatca tggaaagaag cagcttcgag aagaatccca
7441 tcgactttct ggaagccaag ggctacaaag aagtgaaaaa ggacctgatc atcaagctgc
7501 ctaagtactc cctgttcgag ctggaaaacg gccggaagag aatgctggcc tctgccggcg
7561 aactgcagaa gggaaacgaa ctggccctgc cctccaaata tgtgaacttc ctgtacctgg
7621 ccagccacta tgagaagctg aagggctccc ccgaggataa tgagcagaaa cagctgtttg
7681 tggaacagca caagcactac ctggacgaga tcatcgagca gatcagcgag ttctccaaga
7741 gagtgatcct ggccgacgct aatctggaca aagtgctgtc cgcctacaac aagcaccggg
7801 ataagcccat cagagagcag gccgagaata tcatccacct gtttaccctg accaatctgg
7861 gagcccctgc cgccttcaag tactttgaca ccaccatcga ccggaagagg tacaccagca
7921 ccaaagaggt gctggacgcc accctgatcc accagagcat caccggcctg tacgagacac
7981 ggatcgacct gtctcagctg ggaggcgaca gcgctggagg aggtggaagc ggaggaggag
8041 gaagcggagg aggaggtagc ggacctaaga aaaagaggaa ggtggcggcc gctggatccg
8101 gacgggctga cgcattggac gattttgatc tggatatgct gggaagtgac gccctcgatg
8161 attttgacct tgacatgctt ggttcggatg cccttgatga ctttgacctc gacatgctcg
8221 gcagtgacgc ccttgatgat ttcgacctgg acatgctgat taacgctagc ggcagtggag
8281 agggcagagg aagtctgcta acatgcggtg acgtcgagga gaatcctggc ccagtgagca
8341 agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa
8401 acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac ggcaagctga
8461 ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca
8521 ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag cagcacgact
8581 tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg
8641 acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca
8701 tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt
8761 acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac ggcatcaagg
8821 tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc gaccactacc
8881 agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagca
8941 cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt
9001 tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa gaattcgata
9061 tcaagcttat cgataatcaa cctctggatt acaaaatttg tgaaagattg actggtattc
9121 ttaactatgt tgctcctttt acgctatgtg gatacgctgc tttaatgcct ttgtatcatg
9181 ctattgcttc ccgtatggct ttcattttct cctccttgta taaatcctgg ttgctgtctc
9241 tttatgagga gttgtggccc gttgtcaggc aacgtggcgt ggtgtgcact gtgtttgctg
9301 acgcaacccc cactggttgg ggcattgcca ccacctgtca gctcctttcc gggactttcg
9361 ctttccccct ccctattgcc acggcggaac tcatcgccgc ctgccttgcc cgctgctgga
9421 caggggctcg gctgttgggc actgacaatt ccgtggtgtt gtcggggaaa tcatcgtcct
9481 ttccttggct gctcgcctgt gttgccacct ggattctgcg cgggacgtcc ttctgctacg
9541 tcccttcggc cctcaatcca gcggaccttc cttcccgcgg cctgctgccg gctctgcggc
9601 ctcttccgcg tcttcgcctt cgccctcaga cgagtcggat ctccctttgg gccgcctccc
9661 cgcatcgata ccgtcgacct cgagacctag aaaaacatgg agcaatcaca agtagcaata
9721 cagcagctac caatgctgat tgtgcctggc tagaagcaca agaggaggag gaggtgggtt
9781 ttccagtcac acctcaggta cctttaagac caatgactta caaggcagct gtagatctta
9841 gccacttttt aaaagaaaag gggggactgg aagggctaat tcactcccaa cgaagacaag
9901 atatccttga tctgtggatc taccacacac aaggctactt ccctgattgg cagaactaca
9961 caccagggcc agggatcaga tatccactga cctttggatg gtgctacaag ctagtaccag
10021 ttgagcaaga gaaggtagaa gaagccaatg aaggagagaa cacccgcttg ttacaccctg
10081 tgagcctgca tgggatggat gacccggaga gagaagtatt agagtggagg tttgacagcc
10141 gcctagcatt tcatcacatg gcccgagagc tgcatccgga ctgtactggg tctctctggt
10201 tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc
10261 aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta
10321 actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagg gcccgtttaa
10381 acccgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc
10441 cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag
10501 gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag
10561 gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc ggtgggctct
10621 atggcttctg aggcggaaag aaccagctgg ggctctaggg ggtatcccca cgcgccctgt
10681 agcggcgcat taagcgcggc gggggggggg ggggggtggg gcaggacagc aagggggagg
10741 attgggaaga caatagcagg catgctgggg atgcggtggg ctctatggct tctgaggcgg
10801 aaagaaccag ctggggctct agggggtatc cccacgcgcc ctgtagcggc gcattaagcg
10861 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg
10921 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc
10981 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
11041 aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc
11101 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac
11161 tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt
11221 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg
11281 tcagttaggg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca
11341 tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag gcagaagtat
11401 gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc
11461 gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat
11521 ttatgcagag gccgaggccg cctctgcctc tgagctattc cagaagtagt gaggaggctt
11581 ttttggaggc ctaggctttt gcaaaaagct cccgggagct tgtatatcca ttttcggatc
11641 tgatcagcac gtgttgacaa ttaatcatcg gcatagtata tcggcatagt ataatacgac
11701 aaggtgagga actaaaccat ggccaagttg accagtgccg ttccggtgct caccgcgcgc
11761 gacgtcgccg gagcggtcga gttctggacc gaccggctcg ggttctcccg ggacttcgtg
11821 gaggacgact tcgccggtgt ggtccgggac gacgtgaccc tgttcatcag cgcggtccag
11881 gaccaggtgg tgccggacaa caccctggcc tgggtgtggg tgcgcggcct ggacgagctg
11941 tacgccgagt ggtcggaggt cgtgtccacg aacttccggg acgcctccgg gccggccatg
12001 accgagatcg gcgagcagcc gtgggggcgg gagttcgccc tgcgcgaccc ggccggcaac
12061 tgcgtgcact tcgtggccga ggagcaggac tgacacgtgc tacgagattt cgattccacc
12121 gccgccttct atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc
12181 ctccagcgcg gggatctcat gctggagttc ttcgcccacc ccaacttgtt tattgcagct
12241 tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc atttttttca
12301 ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctgtataccg
12361 tcgacctcta gctagagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt
12421 tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt
12481 gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg
12541 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
12601 cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
12661 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
12721 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
12781 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
12841 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
12901 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
12961 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
13021 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
13081 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
13141 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
13201 ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg
13261 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
13321 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
13381 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
13441 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
13501 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
13561 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
13621 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
13681 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
13741 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
13801 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
13861 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
13921 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
13981 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
14041 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
14101 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
14161 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
14221 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
14281 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
14341 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
14401 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
14461 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
14521 acatttcccc gaaaagtgcc acctgac
//
