| 质粒名称: | pcDNA3.1-dCas9-Dnmt3a-Dnmt3l-P2A-eGFP |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 128424 |
| 存储实验室: | Bradley Bernstein |
|
载体骨架基本信息 |
|
| 载体名称: | pcDNA3.1 |
| 载体出品公司: | Invitrogen |
| 空载体大小: | 5428 bp |
| 完整质粒大小: | 12000 bp |
| 载体修饰: |
pcDNA3.1-dCas9-Dnmt3a-Dnmt3l-P2A-eGFP Human codon optimized dCas9-Dnmt3a-Dnmt3l
|
| 载体类型: | 哺乳细胞表达，Crispr |
| 筛选标记: | 新霉素（G418） |
| 载体抗性: | 氨苄霉素 |
| 生长温度: | 37 ℃ |
| 宿主菌: | NEB stable |
| 拷贝数: | 高拷贝 |
|
插入基因信息 |
|
| 插入基因名称: | dCas9m4-sv40NLS-DNMT3A-3L-sv40NLS-P2A-eGFP |
| 物种来源: | Synthetic; Streptococcus pyogenes Type II |
| 插入大小: | 6639 bp |
| 启动子: | T7 |
| 标签/融合蛋白: | GFP（C端） |
| 克隆方法: | 限制性内切酶 |
| 5’测序引物 | GAGCGGATAACAATTTCACACAGG |
| 3’测序引物 | CGCAAATGGGCGGTAGGCGTG |
|
文献引用方法 |
|
| 材料和方法部分: | pcDNA3.1-dCas9-Dnmt3a-Dnmt3l-P2A-eGFP was a gift from Bradley Bernstein (Addgene plasmid # 128424 ; http://n2t.net/addgene:128424 ; RRID:Addgene_128424) |
| 参考文献部分: | Epigenome editing strategies for the functional annotation of CTCF insulators. Tarjan DR, Flavahan WA, Bernstein BE. Nat Commun. 2019 Sep 18;10(1):4258. doi: 10.1038/s41467-019-12166-w. 10.1038/s41467-019-12166-w PubMed 31534142 |
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pcDNA3.1-dCas9-Dnmt3a-Dnmt3l-P2A-eGFP载体质粒基本信息
pcDNA3.1-dCas9-Dnmt3a-Dnmt3l-P2A-eGFP质粒图谱载体图谱和pcDNA3.1-dCas9-Dnmt3a-Dnmt3l-P2A-eGFP载体序列质粒序列多克隆位点信息
pcDNA3.1-dCas9-Dnmt3a-Dnmt3l-P2A-eGFP质粒载体简介
pcDNA3.1-dCas9-Dnmt3a-Dnmt3l-P2A-eGFP质粒序列载体序列
LOCUS pcDNA3.1-dCas9-Dnmt3a-Dnmt3l-P2A-eGFP 12070 bp ds-DNA circular SYN 12-MAY-2021
DEFINITION Human Codon Optimized dCas9-DNMT3A3L.
ACCESSION .
VERSION .
KEYWORDS pcDNA3.1-dCas9-Dnmt3a-Dnmt3l-P2A-eGFP
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 12070)
AUTHORS Tarjan DR, Flavahan WA, Bernstein BE
TITLE Epigenome editing strategies for the functional annotation of CTCF
insulators.
JOURNAL Nat Commun. 2019 Sep 18;10(1):4258. doi: 10.1038/s41467-019-12166-w.
PUBMED 31534142
REFERENCE 2 (bases 1 to 12070)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58
http://www.biofeng.com
FEATURES Location/Qualifiers
source 1..12070
/organism="synthetic DNA construct"
/mol_type="other DNA"
enhancer 1..380
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 381..584
/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"
primer_bind 629..648
/label=T7
/note="T7 promoter, forward primer"
promoter 629..647
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
CDS 763..4866
/codon_start=1
/product="catalytically dead mutant of the Cas9
endonuclease from the Streptococcus pyogenes Type II
CRISPR/Cas system"
/label=Cas9m4
/note="RNA-guided DNA-binding protein that lacks
endonuclease activity due to the mutations D10A, D839A,
H840A, and N863A (Mali et al., 2013)"
/translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
LYYLQNGRDMYVDQELDINRLSDYDVAAIVPQSFLKDDSIDNKVLTRSDKARGKSDNVP
SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
DATLIHQSITGLYETRIDLSQLGGD"
RBS 4323..4331
/label=Shine-Dalgarno sequence
/note="full consensus sequence for ribosome-binding sites
upstream of start codons in E. coli; complementary to a
region in the 3' end of the 16S rRNA (Chen et al., 1994)"
CDS 4879..4899
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 6592..6612
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 6628..6684
/codon_start=1
/product="2A peptide from porcine teschovirus-1
polyprotein"
/label=P2A
/note="Eukaryotic ribosomes fail to insert a peptide bond
between the Gly and Pro residues, yielding separate
polypeptides."
/translation="ATNFSLLKQAGDVEENPGP"
CDS 6685..7398
/codon_start=1
/product="green fluorescent protein"
/label=GFP
/translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
FVTAAGITHGMDELYK"
primer_bind complement(6727..6748)
/label=EGFP-N
/note="EGFP, reverse primer"
primer_bind complement(6988..7007)
/label=EXFP-R
/note="For distinguishing EGFP variants, reverse primer"
primer_bind 7335..7356
/label=EGFP-C
/note="EGFP, forward primer"
primer_bind complement(7427..7444)
/label=BGH-rev
/note="Bovine growth hormone terminator, reverse primer.
Also called BGH reverse"
polyA_signal 7433..7657
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
rep_origin 7703..8131
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(7790..7809)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 8000..8021
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind complement(8140..8160)
/label=pBABE 3'
/note="SV40 enhancer, reverse primer for pBABE vectors"
promoter 8145..8474
/label=SV40 promoter
/note="SV40 enhancer and early promoter"
rep_origin 8325..8460
/label=SV40 ori
/note="SV40 origin of replication"
primer_bind 8387..8406
/label=SV40pro-F
/note="SV40 promoter/origin, forward primer"
CDS 8541..9335
/codon_start=1
/gene="aph(3')-II (or nptII)"
/product="aminoglycoside phosphotransferase from Tn5"
/label=NeoR/KanR
/note="confers resistance to neomycin, kanamycin, and G418
(Geneticin(R))"
/translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
primer_bind complement(8595..8614)
/label=Neo-R
/note="Neomycin resistance gene, reverse primer"
primer_bind 9205..9224
/label=Neo-F
/note="Neomycin resistance gene, forward primer"
polyA_signal 9509..9630
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(9546..9565)
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
primer_bind 9600..9619
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
primer_bind complement(9679..9695)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(9679..9695)
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind complement(9692..9714)
/label=M13/pUC Reverse
/note="In lacZ gene"
protein_bind 9703..9719
/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(9727..9757)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 9772..9793
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(9910..9927)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(10081..10669)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(10161..10180)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS complement(10840..11700)
/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 11463..11482
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
promoter complement(11701..11805)
/gene="bla"
/label=AmpR promoter
primer_bind complement(11880..11899)
/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 agctctctgg ctaactagag
601 aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag acccaagctg
661 gctagcgttt aaacttaagc ttggtaccga gctcggatcc actagtccag tgtggtggaa
721 ttctgcagat atccagcaca gtggcggccg ctcgagtcta gaatggacaa gaagtattca
781 atcggcctgg ctatcggaac caacagcgtg gggtgggcag tcatcacaga cgagtataag
841 gtcccctcca agaagtttaa ggtgctgggc aataccgatc gccactccat caagaagaac
901 ctgatcggcg ccctgctgtt cgactctggc gagacagcag aggcaaccag gctgaagagg
961 acagcacgga gaaggtatac ccgccggaag aaccggatct gctacctgca ggagatcttc
1021 agcaatgaga tggccaaggt ggacgattcc ttctttcacc gcctggagga gtctttcctg
1081 gtggaggagg ataagaagca cgagcggcac ccaatctttg gcaacatcgt ggacgaggtg
1141 gcctatcacg agaagtaccc caccatctat cacctgcgga agaagctggt ggattccaca
1201 gacaaggccg atctgagact gatctatctg gccctggccc acatgatcaa gttcaggggc
1261 cactttctga tcgagggcga cctgaatccc gataacagcg atgtggacaa gctgttcatc
1321 cagctggtgc agacctacaa ccagctgttt gaggagaatc ctatcaacgc ctctggagtg
1381 gacgcaaagg caatcctgag cgccagactg tctaagagca gaaggctgga gaatctgatc
1441 gcccagctgc ctggcgagaa gaagaatggc ctgtttggca acctgatcgc cctgtccctg
1501 ggcctgaccc caaatttcaa gtctaacttt gacctggccg aggatgccaa gctgcagctg
1561 tccaaggata catatgacga tgacctggac aacctgctgg cccagatcgg cgaccagtac
1621 gccgatctgt tcctggccgc caagaacctg agcgatgcca tcctgctgtc cgacatcctg
1681 cgcgtgaata cagagatcac caaggcccct ctgtccgcct ctatgatcaa gagatatgat
1741 gagcaccacc aggacctgac cctgctgaag gccctggtga ggcagcagct gccagagaag
1801 tacaaggaga tcttctttga tcagagcaag aacggatacg caggatatat cgacggagga
1861 gcatcccagg aggagttcta caagtttatc aagcctatcc tggagaagat ggacggcacc
1921 gaggagctgc tggtgaagct gaaccgggag gacctgctgc ggaagcagag aacattcgat
1981 aatggcagca tcccccacca gatccacctg ggagagctgc acgcaatcct gcgccggcag
2041 gaggatttct acccttttct gaaggacaat agggagaaga tcgagaagat cctgaccttc
2101 cgcatcccat actatgtggg accactggca aggggaaaca gcagatttgc ctggatgaca
2161 agaaagtccg aggagacaat caccccttgg aatttcgagg aggtggtgga taagggcgcc
2221 tctgcccaga gcttcatcga gaggatgacc aactttgaca agaatctgcc caacgagaag
2281 gtgctgccta agcactctct gctgtacgag tatttcacag tgtataatga gctgaccaag
2341 gtgaagtacg tgacagaggg catgagaaag ccagccttcc tgagcggcga gcagaagaag
2401 gccatcgtgg acctgctgtt taagacaaac aggaaggtga ccgtgaagca gctgaaggag
2461 gactatttca agaagatcga gtgttttgat tccgtggaga tctctggcgt ggaggaccgg
2521 tttaatgcct ccctgggcac ataccacgat ctgctgaaga tcatcaagga taaggacttc
2581 ctggacaatg aggagaacga ggacatcctg gaggatatcg tgctgacact gaccctgttt
2641 gaggatcggg agatgatcga ggagagactg aagacctatg cccacctgtt cgatgacaaa
2701 gtgatgaagc agctgaagag aaggcgctac acaggatggg gcaggctgag ccgcaagctg
2761 atcaacggca tcagagacaa gcagtctggc aagaccatcc tggattttct gaagagcgac
2821 ggcttcgcca acaggaactt catgcagctg atccacgatg actccctgac attcaaggag
2881 gatatccaga aggcacaggt gtccggacag ggcgactctc tgcacgagca catcgccaat
2941 ctggccggca gcccagccat caagaagggc atcctgcaga ccgtgaaggt ggtggacgag
3001 ctggtgaaag tgatgggcag gcacaagccc gagaatatcg tgatcgagat ggcccgcgag
3061 aaccagacca cacagaaggg ccagaagaat agcagggagc gcatgaagcg catcgaggag
3121 ggcatcaagg agctgggctc ccagatcctg aaggagcacc ccgtggagaa tacccagctg
3181 cagaacgaga agctgtatct gtactatctg cagaacggcc gggacatgta cgtggatcag
3241 gagctggaca tcaatagact gtctgattat gacgtggccg ccatcgtgcc ccagtctttc
3301 ctgaaggatg acagcatcga caacaaggtg ctgacacgga gcgataaggc cagaggcaag
3361 agcgacaatg tgccttccga ggaggtggtg aagaagatga agaattactg gcggcagctg
3421 ctgaacgcca agctgatcac ccagcggaag tttgataacc tgacaaaggc agagagagga
3481 ggcctgtccg agctggacaa ggcaggcttc atcaagaggc agctggtgga gacacgccag
3541 atcaccaagc acgtggccca gatcctggat tctcgcatga ataccaagta cgacgagaac
3601 gataagctga tccgggaggt gaaagtgatc accctgaaga gcaagctggt gtccgacttc
3661 cggaaggact tccagtttta taaggtgcgc gagatcaaca attatcacca cgcccacgac
3721 gcatacctga acgcagtggt gggaaccgcc ctgatcaaga agtacccaaa gctggagagc
3781 gagttcgtgt acggcgatta taaggtgtac gacgtgagaa agatgatcgc caagtccgag
3841 caggagatcg gcaaggccac agccaagtat ttcttttact ctaacatcat gaacttcttt
3901 aagacagaga tcaccctggc aaacggagag atcaggaagc ggcccctgat cgagacaaat
3961 ggcgagacag gcgagatcgt gtgggataag ggccgggact ttgccaccgt gagaaaggtg
4021 ctgagcatgc cacaggtgaa catcgtgaag aagacagagg tgcagaccgg cggcttctcc
4081 aaggagtcta tcctgcccaa gaggaattcc gataagctga tcgcccgcaa gaaggattgg
4141 gaccccaaga agtatggcgg cttcgactcc cctaccgtgg cctactctgt gctggtggtg
4201 gccaaggtgg agaagggcaa gtctaagaag ctgaagagcg tgaaggagct gctgggcatc
4261 acaatcatgg agaggagctc cttcgagaag aaccccatcg attttctgga ggccaagggc
4321 tataaggagg tgaagaagga cctgatcatc aagctgccta agtactccct gtttgagctg
4381 gagaatggaa ggaagaggat gctggcatct gccggagagc tgcagaaggg caacgagctg
4441 gccctgccaa gcaagtacgt gaatttcctg tatctggcca gccactacga gaagctgaag
4501 ggctcccccg aggataacga gcagaagcag ctgtttgtgg agcagcacaa gcactatctg
4561 gacgagatca tcgagcagat cagcgagttc tccaagagag tgatcctggc cgatgccaac
4621 ctggacaagg tgctgtccgc ctacaataag cacagggaca agcctatccg cgagcaggcc
4681 gagaacatca tccacctgtt cacactgacc aatctgggcg ccccagccgc cttcaagtat
4741 tttgatacca caatcgaccg gaagagatac acatctacca aggaggtgct ggatgccacc
4801 ctgatccacc agagcatcac aggcctgtac gagacaagga tcgacctgag ccagctggga
4861 ggcgattccc gggcagatcc caagaagaag cggaaggtgg aggcaagcgg atccggaagg
4921 gcatctcctg gaatcccagg aagcacccgc aaccacgatc aggagtttga cccccctaag
4981 gtgtacccac ccgtgccagc cgagaagagg aagcccatcc gcgtgctgtc cctgttcgac
5041 ggcatcgcca caggcctgct ggtgctgaag gatctgggca tccaggtgga cagatatatc
5101 gcctccgagg tgtgcgagga ttctatcacc gtgggcatgg tgaggcacca gggcaagatc
5161 atgtacgtgg gcgacgtgcg cagcgtgaca cagaagcaca tccaggagtg gggacccttc
5221 gacctggtca tcggaggcag cccctgtaat gacctgtcca tcgtgaaccc tgcaaggaag
5281 ggcctgtatg agggaaccgg cagactgttc tttgagttct acaggctgct gcacgacgcc
5341 cgccctaagg agggcgatga caggccattc ttttggctgt ttgagaacgt ggtggccatg
5401 ggcgtgagcg acaagcggga tatctccaga ttcctggagt ctaatcccgt gatgatcgat
5461 gcaaaggagg tgtctgccgc acacagggca aggtactttt ggggaaatct gcctggcatg
5521 aaccgcccac tggccagcac cgtgaacgac aagctggagc tgcaggagtg cctggagcac
5581 ggaaggatcg ccaagttctc caaggtgcgg acaatcacca caagatctaa cagcatcaag
5641 cagggcaagg atcagcactt ccccgtgttc atgaatgaga aggaggacat cctgtggtgt
5701 accgagatgg agcgcgtgtt cggctttcca gtgcactata cagacgtgag caatatgagc
5761 cggctggcaa ggcagagact gctgggccgg tcctggtctg tgccagtgat cagacacctg
5821 ttcgcccccc tgaaggagta ctttgcctgc gtgtctagcg gcaactctaa tgccaacagc
5881 agaggccctt ccttttcctc tggcctggtg ccactgtctc tgaggggcag ccacatgggc
5941 cccatggaga tctacaagac cgtgtccgcc tggaagaggc agcctgtgcg cgtgctgtct
6001 ctgttccgca acatcgacaa ggtgctgaag agcctgggct ttctggagag cggatccgga
6061 tctggaggag gcaccctgaa gtatgtggag gatgtgacaa atgtggtgcg gagagacgtg
6121 gagaagtggg gccccttcga tctggtgtac ggatccaccc agccactggg aagctcctgc
6181 gataggtgtc caggatggta tatgttccag tttcacagaa tcctgcagta cgcactgcca
6241 aggcaggaga gccagcgccc tttcttttgg atctttatgg acaacctgct gctgacagag
6301 gatgaccagg agacaacaac ccgcttcctg cagacagagg cagtgaccct gcaggatgtg
6361 aggggacgcg actatcagaa tgccatgcgg gtgtggtcta acatccctgg cctgaagagc
6421 aagcacgccc ccctgacccc taaggaggag gagtacctgc aggcccaggt gcggagcaga
6481 tccaagctgg atgcccctaa ggtggacctg ctggtgaaga attgtctgct gccactgcgg
6541 gagtacttca agtactttag tcagaatagc ctgccactgt cacgagccga ccccaagaag
6601 aaacggaagg tgtctagagg aagcggagct actaacttca gcctgctgaa gcaggctgga
6661 gacgtggagg agaaccctgg acctatgagc aagggcgagg agctgttcac cggggtggtg
6721 cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt gtccggcgag
6781 ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac caccggcaag
6841 ctgcccgtgc cctggcccac cctcgtgacc accctgacct acggcgtgca gtgcttcagc
6901 cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac
6961 gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg cgccgaggtg
7021 aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag
7081 gacggcaaca tcctggggca caagctggag tacaactaca acagccacaa cgtctatatc
7141 atggccgaca agcagaagaa cggcatcaag gtgaacttca agatccgcca caacatcgag
7201 gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg cgacggcccc
7261 gtgctgctgc ccgacaacca ctacctgagc acccagtccg ccctgagcaa agaccccaac
7321 gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat cactcacggc
7381 atggacgagc tgtacaagta agggcccgtt taaacccgct gatcagcctc gactgtgcct
7441 tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt
7501 gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg
7561 tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac
7621 aatagcaggc atgctgggga tgcggtgggc tctatggctt ctgaggcgga aagaaccagc
7681 tggggctcta gggggtatcc ccacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg
7741 gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct
7801 ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg
7861 ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag
7921 ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg
7981 gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc
8041 tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat
8101 gagctgattt aacaaaaatt taacgcgaat taattctgtg gaatgtgtgt cagttagggt
8161 gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt
8221 cagcaaccag gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc
8281 atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc
8341 cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg
8401 ccgaggccgc ctctgcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc
8461 taggcttttg caaaaagctc ccgggagctt gtatatccat tttcggatct gatcaagaga
8521 caggatgagg atcgtttcgc atgattgaac aagatggatt gcacgcaggt tctccggccg
8581 cttgggtgga gaggctattc ggctatgact gggcacaaca gacaatcggc tgctctgatg
8641 ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct ttttgtcaag accgacctgt
8701 ccggtgccct gaatgaactg caggacgagg cagcgcggct atcgtggctg gccacgacgg
8761 gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc gggaagggac tggctgctat
8821 tgggcgaagt gccggggcag gatctcctgt catctcacct tgctcctgcc gagaaagtat
8881 ccatcatggc tgatgcaatg cggcggctgc atacgcttga tccggctacc tgcccattcg
8941 accaccaagc gaaacatcgc atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg
9001 atcaggatga tctggacgaa gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc
9061 tcaaggcgcg catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc
9121 cgaatatcat ggtggaaaat ggccgctttt ctggattcat cgactgtggc cggctgggtg
9181 tggcggaccg ctatcaggac atagcgttgg ctacccgtga tattgctgaa gagcttggcg
9241 gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca
9301 tcgccttcta tcgccttctt gacgagttct tctgagcggg actctggggt tcgaaatgac
9361 cgaccaagcg acgcccaacc tgccatcacg agatttcgat tccaccgccg ccttctatga
9421 aaggttgggc ttcggaatcg ttttccggga cgccggctgg atgatcctcc agcgcgggga
9481 tctcatgctg gagttcttcg cccaccccaa cttgtttatt gcagcttata atggttacaa
9541 ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg
9601 tggtttgtcc aaactcatca atgtatctta tcatgtctgt ataccgtcga cctctagcta
9661 gagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat
9721 tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag
9781 ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
9841 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc
9901 ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc
9961 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
10021 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
10081 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
10141 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
10201 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
10261 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
10321 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
10381 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
10441 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
10501 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac
10561 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
10621 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
10681 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
10741 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
10801 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
10861 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
10921 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
10981 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
11041 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
11101 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg
11161 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
11221 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
11281 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
11341 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
11401 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg
11461 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
11521 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
11581 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
11641 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
11701 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
11761 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
11821 agtgccacct gacgtcgacg gatcgggaga tctcccgatc ccctatggtg cactctcagt
11881 acaatctgct ctgatgccgc atagttaagc cagtatctgc tccctgcttg tgtgttggag
11941 gtcgctgagt agtgcgcgag caaaatttaa gctacaacaa ggcaaggctt gaccgacaat
12001 tgcatgaaga atctgcttag ggttaggcgt tttgcgctgc ttcgcgatgt acgggccaga
12061 tatacgcgtt
//
