| 質粒名稱: | pdCas9-DNMT3A-PuroR_v2 |
|---|---|
| 出品公司: | Addgene |
| 目錄編號: | 74407 |
| 存儲實驗室: | Vlatka Zoldos |
|
載體骨架基本信息 |
|
| 載體名稱: | PX462 |
| 載體出品公司: | Clontech |
| 空載體大小: | 4246bp |
| 完整質粒大小: | 10152bp |
| 載體類型: | 哺乳細胞表達, CRISPR |
| 篩選標記: | Puromycin |
| 載體抗性: | 氨芐霉素 |
| 生長溫度: | 37 ℃ |
| 宿主菌: | DH5a |
| 拷貝數: | 未知 |
|
插入基因信息 |
|
| 插入基因名稱: | dCas9-DNMT3A、SpdCas9-DNMT3A-T2A-PuroR、S. pyogenes dCas9 fused with the catalytic domain of human DNMT3A (amino acids P602-V912) and T2A-PuroR_v2、dCas9-DNMT3A-PuroR |
| 物種來源: | H. sapiens (human), Synthetic; S. pyogenes |
| 插入大小: | 5906bp |
| 基因突變: |
D10A and H840A in S.pyogenes Cas9
GenBank IDAKA60242.1 NP_072046.2
|
| 啟動子: | CBh |
| 標簽/融合蛋白: |
3xFLAG (N terminal on insert)
SV40 NLS (N terminal on insert)
T2A-PuroR (C terminal on insert)
|
| 克隆方法: | 限制性內切酶 |
| 5’克隆位點: | BamHI (not destroyed) |
| 3’克隆位點: | FseI (not destroyed) |
| 5’測序引物 | GS-linker For 5' GAAGAGGTACACCAGCACCAAAG 3' |
| 3’測序引物 |
BGH Rev
(Common Sequencing Primers)
|
|
文獻引用方法 |
|
| 材料和方法部分: | pdCas9-DNMT3A-PuroR_v2 was a gift from Vlatka Zoldos (Addgene plasmid # 74407 ; //n2t.net/addgene:74407 ; RRID:Addgene_74407) |
| 參考文獻部分: | Repurposing the CRISPR-Cas9 system for targeted DNA methylation. Vojta A, Dobrinic P, Tadic V, Bockor L, Korac P, Julg B, Klasic M, Zoldos V. Nucleic Acids Res. 2016 Mar 11. pii: gkw159. 10.1093/nar/gkw159 PubMed 26969735 |
買家導航
pdCas9-DNMT3A-PuroR_v2載體質粒基本信息
pdCas9-DNMT3A-PuroR_v2質粒圖譜載體圖譜和pdCas9-DNMT3A-PuroR_v2載體序列質粒序列多克隆位點信息
pdCas9-DNMT3A-PuroR_v2質粒載體簡介
pdCas9-DNMT3A-PuroR_v2質粒序列載體序列
LOCUS pdCas9-DNMT3A-PuroR_v2 10152 bp DNA circular SYN 14-APR-2025
DEFINITION Expression of human codon-optimized dCas9-DNMT3A fusion with
T2A-PuroR (v2) for targeted DNA methylation in mammalian cells;
cloning backbone for sgRNA.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 10152)
AUTHORS Vojta A, Dobrinic P, Tadic V, Bockor L, Korac P, Julg B, Klasic M,
Zoldos V
TITLE Repurposing the CRISPR-Cas9 system for targeted DNA methylation.
JOURNAL Nucleic Acids Res. 2016 Mar 11. pii: gkw159.
PUBMED 26969735
REFERENCE 2 (bases 1 to 10152)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Apr 14, 2025 from SnapGene Server 8.0.1
//gt12365.cn
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Nucleic
Acids Res. 2016 Mar 11. pii: gkw159."
COMMENT Sequence Label: pdCas9-DNMT3A-PuroR_v2
FEATURES Location/Qualifiers
source 1..10152
/mol_type="other DNA"
/organism="synthetic DNA construct"
promoter 1..249
/label=U6 promoter
/note="RNA polymerase III promoter for human U6 snRNA
(Domitrovich & Kunkel, 2003)"
primer_bind 1..21
/label=hU6-F
/note="Human U6 promoter, forward primer"
primer_bind 172..191
/label=LKO.1 5'
/note="Human U6 promoter, forward primer"
misc_RNA 268..343
/label=gRNA scaffold
/note="guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system"
enhancer 440..725
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer;
contains an 18-bp deletion relative to the standard CMV
enhancer"
promoter 727..1004
/label=chicken beta-actin promoter
intron 1005..1233
/label=hybrid intron
/note="hybrid between chicken beta-actin (CBA) and minute
virus of mice (MMV) introns (Gray et al., 2011)"
regulatory 1245..1254
/label=Kozak sequence
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
/regulatory_class="other"
CDS 1254..1319
/codon_start=1
/product="three tandem FLAG(R) epitope tags, followed by an
enterokinase cleavage site"
/label=3xFLAG
/translation="DYKDHDGDYKDHDIDYKDDDDK"
CDS 1326..1346
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS join(1371..1394,1395..1397,1398..3884,3885..3887,
3888..5471)
/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 1371..5471
/codon_start=1
/product="Cas9 (Csn1) endonuclease from the Streptococcus
pyogenes Type II CRISPR/Cas system"
/label=Cas9
/note="generates RNA-guided double strand breaks in DNA"
/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 5472..5519
/codon_start=1
/product="bipartite nuclear localization signal from
nucleoplasmin"
/label=nucleoplasmin NLS
/translation="KRPAATKKAGQAKKKK"
CDS 5547..6479
/codon_start=1
/product="catalytic domain of DNA methyltransferase 3A"
/label=DNMT3A catalytic domain
/translation="PSRLQMFFANNHDQEFDPPKVYPPVPAEKRKPIRVLSLFDGIATG
LLVLKDLGIQVDRYIASEVCEDSITVGMVRHQGKIMYVGDVRSVTQKHIQEWGPFDLVI
GGSPCNDLSIVNPARKGLYEGTGRLFFEFYRLLHDARPKEGDDRPFFWLFENVVAMGVS
DKRDISRFLESNPVMIDAKEVSAAHRARYFWGNLPGMNRPLASTVNDKLELQECLEHGR
IAKFSKVRTITTRSNSIKQGKDQHFPVFMNEKEDILWCTEMERVFGFPVHYTDVSNMSR
LARQRLLGRSWSVPVIRHLFAPLKEYFACV"
CDS 6507..6560
/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 6561..7160
/codon_start=1
/gene="pac from Streptomyces alboniger"
/product="puromycin N-acetyltransferase"
/label=PuroR
/note="confers resistance to puromycin"
/translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
primer_bind complement(6561..6580)
/label=Puro-R
/note="Puromycin resistance gene, reverse primer. Also
called puro-variant-R"
primer_bind complement(7193..7210)
/label=BGH-rev
/note="Bovine growth hormone terminator, reverse primer.
Also called BGH reverse"
polyA_signal 7199..7406
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
repeat_region 7415..7555
/label=AAV2 ITR
/note="inverted terminal repeat of adeno-associated virus
serotype 2"
repeat_region 7415..7544
/label=AAV2 ITR
rep_origin 7630..8085
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(7717..7736)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 7927..7948
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind complement(8102..8121)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 8221..8243
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
primer_bind complement(8281..8299)
/label=pBRforEco
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
promoter 8367..8471
/gene="bla"
/label=AmpR promoter
CDS join(8472..8540,8541..9332)
/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 complement(8690..8709)
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
rep_origin 9503..10091
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 9992..10011
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
ORIGIN
1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
61 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
241 cgaaacaccg ggtcttcgag aagacctgtt ttagagctag aaatagcaag ttaaaataag
301 gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttg ttttagagct
361 agaaatagca agttaaaata aggctagtcc gtttttagcg cgtgcgccaa ttctgcagac
421 aaatggctct agaggtaccc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
481 ccaacgaccc ccgcccattg acgtcaatag taacgccaat agggactttc cattgacgtc
541 aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc
601 caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tgtgcccagt
661 acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta
721 ccatggtcga ggtgagcccc acgttctgct tcactctccc catctccccc ccctccccac
781 ccccaatttt gtatttattt attttttaat tattttgtgc agcgatgggg gcgggggggg
841 ggggggggcg cgcgccaggc ggggcggggc ggggcgaggg gcggggcggg gcgaggcgga
901 gaggtgcggc ggcagccaat cagagcggcg cgctccgaaa gtttcctttt atggcgaggc
961 ggcggcggcg gcggccctat aaaaagcgaa gcgcgcggcg ggcgggagtc gctgcgacgc
1021 tgccttcgcc ccgtgccccg ctccgccgcc gcctcgcgcc gcccgccccg gctctgactg
1081 accgcgttac tcccacaggt gagcgggcgg gacggccctt ctcctccggg ctgtaattag
1141 ctgagcaaga ggtaagggtt taagggatgg ttggttggtg gggtattaat gtttaattac
1201 ctggagcacc tgcctgaaat cacttttttt caggttggac cggtgccacc atggactata
1261 aggaccacga cggagactac aaggatcatg atattgatta caaagacgat gacgataaga
1321 tggccccaaa gaagaagcgg aaggtcggta tccacggagt cccagcagcc gacaagaagt
1381 acagcatcgg cctggccatc ggcaccaact ctgtgggctg ggccgtgatc accgacgagt
1441 acaaggtgcc cagcaagaaa ttcaaggtgc tgggcaacac cgaccggcac agcatcaaga
1501 agaacctgat cggagccctg ctgttcgaca gcggcgaaac agccgaggcc acccggctga
1561 agagaaccgc cagaagaaga tacaccagac ggaagaaccg gatctgctat ctgcaagaga
1621 tcttcagcaa cgagatggcc aaggtggacg acagcttctt ccacagactg gaagagtcct
1681 tcctggtgga agaggataag aagcacgagc ggcaccccat cttcggcaac atcgtggacg
1741 aggtggccta ccacgagaag taccccacca tctaccacct gagaaagaaa ctggtggaca
1801 gcaccgacaa ggccgacctg cggctgatct atctggccct ggcccacatg atcaagttcc
1861 ggggccactt cctgatcgag ggcgacctga accccgacaa cagcgacgtg gacaagctgt
1921 tcatccagct ggtgcagacc tacaaccagc tgttcgagga aaaccccatc aacgccagcg
1981 gcgtggacgc caaggccatc ctgtctgcca gactgagcaa gagcagacgg ctggaaaatc
2041 tgatcgccca gctgcccggc gagaagaaga atggcctgtt cggcaacctg attgccctga
2101 gcctgggcct gacccccaac ttcaagagca acttcgacct ggccgaggat gccaaactgc
2161 agctgagcaa ggacacctac gacgacgacc tggacaacct gctggcccag atcggcgacc
2221 agtacgccga cctgtttctg gccgccaaga acctgtccga cgccatcctg ctgagcgaca
2281 tcctgagagt gaacaccgag atcaccaagg cccccctgag cgcctctatg atcaagagat
2341 acgacgagca ccaccaggac ctgaccctgc tgaaagctct cgtgcggcag cagctgcctg
2401 agaagtacaa agagattttc ttcgaccaga gcaagaacgg ctacgccggc tacattgacg
2461 gcggagccag ccaggaagag ttctacaagt tcatcaagcc catcctggaa aagatggacg
2521 gcaccgagga actgctcgtg aagctgaaca gagaggacct gctgcggaag cagcggacct
2581 tcgacaacgg cagcatcccc caccagatcc acctgggaga gctgcacgcc attctgcggc
2641 ggcaggaaga tttttaccca ttcctgaagg acaaccggga aaagatcgag aagatcctga
2701 ccttccgcat cccctactac gtgggccctc tggccagggg aaacagcaga ttcgcctgga
2761 tgaccagaaa gagcgaggaa accatcaccc cctggaactt cgaggaagtg gtggacaagg
2821 gcgcttccgc ccagagcttc atcgagcgga tgaccaactt cgataagaac ctgcccaacg
2881 agaaggtgct gcccaagcac agcctgctgt acgagtactt caccgtgtat aacgagctga
2941 ccaaagtgaa atacgtgacc gagggaatga gaaagcccgc cttcctgagc ggcgagcaga
3001 aaaaggccat cgtggacctg ctgttcaaga ccaaccggaa agtgaccgtg aagcagctga
3061 aagaggacta cttcaagaaa atcgagtgct tcgactccgt ggaaatctcc ggcgtggaag
3121 atcggttcaa cgcctccctg ggcacatacc acgatctgct gaaaattatc aaggacaagg
3181 acttcctgga caatgaggaa aacgaggaca ttctggaaga tatcgtgctg accctgacac
3241 tgtttgagga cagagagatg atcgaggaac ggctgaaaac ctatgcccac ctgttcgacg
3301 acaaagtgat gaagcagctg aagcggcgga gatacaccgg ctggggcagg ctgagccgga
3361 agctgatcaa cggcatccgg gacaagcagt ccggcaagac aatcctggat ttcctgaagt
3421 ccgacggctt cgccaacaga aacttcatgc agctgatcca cgacgacagc ctgaccttta
3481 aagaggacat ccagaaagcc caggtgtccg gccagggcga tagcctgcac gagcacattg
3541 ccaatctggc cggcagcccc gccattaaga agggcatcct gcagacagtg aaggtggtgg
3601 acgagctcgt gaaagtgatg ggccggcaca agcccgagaa catcgtgatc gaaatggcca
3661 gagagaacca gaccacccag aagggacaga agaacagccg cgagagaatg aagcggatcg
3721 aagagggcat caaagagctg ggcagccaga tcctgaaaga acaccccgtg gaaaacaccc
3781 agctgcagaa cgagaagctg tacctgtact acctgcagaa tgggcgggat atgtacgtgg
3841 accaggaact ggacatcaac cggctgtccg actacgatgt ggacgctatc gtgcctcaga
3901 gctttctgaa ggacgactcc atcgacaaca aggtgctgac cagaagcgac aagaaccggg
3961 gcaagagcga caacgtgccc tccgaagagg tcgtgaagaa gatgaagaac tactggcggc
4021 agctgctgaa cgccaagctg attacccaga gaaagttcga caatctgacc aaggccgaga
4081 gaggcggcct gagcgaactg gataaggccg gcttcatcaa gagacagctg gtggaaaccc
4141 ggcagatcac aaagcacgtg gcacagatcc tggactcccg gatgaacact aagtacgacg
4201 agaatgacaa gctgatccgg gaagtgaaag tgatcaccct gaagtccaag ctggtgtccg
4261 atttccggaa ggatttccag ttttacaaag tgcgcgagat caacaactac caccacgccc
4321 acgacgccta cctgaacgcc gtcgtgggaa ccgccctgat caaaaagtac cctaagctgg
4381 aaagcgagtt cgtgtacggc gactacaagg tgtacgacgt gcggaagatg atcgccaaga
4441 gcgagcagga aatcggcaag gctaccgcca agtacttctt ctacagcaac atcatgaact
4501 ttttcaagac cgagattacc ctggccaacg gcgagatccg gaagcggcct ctgatcgaga
4561 caaacggcga aaccggggag atcgtgtggg ataagggccg ggattttgcc accgtgcgga
4621 aagtgctgag catgccccaa gtgaatatcg tgaaaaagac cgaggtgcag acaggcggct
4681 tcagcaaaga gtctatcctg cccaagagga acagcgataa gctgatcgcc agaaagaagg
4741 actgggaccc taagaagtac ggcggcttcg acagccccac cgtggcctat tctgtgctgg
4801 tggtggccaa agtggaaaag ggcaagtcca agaaactgaa gagtgtgaaa gagctgctgg
4861 ggatcaccat catggaaaga agcagcttcg agaagaatcc catcgacttt ctggaagcca
4921 agggctacaa agaagtgaaa aaggacctga tcatcaagct gcctaagtac tccctgttcg
4981 agctggaaaa cggccggaag agaatgctgg cctctgccgg cgaactgcag aagggaaacg
5041 aactggccct gccctccaaa tatgtgaact tcctgtacct ggccagccac tatgagaagc
5101 tgaagggctc ccccgaggat aatgagcaga aacagctgtt tgtggaacag cacaagcact
5161 acctggacga gatcatcgag cagatcagcg agttctccaa gagagtgatc ctggccgacg
5221 ctaatctgga caaagtgctg tccgcctaca acaagcaccg ggataagccc atcagagagc
5281 aggccgagaa tatcatccac ctgtttaccc tgaccaatct gggagcccct gccgccttca
5341 agtactttga caccaccatc gaccggaaga ggtacaccag caccaaagag gtgctggacg
5401 ccaccctgat ccaccagagc atcaccggcc tgtacgagac acggatcgac ctgtctcagc
5461 tgggaggcga caaaaggccg gcggccacga aaaaggccgg acaggccaaa aagaaaaagc
5521 tcgagggcgg aggcgggagc ggatccccct cccggctcca gatgttcttc gctaataacc
5581 acgaccagga atttgaccct ccaaaggttt acccacctgt cccagctgag aagaggaagc
5641 ccatccgggt gctgtctctc tttgatggaa tcgctacagg gctcctggtg ctgaaggact
5701 tgggcattca ggtggaccgc tacattgcct cggaggtgtg tgaggactcc atcacggtgg
5761 gcatggtgcg gcaccagggg aagatcatgt acgtcgggga cgtccgcagc gtcacacaga
5821 agcatatcca ggagtggggc ccattcgatc tggtgattgg gggcagtccc tgcaatgacc
5881 tctccatcgt caaccctgct cgcaagggcc tctacgaggg cactggccgg ctcttctttg
5941 agttctaccg cctcctgcat gatgcgcggc ccaaggaggg agatgatcgc cccttcttct
6001 ggctctttga gaatgtggtg gccatgggcg ttagtgacaa gagggacatc tcgcgatttc
6061 tcgagtccaa ccctgtgatg attgatgcca aagaagtgtc agctgcacac agggcccgct
6121 acttctgggg taaccttccc ggtatgaaca ggccgttggc atccactgtg aatgataagc
6181 tggagctgca ggagtgtctg gagcatggca ggatagccaa gttcagcaaa gtgaggacca
6241 ttactacgag gtcaaactcc ataaagcagg gcaaagacca gcattttcct gtgttcatga
6301 atgagaaaga ggacatctta tggtgcactg aaatggaaag ggtatttggt ttcccagtcc
6361 actatactga cgtctccaac atgagccgct tggcgaggca gagactgctg ggccggtcat
6421 ggagcgtgcc agtcatccgc cacctcttcg ctccgctgaa ggagtatttt gcgtgtgtgt
6481 ccggccggcc cgaattcggc agtggagagg gcagaggaag tctgctaaca tgcggtgacg
6541 tcgaggagaa tcctggccca atgaccgagt acaagcccac ggtgcgcctc gccacccgcg
6601 acgacgtccc cagggccgta cgcaccctcg ccgccgcgtt cgccgactac cccgccacgc
6661 gccacaccgt cgatccggac cgccacatcg agcgggtcac cgagctgcaa gaactcttcc
6721 tcacgcgcgt cgggctcgac atcggcaagg tgtgggtcgc ggacgacggc gccgcggtgg
6781 cggtctggac cacgccggag agcgtcgaag cgggggcggt gttcgccgag atcggcccgc
6841 gcatggccga gttgagcggt tcccggctgg ccgcgcagca acagatggaa ggcctcctgg
6901 cgccgcaccg gcccaaggag cccgcgtggt tcctggccac cgtcggcgtc tcgcccgacc
6961 accagggcaa gggtctgggc agcgccgtcg tgctccccgg agtggaggcg gccgagcgcg
7021 ccggggtgcc cgccttcctg gagacctccg cgccccggaa cctccccttc tacgagcggc
7081 tcggcttcac cgtcaccgcc gacgtcgagg tgcccgaagg accgcgcacc tggtgcatga
7141 cccgcaagcc cggtgcctga gaattcaatt ctaactagag ctcgctgatc agcctcgact
7201 gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg
7261 gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg
7321 agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg
7381 gaagagaata gcaggcatgc tggggagcgg ccgcaggaac ccctagtgat ggagttggcc
7441 actccctctc tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc
7501 ccgggctttg cccgggcggc ctcagtgagc gagcgagcgc gcagctgcct gcaggggcgc
7561 ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat acgtcaaagc
7621 aaccatagta cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca
7681 gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct
7741 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt
7801 tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgatttgggt gatggttcac
7861 gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct
7921 ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg ggctattctt
7981 ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac
8041 aaaaatttaa cgcgaatttt aacaaaatat taacgtttac aattttatgg tgcactctca
8101 gtacaatctg ctctgatgcc gcatagttaa gccagccccg acacccgcca acacccgctg
8161 acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct
8221 ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg
8281 gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt
8341 caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
8401 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
8461 aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat
8521 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc
8581 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga
8641 gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg
8701 cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc
8761 agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag
8821 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc
8881 tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg
8941 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg
9001 acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac
9061 ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac
9121 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg
9181 agcgtggaag ccgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg
9241 tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg
9301 agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac
9361 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg
9421 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
9481 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
9541 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
9601 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt
9661 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
9721 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
9781 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
9841 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
9901 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
9961 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
10021 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
10081 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
10141 ttgctcacat gt
//