| 质粒名称: | pX458M-53BP1-DN1S |
|---|---|
| 出品公司: | 爱迪基因 |
| 目录编号: | 131045 |
| 存储实验室: | Punam Malik |
|
载体骨架基本信息 |
|
| 载体名称: | PX458M |
| 载体抗性: | 氨苄青霉素 |
| 空载体大小: | 9299 bp |
| 完整质粒大小: | 10583 bp |
| 载体类型: | 哺乳细胞表达,CRISPR |
| 生长条件: | 37 ℃ |
|
插入基因信息 |
|
| 插入基因名称: | SpCas9-53BP1-DN1S |
| 物种来源: | H. sapiens (human) |
| 插入大小: | 1284 bp |
| 基因突变: | Only expressing residues 1231-1644 of complete 53BP1 CDS. |
| GenBank编号 | NM_005657.2 |
| 启动子 | CBH |
| 标签/融合蛋白: |
GFP（主干上的N终端）
SpCas9（主干上的C终端）
|
| 克隆方法: | 限制性内切酶 |
| 5' 克隆位点: | SanDI(未破坏) |
| 3' 克隆位点: | FseI (未破坏) |
| 5' 测序引物 | GATCACAAAGCACGTGGCACAG |
| 3' 测序引物 | GAACAGCTCCTCGCCCTTGC |
|
文献引用方法 |
|
| 材料和方法部分: | pX458M-53BP1-DN1S was a gift from Punam Malik (Addgene plasmid # 131045 ; http://n2t.net/addgene:131045 ; RRID:Addgene_131045) |
| 参考文献部分: | CRISPR-Cas9 fusion to dominant-negative 53BP1 enhances HDR and inhibits NHEJ specifically at Cas9 target sites. Jayavaradhan R, Pillis DM, Goodman M, Zhang F, Zhang Y, Andreassen PR, Malik P. Nat Commun. 2019 Jun 28;10(1):2866. doi: 10.1038/s41467-019-10735-7. 10.1038/s41467-019-10735-7 PubMed 31253785 |
买家导航
pX458M-53BP1-DN1S载体质粒基本信息
pX458M-53BP1-DN1S质粒图谱载体图谱和pX458M-53BP1-DN1S载体序列质粒序列多克隆位点信息
pX458M-53BP1-DN1S质粒载体简介
pX458M-53BP1-DN1S质粒序列载体序列
LOCUS pX458M-53BP1-DN1S 10582 bp ds-DNA circular SYN 12-MAY-2021
DEFINITION Plasmid encoding Cas9-53BP1-DN1S fusion with BbsI restriction sites
for insertion of guide RNA sequence..
ACCESSION .
VERSION .
KEYWORDS pX458M-53BP1-DN1S
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 10582)
AUTHORS Jayavaradhan R, Pillis DM, Goodman M, Zhang F, Zhang Y, Andreassen
PR, Malik P
TITLE CRISPR-Cas9 fusion to dominant-negative 53BP1 enhances HDR and
inhibits NHEJ specifically at Cas9 target sites.
JOURNAL Nat Commun. 2019 Jun 28;10(1):2866. doi: 10.1038/s41467-019-10735-7.
PUBMED 31253785
REFERENCE 2 (bases 1 to 10582)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..10582
/organism="synthetic DNA construct"
/mol_type="other DNA"
promoter 1..241
/label=U6 promoter
/note="RNA polymerase III promoter for human U6 snRNA"
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"
enhancer 450..735
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer;
contains an 18-bp deletion relative to the standard CMV
enhancer"
promoter 737..1014
/label=chicken beta-actin promoter
intron 1015..1242
/label=hybrid intron
/note="hybrid between chicken beta-actin (CBA) and minute
virus of mice (MMV) introns (Gray et al., 2011)"
regulatory 1254..1263
/regulatory_class="other"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
CDS 1263..1328
/codon_start=1
/product="three tandem FLAG(R) epitope tags, followed by an
enterokinase cleavage site"
/label=3xFLAG
/translation="DYKDHDGDYKDHDIDYKDDDDK"
CDS 1335..1355
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 1380..5480
/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="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
ATLIHQSITGLYETRIDLSQLGGD"
CDS 6765..6812
/codon_start=1
/product="bipartite nuclear localization signal from
nucleoplasmin"
/label=nucleoplasmin NLS
/translation="KRPAATKKAGQAKKKK"
CDS 6828..6881
/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 6882..7595
/codon_start=1
/product="enhanced GFP"
/label=EGFP
/translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
FVTAAGITLGMDELYK"
CDS 6882..7595
/codon_start=1
/product="the original enhanced GFP (Yang et al., 1996)"
/label=EGFP
/note="mammalian codon-optimized"
/translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
FVTAAGITLGMDELYK"
primer_bind complement(6924..6945)
/label=EGFP-N
/note="EGFP, reverse primer"
primer_bind complement(7185..7204)
/label=EXFP-R
/note="For distinguishing EGFP variants, reverse primer"
primer_bind 7532..7553
/label=EGFP-C
/note="EGFP, forward primer"
primer_bind complement(7623..7640)
/label=BGH-rev
/note="Bovine growth hormone terminator, reverse primer.
Also called BGH reverse"
polyA_signal 7629..7836
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
repeat_region 7845..7985
/label=AAV2 ITR
/note="inverted terminal repeat of adeno-associated virus
serotype 2"
repeat_region 7845..7974
/label=AAV2 ITR
rep_origin 8060..8515
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(8147..8166)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 8357..8378
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind complement(8532..8551)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 8651..8673
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
primer_bind complement(8711..8729)
/label=pBRforEco
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
promoter 8797..8901
/gene="bla"
/label=AmpR promoter
CDS 8902..9762
/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(9120..9139)
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
rep_origin 9933..10521
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 10422..10441
/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 taagagctat gctggaaaca gcatagcaag
301 tttaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttg
361 ttttagagct agaaatagca agttaaaata aggctagtcc gtttttagcg cgtgcgccaa
421 ttctgcagac aaatggctct agaggtaccc gttacataac ttacggtaaa tggcccgcct
481 ggctgaccgc ccaacgaccc ccgcccattg acgtcaatag taacgccaat agggactttc
541 cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg
601 tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat
661 tgtgcccagt acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc
721 atcgctatta ccatggtcga ggtgagcccc acgttctgct tcactctccc catctccccc
781 ccctccccac ccccaatttt gtatttattt attttttaat tattttgtgc agcgatgggg
841 gcgggggggg ggggggggcg cgcgccaggc ggggcggggc ggggcgaggg gcggggcggg
901 gcgaggcgga gaggtgcggc ggcagccaat cagagcggcg cgctccgaaa gtttcctttt
961 atggcgaggc ggcggcggcg gcggccctat aaaaagcgaa gcgcgcggcg ggcgggagtc
1021 gctgcgcgct gccttcgccc cgtgccccgc tccgccgccg cctcgcgccg cccgccccgg
1081 ctctgactga ccgcgttact cccacaggtg agcgggcggg acggcccttc tcctccgggc
1141 tgtaattagc tgagcaagag gtaagggttt aagggatggt tggttggtgg ggtattaatg
1201 tttaattacc tggagcacct gcctgaaatc actttttttc aggttggacc ggtgccacca
1261 tggactataa ggaccacgac ggagactaca aggatcatga tattgattac aaagacgatg
1321 acgataagat ggccccaaag aagaagcgga aggtcggtat ccacggagtc ccagcagccg
1381 acaagaagta cagcatcggc ctggacatcg gcaccaactc tgtgggctgg gccgtgatca
1441 ccgacgagta caaggtgccc agcaagaaat tcaaggtgct gggcaacacc gaccggcaca
1501 gcatcaagaa gaacctgatc ggagccctgc tgttcgacag cggcgaaaca gccgaggcca
1561 cccggctgaa gagaaccgcc agaagaagat acaccagacg gaagaaccgg atctgctatc
1621 tgcaagagat cttcagcaac gagatggcca aggtggacga cagcttcttc cacagactgg
1681 aagagtcctt cctggtggaa gaggataaga agcacgagcg gcaccccatc ttcggcaaca
1741 tcgtggacga ggtggcctac cacgagaagt accccaccat ctaccacctg agaaagaaac
1801 tggtggacag caccgacaag gccgacctgc ggctgatcta tctggccctg gcccacatga
1861 tcaagttccg gggccacttc ctgatcgagg gcgacctgaa ccccgacaac agcgacgtgg
1921 acaagctgtt catccagctg gtgcagacct acaaccagct gttcgaggaa aaccccatca
1981 acgccagcgg cgtggacgcc aaggccatcc tgtctgccag actgagcaag agcagacggc
2041 tggaaaatct gatcgcccag ctgcccggcg agaagaagaa tggcctgttc ggaaacctga
2101 ttgccctgag cctgggcctg acccccaact tcaagagcaa cttcgacctg gccgaggatg
2161 ccaaactgca gctgagcaag gacacctacg acgacgacct ggacaacctg ctggcccaga
2221 tcggcgacca gtacgccgac ctgtttctgg ccgccaagaa cctgtccgac gccatcctgc
2281 tgagcgacat cctgagagtg aacaccgaga tcaccaaggc ccccctgagc gcctctatga
2341 tcaagagata cgacgagcac caccaggacc tgaccctgct gaaagctctc gtgcggcagc
2401 agctgcctga gaagtacaaa gagattttct tcgaccagag caagaacggc tacgccggct
2461 acattgacgg cggagccagc caggaagagt tctacaagtt catcaagccc atcctggaaa
2521 agatggacgg caccgaggaa ctgctcgtga agctgaacag agaggacctg ctgcggaagc
2581 agcggacctt cgacaacggc agcatccccc accagatcca cctgggagag ctgcacgcca
2641 ttctgcggcg gcaggaagat ttttacccat tcctgaagga caaccgggaa aagatcgaga
2701 agatcctgac cttccgcatc ccctactacg tgggccctct ggccagggga aacagcagat
2761 tcgcctggat gaccagaaag agcgaggaaa ccatcacccc ctggaacttc gaggaagtgg
2821 tggacaaggg cgcttccgcc cagagcttca tcgagcggat gaccaacttc gataagaacc
2881 tgcccaacga gaaggtgctg cccaagcaca gcctgctgta cgagtacttc accgtgtata
2941 acgagctgac caaagtgaaa tacgtgaccg agggaatgag aaagcccgcc ttcctgagcg
3001 gcgagcagaa aaaggccatc gtggacctgc tgttcaagac caaccggaaa gtgaccgtga
3061 agcagctgaa agaggactac ttcaagaaaa tcgagtgctt cgactccgtg gaaatctccg
3121 gcgtggaaga tcggttcaac gcctccctgg gcacatacca cgatctgctg aaaattatca
3181 aggacaagga cttcctggac aatgaggaaa acgaggacat tctggaagat atcgtgctga
3241 ccctgacact gtttgaggac agagagatga tcgaggaacg gctgaaaacc tatgcccacc
3301 tgttcgacga caaagtgatg aagcagctga agcggcggag atacaccggc tggggcaggc
3361 tgagccggaa gctgatcaac ggcatccggg acaagcagtc cggcaagaca atcctggatt
3421 tcctgaagtc cgacggcttc gccaacagaa acttcatgca gctgatccac gacgacagcc
3481 tgacctttaa agaggacatc cagaaagccc aggtgtccgg ccagggcgat agcctgcacg
3541 agcacattgc caatctggcc ggcagccccg ccattaagaa gggcatcctg cagacagtga
3601 aggtggtgga cgagctcgtg aaagtgatgg gccggcacaa gcccgagaac atcgtgatcg
3661 aaatggccag agagaaccag accacccaga agggacagaa gaacagccgc gagagaatga
3721 agcggatcga agagggcatc aaagagctgg gcagccagat cctgaaagaa caccccgtgg
3781 aaaacaccca gctgcagaac gagaagctgt acctgtacta cctgcagaat gggcgggata
3841 tgtacgtgga ccaggaactg gacatcaacc ggctgtccga ctacgatgtg gaccatatcg
3901 tgcctcagag ctttctgaag gacgactcca tcgacaacaa ggtgctgacc agaagcgaca
3961 agaaccgggg caagagcgac aacgtgccct ccgaagaggt cgtgaagaag atgaagaact
4021 actggcggca gctgctgaac gccaagctga ttacccagag aaagttcgac aatctgacca
4081 aggccgagag aggcggcctg agcgaactgg ataaggccgg cttcatcaag agacagctgg
4141 tggaaacccg gcagatcaca aagcacgtgg cacagatcct ggactcccgg atgaacacta
4201 agtacgacga gaatgacaag ctgatccggg aagtgaaagt gatcaccctg aagtccaagc
4261 tggtgtccga tttccggaag gatttccagt tttacaaagt gcgcgagatc aacaactacc
4321 accacgccca cgacgcctac ctgaacgccg tcgtgggaac cgccctgatc aaaaagtacc
4381 ctaagctgga aagcgagttc gtgtacggcg actacaaggt gtacgacgtg cggaagatga
4441 tcgccaagag cgagcaggaa atcggcaagg ctaccgccaa gtacttcttc tacagcaaca
4501 tcatgaactt tttcaagacc gagattaccc tggccaacgg cgagatccgg aagcggcctc
4561 tgatcgagac aaacggcgaa accggggaga tcgtgtggga taagggccgg gattttgcca
4621 ccgtgcggaa agtgctgagc atgccccaag tgaatatcgt gaaaaagacc gaggtgcaga
4681 caggcggctt cagcaaagag tctatcctgc ccaagaggaa cagcgataag ctgatcgcca
4741 gaaagaagga ctgggaccct aagaagtacg gcggcttcga cagccccacc gtggcctatt
4801 ctgtgctggt ggtggccaaa gtggaaaagg gcaagtccaa gaaactgaag agtgtgaaag
4861 agctgctggg gatcaccatc atggaaagaa gcagcttcga gaagaatccc atcgactttc
4921 tggaagccaa gggctacaaa gaagtgaaaa aggacctgat catcaagctg cctaagtact
4981 ccctgttcga gctggaaaac ggccggaaga gaatgctggc ctctgccggc gaactgcaga
5041 agggaaacga actggccctg ccctccaaat atgtgaactt cctgtacctg gccagccact
5101 atgagaagct gaagggctcc cccgaggata atgagcagaa acagctgttt gtggaacagc
5161 acaagcacta cctggacgag atcatcgagc agatcagcga gttctccaag agagtgatcc
5221 tggccgacgc taatctggac aaagtgctgt ccgcctacaa caagcaccgg gataagccca
5281 tcagagagca ggccgagaat atcatccacc tgtttaccct gaccaatctg ggagcccctg
5341 ccgccttcaa gtactttgac accaccatcg accggaagag gtacaccagc accaaagagg
5401 tgctggacgc caccctgatc caccagagca tcaccggcct gtacgagaca cggatcgacc
5461 tgtctcagct gggaggcgac acagggtcca caggatccac aggcagcaca gggagcatgg
5521 gaccacatgg ccatgtctta catcgtcaca tgagaacaat ccgggaagta cgcacacttg
5581 tcactcgtgt cattacagat gtgtattatg tggatggaac agaagtagaa agaaaagtaa
5641 ctgaggagac tgaagagcca attgtagagt gtcaggagtg tgaaactgaa gtttcccctt
5701 cacagactgg gggctcctca ggtgacctgg gggatatcag ctccttctcc tccaaggcat
5761 ccagcttaca ccgcacatca agtgggacaa gtctctcagc tatgcacagc agtggaagct
5821 cagggaaagg agccggacca ctcagaggga aaaccagcgg gacagaaccc gcagattttg
5881 ccttacccag ctcccgagga ggcccaggaa aactgagtcc tagaaaaggg gtcagtcaga
5941 cagggacgcc agtgtgtgag gaggatggtg atgcaggcct tggcatcaga cagggaggga
6001 aggctccagt cacgcctcgt gggcgtgggc gaaggggccg cccaccttct cggaccactg
6061 gaaccagaga aacagctgtg cctggcccct tgggcataga ggacatttca cctaacttgt
6121 caccagatga taaatccttc agccgtgtcg tgccccgagt gccagactcc accagacgaa
6181 cagatgtggg tgctggtgct ttgcgtcgta gtgactctcc agaaattcct ttccaggctg
6241 ctgctggccc ttctgatggc ttagatgcct cctctccagg aaatagcttt gtagggctcc
6301 gtgttgtagc caagtggtca tccaatggct acttttactc tgggaaaatc acacgagatg
6361 tcggagctgg gaagtataaa ttgctctttg atgatgggta cgaatgtgat gtgttgggca
6421 aagacattct gttatgtgac cccatcccgc tggacactga agtgacggcc ctctcggagg
6481 atgagtattt cagtgcagga gtggtgaaag gacataggaa ggagtctggg gaactgtact
6541 acagcattga aaaagaaggc caaagaaagt ggtataagcg aatggctgtc atcctgtcct
6601 tggagcaagg aaacagactg agagagcagt atgggcttgg cccctatgaa gcagtaacac
6661 ctcttacaaa ggcagcagat atcagcttag acaatttggt ggaagggaag cggaaacgga
6721 gatctaacgt cagctcccca gccaccccta ctgcctcctc gagcaaaagg ccggcggcca
6781 cgaaaaaggc cggccaggca aaaaagaaaa aggaattcgg cagtggagag ggcagaggaa
6841 gtctgctaac atgcggtgac gtcgaggaga atcctggccc agtgagcaag ggcgaggagc
6901 tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt
6961 tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc ctgaagttca
7021 tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ctgacctacg
7081 gcgtgcagtg cttcagccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg
7141 ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac ggcaactaca
7201 agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg
7261 gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac aactacaaca
7321 gccacaacgt ctatatcatg gccgacaagc agaagaacgg catcaaggtg aacttcaaga
7381 tccgccacaa catcgaggac ggcagcgtgc agctcgccga ccactaccag cagaacaccc
7441 ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagcacc cagtccgccc
7501 tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc gtgaccgccg
7561 ccgggatcac tctcggcatg gacgagctgt acaaggaatt ctaactagag ctcgctgatc
7621 agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc
7681 cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc
7741 gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg
7801 ggaggattgg gaagagaata gcaggcatgc tggggagcgg ccgcaggaac ccctagtgat
7861 ggagttggcc actccctctc tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt
7921 cgcccgacgc ccgggctttg cccgggcggc ctcagtgagc gagcgagcgc gcagctgcct
7981 gcaggggcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat
8041 acgtcaaagc aaccatagta cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg
8101 gttacgcgca gcgtgaccgc tacacttgcc agcgccttag cgcccgctcc tttcgctttc
8161 ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc
8221 cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgatttgggt
8281 gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag
8341 tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa ctctatctcg
8401 ggctattctt ttgatttata agggattttg ccgatttcgg tctattggtt aaaaaatgag
8461 ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgtttac aattttatgg
8521 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagccccg acacccgcca
8581 acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct
8641 gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg
8701 agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt
8761 tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt
8821 ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa
8881 taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt
8941 tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat
9001 gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag
9061 atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg
9121 ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata
9181 cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat
9241 ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc
9301 aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg
9361 ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac
9421 gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact
9481 ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa
9541 gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct
9601 ggagccggtg agcgtggaag ccgcggtatc attgcagcac tggggccaga tggtaagccc
9661 tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga
9721 cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac
9781 tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag
9841 atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
9901 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
9961 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
10021 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt
10081 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
10141 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
10201 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
10261 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
10321 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
10381 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
10441 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
10501 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
10561 tgctggcctt ttgctcacat gt
//
