| 质粒名称: | pJSC131 - Bacterial expression plasmid for SpCas9-HF1, REC3 FRET variant |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 101212 |
| 存储实验室: | Jennifer Doudna, Keith Joung |
|
载体骨架基本信息 |
|
| 载体名称: | pCT10 |
| 载体出品公司: | Doudna lab |
| 空载体大小: | -- |
| 完整质粒大小: | 10073 bp |
| 载体修饰: | -- |
| 载体类型: | 细菌表达 |
| 筛选标记: | -- |
| 载体抗性: | 氨苄青霉素，100μg/mL |
| 生长温度: | 37 ℃ |
| 宿主菌: | DH5a |
| 拷贝数: | 高拷贝 |
|
插入基因信息 |
|
| 插入基因名称: | SpCas9 variant C80S/C574S/S701C/S960C/N497A/R661A/Q695A/Q926A |
| 物种来源: | 化脓性链球菌 |
| 插入大小: | -- |
| 基因突变: | C80S, C574S, S701C, S960C, N497A, R661A, Q695A and Q926A |
| GenBank编号 | -- |
| 启动子: | T7 |
| 标签/融合蛋白: | 10xHis（主干上的N端子）MBP（主干上的N终端）TEV站点（主干上的N终端） |
| 克隆方法: | Gibson Cloning |
| 5’克隆位点: | -- |
| 3’克隆位点: | -- |
| 5’测序引物 | T7 |
| 3’测序引物 | T7-term |
|
文献引用方法 |
|
| 材料和方法部分: | pJSC131 - Bacterial expression plasmid for SpCas9-HF1, REC3 FRET variant was a gift from Jennifer Doudna & Keith Joung (Addgene plasmid # 101212 ; http://n2t.net/addgene:101212 ; RRID:Addgene_101212) |
| 参考文献部分: | Enhanced proofreading governs CRISPR-Cas9 targeting accuracy. Chen JS, Dagdas YS, Kleinstiver BP, Welch MM, Sousa AA, Harrington LB, Sternberg SH, Joung JK, Yildiz A, Doudna JA. Nature. 2017 Sep 20. doi: 10.1038/nature24268. 10.1038/nature24268 PubMed 28931002 |
pJSC131 - Bacterial expression plasmid for SpCas9-HF1, REC3
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载体基本信息
载体图谱质粒图谱和多克隆位点信息
载体简介
载体序列
LOCUS pJSC131 - Bacterial expression plasmid for SpCas9-HF1, REC3 FRET variant 10073 bp ds-DNA circular SYN 13-MAY-2021
DEFINITION Bacterial expression plasmid for SpCas9-HF1, REC3 FRET variant.
ACCESSION .
VERSION .
KEYWORDS pJSC131 - Bacterial expression plasmid for SpCas9-HF1, REC3 FR...
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 10073)
AUTHORS Chen JS, Dagdas YS, Kleinstiver BP, Welch MM, Sousa AA, Harrington
LB, Sternberg SH, Joung JK, Yildiz A, Doudna JA
TITLE Enhanced proofreading governs CRISPR-Cas9 targeting accuracy.
JOURNAL Nature. 2017 Sep 20. doi: 10.1038/nature24268.
PUBMED 28931002
REFERENCE 2 (bases 1 to 10073)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58
http://www.biofeng.com
FEATURES Location/Qualifiers
source 1..10073
/organism="synthetic DNA construct"
/mol_type="other DNA"
primer_bind 946..965
/label=pBRrevBam
/note="pBR322 vectors, tet region, downstream of BamHI,
reverse primer"
primer_bind 1049..1068
/label=T7
/note="T7 promoter, forward primer"
promoter 1049..1067
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
RBS 1156..1178
/note="efficient ribosome binding site from bacteriophage
T7 gene 10 (Olins and Rangwala, 1989)"
CDS 1198..1227
/codon_start=1
/product="10xHis affinity tag"
/label=10xHis
/translation="HHHHHHHHHH"
CDS 1237..2337
/codon_start=1
/gene="malE (mutated)"
/product="maltose binding protein from E. coli"
/label=MBP
/note="This version of the gene does not encode a signal
sequence, so MBP will remain in the cytosol."
/translation="MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE
EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL
IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA
ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE
TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL
ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF
WYAVRTAVINAASGRQTVDEALKDAQT"
primer_bind 2311..2334
/label=MBP-F
/note="Maltose binding protein, forward primer"
CDS 2392..2412
/codon_start=1
/product="tobacco etch virus (TEV) protease recognition and
cleavage site"
/label=TEV site
/translation="ENLYFQS"
CDS 2419..6525
/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="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
NLIGALLFDSGETAEATRLKRTARRRYTRRKNRISYLQEIFSNEMAKVDDSFFHRLEES
FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTAFDKNLPNEKVLPKHSLLYEY
FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIESFD
SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
LKTYAHLFDDKVMKQLKRRRYTGWGALSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
ALIHDDCLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRAITKH
VAQILDSRMNTKYDENDKLIREVKVITLKCKLVSDFRKDFQFYKVREINNYHHAHDAYL
NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
DATLIHQSITGLYETRIDLSQLGGD"
primer_bind complement(6651..6669)
/label=T7 Term
/note="T7 terminator, reverse primer"
terminator 6665..6712
/label=T7 terminator
/note="transcription terminator for bacteriophage T7 RNA
polymerase"
promoter complement(7078..7106)
/label=tet promoter
/note="E. coli promoter for tetracycline efflux protein
gene"
primer_bind complement(7133..7151)
/label=pBRforEco
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
promoter 7219..7323
/gene="bla"
/label=AmpR promoter
CDS 7324..8184
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/label=AmpR
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
primer_bind complement(7542..7561)
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
rep_origin 8355..8943
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 8844..8863
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
primer_bind 9097..9114
/label=L4440
/note="L4440 vector, forward primer"
misc_feature 9129..9268
/label=bom
/note="basis of mobility region from pBR322"
primer_bind complement(9194..9213)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 9354..9376
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
CDS complement(9370..9561)
/codon_start=1
/gene="rop"
/product="Rop protein, which maintains plasmids at low copy
number"
/label=rop
/translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
DELYRSCLARFGDDGENL"
ORIGIN
1 agatggcgga cgcgatggat atgttctgcc aagggttggt ttgcgcattc acagttctcc
61 gcaagaattg attggctcca attcttggag tggtgaatcc gttagcgagg tgccgccggc
121 ttccattcag gtcgaggtgg cccggctcca tgcaccgcga cgcaacgcgg ggaggcagac
181 aaggtatagg gcggcgccta caatccatgc caacccgttc catgtgctcg ccgaggcggc
241 ataaatcgcc gtgacgatca gcggtccaat gatcgaagtt aggctggtaa gagccgcgag
301 cgatccttga agctgtccct gatggtcgtc atctacctgc ctggacagca tggcctgcaa
361 cgcgggcatc ccgatgccgc cggaagcgag aagaatcata atggggaagg ccatccagcc
421 tcgcgtcgcg aacgccagca agacgtagcc cagcgcgtcg gccgccatgc cggcgataat
481 ggcctgcttc tcgccgaaac gtttggtggc gggaccagtg acgaaggctt gagcgagggc
541 gtgcaagatt ccgaataccg caagcgacag gccgatcatc gtcgcgctcc agcgaaagcg
601 gtcctcgccg aaaatgaccc agagcgctgc cggcacctgt cctacgagtt gcatgataaa
661 gaagacagtc ataagtgcgg cgacgatagt catgccccgc gcccaccgga aggagctgac
721 tgggttgaag gctctcaagg gcatcggtcg acgctctccc ttatgcgact cctgcattag
781 gaagcagccc agtagtaggt tgaggccgtt gagcaccgcc gccgcaagga atggtgcatg
841 caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac ccacgccgaa
901 acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat
961 ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta
1021 gaggatcgag atctcgatcc cgcgaaatta atacgactca ctatagggag accacaacgg
1081 tttccctcta gtgccggctc cggagagctc tttaattaag cggccgccct gcaggactcg
1141 agttctagaa ataattttgt ttaactttaa gaaggagata tacatatgaa atcttctcac
1201 catcaccatc accatcacca tcaccatggt tcttctatga aaatcgaaga aggtaaactg
1261 gtaatctgga ttaacggcga taaaggctat aacggtctcg ctgaagtcgg taagaaattc
1321 gagaaagata ccggaattaa agtcaccgtt gagcatccgg ataaactgga agagaaattc
1381 ccacaggttg cggcaactgg cgatggccct gacattatct tctgggcaca cgaccgcttt
1441 ggtggctacg ctcaatctgg cctgttggct gaaatcaccc cggacaaagc gttccaggac
1501 aagctgtatc cgtttacctg ggatgccgta cgttacaacg gcaagctgat tgcttacccg
1561 atcgctgttg aagcgttatc gctgatttat aacaaagatc tgctgccgaa cccgccaaaa
1621 acctgggaag agatcccggc gctggataaa gaactgaaag cgaaaggtaa gagcgcgctg
1681 atgttcaacc tgcaagaacc gtacttcacc tggccgctga ttgctgctga cgggggttat
1741 gcgttcaagt atgaaaacgg caagtacgac attaaagacg tgggcgtgga taacgctggc
1801 gcaaaagcgg gtctgacctt cctggttgac ctgattaaaa acaaacacat gaatgcagac
1861 accgattact ccatcgcaga agctgccttt aataaaggcg aaacagcgat gaccatcaac
1921 ggcccgtggg catggtccaa catcgacacc agcaaagtga attatggtgt aacggtactg
1981 ccgaccttca agggtcaacc atccaaaccg ttcgttggcg tgctgagcgc aggtattaac
2041 gccgccagtc cgaacaaaga gctggcaaaa gagttcctcg aaaactatct gctgactgat
2101 gaaggtctgg aagcggttaa taaagacaaa ccgctgggtg ccgtagcgct gaagtcttac
2161 gaggaagagt tggcgaaaga tccacgtatt gccgccacta tggaaaacgc ccagaaaggt
2221 gaaatcatgc cgaacatccc gcagatgtcc gctttctggt atgccgtgcg tactgcggtg
2281 atcaacgccg ccagcggtcg tcagactgtc gatgaagccc tgaaagacgc gcagactaat
2341 tcgagctcga acaacaacaa caataacaat aacaacaacc tcgggatcga ggaaaacctg
2401 tacttccaat ccaatgcaat ggataagaaa tactcaatag gcttagatat cggcacaaat
2461 agcgtcggat gggcggtgat cactgatgaa tataaggttc cgtctaaaaa gttcaaggtt
2521 ctgggaaata cagaccgcca cagtatcaaa aaaaatctta taggggctct tttatttgac
2581 agtggagaga cagcggaagc gactcgtctc aaacggacag ctcgtagaag gtatacacgt
2641 cggaagaatc gtatttctta tctacaggag attttttcaa atgagatggc gaaagtagat
2701 gatagtttct ttcatcgact tgaagagtct tttttggtgg aagaagacaa gaagcatgaa
2761 cgtcatccta tttttggaaa tatagtagat gaagttgctt atcatgagaa atatccaact
2821 atctatcatc tgcgaaaaaa attggtagat tctactgata aagcggattt gcgcttaatc
2881 tatttggcct tagcgcatat gattaagttt cgtggtcatt ttttgattga gggagattta
2941 aatcctgata atagtgatgt ggacaaacta tttatccagt tggtacaaac ctacaatcaa
3001 ttatttgaag aaaaccctat taacgcaagt ggagtagatg ctaaagcgat tctttctgca
3061 cgattgagta aatcaagacg attagaaaat ctcattgctc agctccccgg tgagaagaaa
3121 aatggcttat ttgggaatct cattgctttg tcattgggtt tgacccctaa ttttaaatca
3181 aattttgatt tggcagaaga tgctaaatta cagctttcaa aagatactta cgatgatgat
3241 ttagataatt tattggcgca aattggagat caatatgctg atttgttttt ggcagctaag
3301 aatttatcag atgctatttt actttcagat atcctaagag taaatactga aataactaag
3361 gctcccctat cagcttcaat gattaaacgc tacgatgaac atcatcaaga cttgactctt
3421 ttaaaagctt tagttcgaca acaacttcca gaaaagtata aagaaatctt ttttgatcaa
3481 tcaaaaaacg gatatgcagg ttatattgat gggggagcta gccaagaaga attttataaa
3541 tttatcaaac caattttaga aaaaatggat ggtactgagg aattattggt gaaactaaat
3601 cgtgaagatt tgctgcgcaa gcaacggacc tttgacaacg gctctattcc ccatcaaatt
3661 cacttgggtg agctgcatgc tattttgaga agacaagaag acttttatcc atttttaaaa
3721 gacaatcgtg agaagattga aaaaatcttg acttttcgaa ttccttatta tgttggtcca
3781 ttggcgcgtg gcaatagtcg ttttgcatgg atgactcgga agtctgaaga aacaattacc
3841 ccatggaatt ttgaagaagt tgtcgataaa ggtgcttcag ctcaatcatt tattgaacgc
3901 atgacagcgt ttgataaaaa tcttccaaat gaaaaagtac taccaaaaca tagtttgctt
3961 tatgagtatt ttacggttta taacgaattg acaaaggtca aatatgttac tgaaggaatg
4021 cgaaaaccag catttctttc aggtgaacag aagaaagcca ttgttgattt actcttcaaa
4081 acaaatcgaa aagtaaccgt taagcaatta aaagaagatt atttcaaaaa aatagaatct
4141 tttgatagtg ttgaaatttc aggagttgaa gatagattta atgcttcatt aggtacctac
4201 catgatttgc taaaaattat taaagataaa gattttttgg ataatgaaga aaatgaagat
4261 atcttagagg atattgtttt aacattgacc ttatttgaag atagggagat gattgaggaa
4321 agacttaaaa catatgctca cctctttgat gataaggtga tgaaacagct taaacgtcgc
4381 cgttatactg gttggggagc gttgtctcga aaattgatta atggtattag ggataagcaa
4441 tctggcaaaa caatattaga ttttttgaaa tcagatggtt ttgccaatcg caattttatg
4501 gccctgatcc atgatgattg tttgacattt aaagaagaca ttcaaaaagc acaagtgtct
4561 ggacaaggcg atagtttaca tgaacatatt gcaaatttag ctggtagccc tgctattaaa
4621 aaaggtattt tacagactgt aaaagttgtt gatgaattgg tcaaagtaat ggggcggcat
4681 aagccagaaa atatcgttat tgaaatggca cgtgaaaatc agacaactca aaagggccag
4741 aaaaattcgc gagagcgtat gaaacgaatc gaagaaggta tcaaagaatt aggaagtcag
4801 attcttaaag agcatcctgt tgaaaatact caattgcaaa atgaaaagct ctatctctat
4861 tatctccaaa atggaagaga catgtatgtg gaccaagaat tagatattaa tcgtttaagt
4921 gattatgatg tcgatcacat tgttccacaa agtttcctta aagacgattc aatagacaat
4981 aaggtcttaa cgcgttctga taaaaatcgt ggtaaatcgg ataacgttcc aagtgaagaa
5041 gtagtcaaaa agatgaaaaa ctattggaga caacttctaa acgccaagtt aatcactcaa
5101 cgtaagtttg ataatttaac gaaagctgaa cgtggaggtt tgagtgaact tgataaagct
5161 ggttttatca aacgccaatt ggttgaaact cgcgccatca ctaagcatgt ggcacaaatt
5221 ttggatagtc gcatgaatac taaatacgat gaaaatgata aacttattcg agaggttaaa
5281 gtgattacct taaaatgtaa attagtttct gacttccgaa aagatttcca attctataaa
5341 gtacgtgaga ttaacaatta ccatcatgcc catgatgcgt atctaaatgc cgtcgttgga
5401 actgctttga ttaagaaata tccaaaactt gaatcggagt ttgtctatgg tgattataaa
5461 gtttatgatg ttcgtaaaat gattgctaag tctgagcaag aaataggcaa agcaaccgca
5521 aaatatttct tttactctaa tatcatgaac ttcttcaaaa cagaaattac acttgcaaat
5581 ggagagattc gcaaacgccc tctaatcgaa actaatgggg aaactggaga aattgtctgg
5641 gataaagggc gagattttgc cacagtgcgc aaagtattgt ccatgcccca agtcaatatt
5701 gtcaagaaaa cagaagtaca gacaggcgga ttctccaagg agtcaatttt accaaaaaga
5761 aattcggaca agcttattgc tcgtaaaaaa gactgggatc caaaaaaata tggtggtttt
5821 gatagtccaa cggtagctta ttcagtccta gtggttgcta aggtggaaaa agggaaatcg
5881 aagaagttaa aatccgttaa agagttacta gggatcacaa ttatggaaag aagttccttt
5941 gaaaaaaatc cgattgactt tttagaagct aaaggatata aggaagttaa aaaagactta
6001 atcattaaac tacctaaata tagtcttttt gagttagaaa acggtcgtaa acggatgctg
6061 gctagtgccg gagaattaca aaaaggaaat gagctggctc tgccaagcaa atatgtgaat
6121 tttttatatt tagctagtca ttatgaaaag ttgaagggta gtccagaaga taacgaacaa
6181 aaacaattgt ttgtggagca gcataagcat tatttagatg agattattga gcaaatcagt
6241 gaattttcta agcgtgttat tttagcagat gccaatttag ataaagttct tagtgcatat
6301 aacaaacata gagacaaacc aatacgtgaa caagcagaaa atattattca tttatttacg
6361 ttgacgaatc ttggagctcc cgctgctttt aaatattttg atacaacaat tgatcgtaaa
6421 cgatatacgt ctacaaaaga agttttagat gccactctta tccatcaatc catcactggt
6481 ctttatgaaa cacgcattga tttgagtcag ctaggaggtg actaataaca ttggaagtgg
6541 ataacggatc cgcgatcgcg gcgcgccacc tggtggccgg ccggtaccac gcgtgcgcgc
6601 tgatccggct gctaacaaag cccgaaagga agctgagttg gctgctgcca ccgctgagca
6661 ataactagca taaccccttg gggcctctaa acgggtcttg aggggttttt tgctgaaagg
6721 aggaactata tccggatatc cacaggacgg gtgtggtcgc catgatcgcg tagtcgatag
6781 tggctccaag tagcgaagcg agcaggactg ggcggcggcc aaagcggtcg gacagtgctc
6841 cgagaacggg tgcgcataga aattgcatca acgcatatag cgctagcagc acgccatagt
6901 gactggcgat gctgtcggaa tggacgatat cccgcaagag gcccggcagt accggcataa
6961 ccaagcctat gcctacagca tccagggtga cggtgccgag gatgacgatg agcgcattgt
7021 tagatttcat acacggtgcc tgactgcgtt agcaatttaa ctgtgataaa ctaccgcatt
7081 aaagcttatc gatgataagc tgtcaaacat gagaattctt gaagacgaaa gggcctcgtg
7141 atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc
7201 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
7261 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataacattg aaaaaggaag
7321 agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt
7381 cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt
7441 gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagttttcgc
7501 cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta
7561 tcccgtgttg acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac
7621 ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa
7681 ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg
7741 atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc
7801 cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg
7861 atgcctgcag caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta
7921 gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg
7981 cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg
8041 tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc
8101 tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt
8161 gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt
8221 gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc
8281 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
8341 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
8401 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
8461 aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag
8521 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
8581 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
8641 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
8701 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
8761 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
8821 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
8881 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
8941 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
9001 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga
9061 gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg
9121 gaagagcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcaat
9181 ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat acactccgct
9241 atcgctacgt gactgggtca tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc
9301 ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag
9361 ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgaggcagc tgcggtaaag
9421 ctcatcagcg tggtcgtgaa gcgattcaca gatgtctgcc tgttcatccg cgtccagctc
9481 gttgagtttc tccagaagcg ttaatgtctg gcttctgata aagcgggcca tgttaagggc
9541 ggttttttcc tgtttggtca ctgatgcctc cgtgtaaggg ggatttctgt tcatgggggt
9601 aatgataccg atgaaacgag agaggatgct cacgatacgg gttactgatg atgaacatgc
9661 ccggttactg gaacgttgtg agggtaaaca actggcggta tggatgcggc gggaccagag
9721 aaaaatcact cagggtcaat gccagcgctt cgttaataca gatgtaggtg ttccacaggg
9781 tagccagcag catcctgcga tgcagatccg gaacataatg gtgcagggcg ctgacttccg
9841 cgtttccaga ctttacgaaa cacggaaacc gaagaccatt catgttgttg ctcaggtcgc
9901 agacgttttg cagcagcagt cgcttcacgt tcgctcgcgt atcggtgatt cattctgcta
9961 accagtaagg caaccccgcc agcctagccg ggtcctcaac gacaggagca cgatcatgcg
10021 cacccgtggc caggacccaa cgctgcccga gatgcgccgc gtgcggctgc tgg
//
