| 质粒名称: | pJSC282 - Bacterial expression plasmid for SpCas9 Cluster 1 (HypaCas9), REC3 FRET variant |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 101231 |
| 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/N692A/M694A/Q695A/H698A |
| 物种来源: | 化脓性链球菌 |
| 插入大小: | -- |
| 基因突变: | C80S, C574S, S701C, S960C, N692A, M694A, Q695A and H698A |
| 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: | pJSC282 - Bacterial expression plasmid for SpCas9 Cluster 1 (HypaCas9), REC3 FRET variant was a gift from Jennifer Doudna & Keith Joung (Addgene plasmid # 101231 ; http://n2t.net/addgene:101231 ; RRID:Addgene_101231) |
| 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 |
pJSC282 - Bacterial expression plasmid for SpCas9 Cluster 1
买家导航
pJSC282 - Bacterial expression plasmid for SpCas9 Cluster 1载体质粒基本信息
pJSC282 - Bacterial expression plasmid for SpCas9 Cluster 1质粒图谱载体图谱和pJSC282 - Bacterial expression plasmid for SpCas9 Cluster 1载体序列质粒序列多克隆位点信息
pJSC282 - Bacterial expression plasmid for SpCas9 Cluster 1质粒载体简介
pJSC282 - Bacterial expression plasmid for SpCas9 Cluster 1质粒序列载体序列
LOCUS pJSC282 - Bacterial expression plasmid for SpCas9 Cluster 1 (HypaCas9), REC3 FRET variant 10073 bp ds-DNA circular SYN 13-MAY-2021
DEFINITION Bacterial expression plasmid for SpCas9 Cluster 1 (HypaCas9), REC3
FRET variant.
ACCESSION .
VERSION .
KEYWORDS pJSC282 - Bacterial expression plasmid for SpCas9 Cluster 1
(H...
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 170..189
/label=pBRrevBam
/note="pBR322 vectors, tet region, downstream of BamHI,
reverse primer"
primer_bind 273..292
/label=T7
/note="T7 promoter, forward primer"
promoter 273..291
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
RBS 380..402
/note="efficient ribosome binding site from bacteriophage
T7 gene 10 (Olins and Rangwala, 1989)"
CDS 422..451
/codon_start=1
/product="10xHis affinity tag"
/label=10xHis
/translation="HHHHHHHHHH"
CDS 461..1561
/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 1535..1558
/label=MBP-F
/note="Maltose binding protein, forward primer"
CDS 1616..1636
/codon_start=1
/product="tobacco etch virus (TEV) protease recognition and
cleavage site"
/label=TEV site
/translation="ENLYFQS"
CDS 1643..5749
/codon_start=1
/product="Cas9 endonuclease from the Streptococcus pyogenes
Type II CRISPR/Cas system, mutated to improve targeting
specificity without compromising on-target activity (Chen
et al., 2017)"
/label=HypaCas9
/note="carries the mutations N692A, M694A, Q695A, and
H698A"
/translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
NLIGALLFDSGETAEATRLKRTARRRYTRRKNRISYLQEIFSNEMAKVDDSFFHRLEES
FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIESFD
SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRAFA
ALIADDCLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
VAQILDSRMNTKYDENDKLIREVKVITLKCKLVSDFRKDFQFYKVREINNYHHAHDAYL
NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
DATLIHQSITGLYETRIDLSQLGGD"
primer_bind complement(5875..5893)
/label=T7 Term
/note="T7 terminator, reverse primer"
terminator 5889..5936
/label=T7 terminator
/note="transcription terminator for bacteriophage T7 RNA
polymerase"
promoter complement(6302..6330)
/label=tet promoter
/note="E. coli promoter for tetracycline efflux protein
gene"
primer_bind complement(6357..6375)
/label=pBRforEco
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
promoter 6443..6547
/gene="bla"
/label=AmpR promoter
CDS 6548..7408
/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(6766..6785)
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
rep_origin 7579..8167
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 8068..8087
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
primer_bind 8321..8338
/label=L4440
/note="L4440 vector, forward primer"
misc_feature 8353..8492
/label=bom
/note="basis of mobility region from pBR322"
primer_bind complement(8418..8437)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 8578..8600
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
CDS complement(8594..8785)
/codon_start=1
/gene="rop"
/product="Rop protein, which maintains plasmids at low copy
number"
/label=rop
/translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
DELYRSCLARFGDDGENL"
ORIGIN
1 ttaggaagca gcccagtagt aggttgaggc cgttgagcac cgccgccgca aggaatggtg
61 catgcaagga gatggcgccc aacagtcccc cggccacggg gcctgccacc atacccacgc
121 cgaaacaagc gctcatgagc ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg
181 cgatataggc gccagcaacc gcacctgtgg cgccggtgat gccggccacg atgcgtccgg
241 cgtagaggat cgagatctcg atcccgcgaa attaatacga ctcactatag ggagaccaca
301 acggtttccc tctagtgccg gctccggaga gctctttaat taagcggccg ccctgcagga
361 ctcgagttct agaaataatt ttgtttaact ttaagaagga gatatacata tgaaatcttc
421 tcaccatcac catcaccatc accatcacca tggttcttct atgaaaatcg aagaaggtaa
481 actggtaatc tggattaacg gcgataaagg ctataacggt ctcgctgaag tcggtaagaa
541 attcgagaaa gataccggaa ttaaagtcac cgttgagcat ccggataaac tggaagagaa
601 attcccacag gttgcggcaa ctggcgatgg ccctgacatt atcttctggg cacacgaccg
661 ctttggtggc tacgctcaat ctggcctgtt ggctgaaatc accccggaca aagcgttcca
721 ggacaagctg tatccgttta cctgggatgc cgtacgttac aacggcaagc tgattgctta
781 cccgatcgct gttgaagcgt tatcgctgat ttataacaaa gatctgctgc cgaacccgcc
841 aaaaacctgg gaagagatcc cggcgctgga taaagaactg aaagcgaaag gtaagagcgc
901 gctgatgttc aacctgcaag aaccgtactt cacctggccg ctgattgctg ctgacggggg
961 ttatgcgttc aagtatgaaa acggcaagta cgacattaaa gacgtgggcg tggataacgc
1021 tggcgcaaaa gcgggtctga ccttcctggt tgacctgatt aaaaacaaac acatgaatgc
1081 agacaccgat tactccatcg cagaagctgc ctttaataaa ggcgaaacag cgatgaccat
1141 caacggcccg tgggcatggt ccaacatcga caccagcaaa gtgaattatg gtgtaacggt
1201 actgccgacc ttcaagggtc aaccatccaa accgttcgtt ggcgtgctga gcgcaggtat
1261 taacgccgcc agtccgaaca aagagctggc aaaagagttc ctcgaaaact atctgctgac
1321 tgatgaaggt ctggaagcgg ttaataaaga caaaccgctg ggtgccgtag cgctgaagtc
1381 ttacgaggaa gagttggcga aagatccacg tattgccgcc actatggaaa acgcccagaa
1441 aggtgaaatc atgccgaaca tcccgcagat gtccgctttc tggtatgccg tgcgtactgc
1501 ggtgatcaac gccgccagcg gtcgtcagac tgtcgatgaa gccctgaaag acgcgcagac
1561 taattcgagc tcgaacaaca acaacaataa caataacaac aacctcggga tcgaggaaaa
1621 cctgtacttc caatccaatg caatggataa gaaatactca ataggcttag atatcggcac
1681 aaatagcgtc ggatgggcgg tgatcactga tgaatataag gttccgtcta aaaagttcaa
1741 ggttctggga aatacagacc gccacagtat caaaaaaaat cttatagggg ctcttttatt
1801 tgacagtgga gagacagcgg aagcgactcg tctcaaacgg acagctcgta gaaggtatac
1861 acgtcggaag aatcgtattt cttatctaca ggagattttt tcaaatgaga tggcgaaagt
1921 agatgatagt ttctttcatc gacttgaaga gtcttttttg gtggaagaag acaagaagca
1981 tgaacgtcat cctatttttg gaaatatagt agatgaagtt gcttatcatg agaaatatcc
2041 aactatctat catctgcgaa aaaaattggt agattctact gataaagcgg atttgcgctt
2101 aatctatttg gccttagcgc atatgattaa gtttcgtggt cattttttga ttgagggaga
2161 tttaaatcct gataatagtg atgtggacaa actatttatc cagttggtac aaacctacaa
2221 tcaattattt gaagaaaacc ctattaacgc aagtggagta gatgctaaag cgattctttc
2281 tgcacgattg agtaaatcaa gacgattaga aaatctcatt gctcagctcc ccggtgagaa
2341 gaaaaatggc ttatttggga atctcattgc tttgtcattg ggtttgaccc ctaattttaa
2401 atcaaatttt gatttggcag aagatgctaa attacagctt tcaaaagata cttacgatga
2461 tgatttagat aatttattgg cgcaaattgg agatcaatat gctgatttgt ttttggcagc
2521 taagaattta tcagatgcta ttttactttc agatatccta agagtaaata ctgaaataac
2581 taaggctccc ctatcagctt caatgattaa acgctacgat gaacatcatc aagacttgac
2641 tcttttaaaa gctttagttc gacaacaact tccagaaaag tataaagaaa tcttttttga
2701 tcaatcaaaa aacggatatg caggttatat tgatggggga gctagccaag aagaatttta
2761 taaatttatc aaaccaattt tagaaaaaat ggatggtact gaggaattat tggtgaaact
2821 aaatcgtgaa gatttgctgc gcaagcaacg gacctttgac aacggctcta ttccccatca
2881 aattcacttg ggtgagctgc atgctatttt gagaagacaa gaagactttt atccattttt
2941 aaaagacaat cgtgagaaga ttgaaaaaat cttgactttt cgaattcctt attatgttgg
3001 tccattggcg cgtggcaata gtcgttttgc atggatgact cggaagtctg aagaaacaat
3061 taccccatgg aattttgaag aagttgtcga taaaggtgct tcagctcaat catttattga
3121 acgcatgaca aactttgata aaaatcttcc aaatgaaaaa gtactaccaa aacatagttt
3181 gctttatgag tattttacgg tttataacga attgacaaag gtcaaatatg ttactgaagg
3241 aatgcgaaaa ccagcatttc tttcaggtga acagaagaaa gccattgttg atttactctt
3301 caaaacaaat cgaaaagtaa ccgttaagca attaaaagaa gattatttca aaaaaataga
3361 atcttttgat agtgttgaaa tttcaggagt tgaagataga tttaatgctt cattaggtac
3421 ctaccatgat ttgctaaaaa ttattaaaga taaagatttt ttggataatg aagaaaatga
3481 agatatctta gaggatattg ttttaacatt gaccttattt gaagataggg agatgattga
3541 ggaaagactt aaaacatatg ctcacctctt tgatgataag gtgatgaaac agcttaaacg
3601 tcgccgttat actggttggg gacgtttgtc tcgaaaattg attaatggta ttagggataa
3661 gcaatctggc aaaacaatat tagatttttt gaaatcagat ggttttgcca atcgcgcttt
3721 tgctgctctg atcgctgatg attgtttgac atttaaagaa gacattcaaa aagcacaagt
3781 gtctggacaa ggcgatagtt tacatgaaca tattgcaaat ttagctggta gccctgctat
3841 taaaaaaggt attttacaga ctgtaaaagt tgttgatgaa ttggtcaaag taatggggcg
3901 gcataagcca gaaaatatcg ttattgaaat ggcacgtgaa aatcagacaa ctcaaaaggg
3961 ccagaaaaat tcgcgagagc gtatgaaacg aatcgaagaa ggtatcaaag aattaggaag
4021 tcagattctt aaagagcatc ctgttgaaaa tactcaattg caaaatgaaa agctctatct
4081 ctattatctc caaaatggaa gagacatgta tgtggaccaa gaattagata ttaatcgttt
4141 aagtgattat gatgtcgatc acattgttcc acaaagtttc cttaaagacg attcaataga
4201 caataaggtc ttaacgcgtt ctgataaaaa tcgtggtaaa tcggataacg ttccaagtga
4261 agaagtagtc aaaaagatga aaaactattg gagacaactt ctaaacgcca agttaatcac
4321 tcaacgtaag tttgataatt taacgaaagc tgaacgtgga ggtttgagtg aacttgataa
4381 agctggtttt atcaaacgcc aattggttga aactcgccaa atcactaagc atgtggcaca
4441 aattttggat agtcgcatga atactaaata cgatgaaaat gataaactta ttcgagaggt
4501 taaagtgatt accttaaaat gtaaattagt ttctgacttc cgaaaagatt tccaattcta
4561 taaagtacgt gagattaaca attaccatca tgcccatgat gcgtatctaa atgccgtcgt
4621 tggaactgct ttgattaaga aatatccaaa acttgaatcg gagtttgtct atggtgatta
4681 taaagtttat gatgttcgta aaatgattgc taagtctgag caagaaatag gcaaagcaac
4741 cgcaaaatat ttcttttact ctaatatcat gaacttcttc aaaacagaaa ttacacttgc
4801 aaatggagag attcgcaaac gccctctaat cgaaactaat ggggaaactg gagaaattgt
4861 ctgggataaa gggcgagatt ttgccacagt gcgcaaagta ttgtccatgc cccaagtcaa
4921 tattgtcaag aaaacagaag tacagacagg cggattctcc aaggagtcaa ttttaccaaa
4981 aagaaattcg gacaagctta ttgctcgtaa aaaagactgg gatccaaaaa aatatggtgg
5041 ttttgatagt ccaacggtag cttattcagt cctagtggtt gctaaggtgg aaaaagggaa
5101 atcgaagaag ttaaaatccg ttaaagagtt actagggatc acaattatgg aaagaagttc
5161 ctttgaaaaa aatccgattg actttttaga agctaaagga tataaggaag ttaaaaaaga
5221 cttaatcatt aaactaccta aatatagtct ttttgagtta gaaaacggtc gtaaacggat
5281 gctggctagt gccggagaat tacaaaaagg aaatgagctg gctctgccaa gcaaatatgt
5341 gaatttttta tatttagcta gtcattatga aaagttgaag ggtagtccag aagataacga
5401 acaaaaacaa ttgtttgtgg agcagcataa gcattattta gatgagatta ttgagcaaat
5461 cagtgaattt tctaagcgtg ttattttagc agatgccaat ttagataaag ttcttagtgc
5521 atataacaaa catagagaca aaccaatacg tgaacaagca gaaaatatta ttcatttatt
5581 tacgttgacg aatcttggag ctcccgctgc ttttaaatat tttgatacaa caattgatcg
5641 taaacgatat acgtctacaa aagaagtttt agatgccact cttatccatc aatccatcac
5701 tggtctttat gaaacacgca ttgatttgag tcagctagga ggtgactaat aacattggaa
5761 gtggataacg gatccgcgat cgcggcgcgc cacctggtgg ccggccggta ccacgcgtgc
5821 gcgctgatcc ggctgctaac aaagcccgaa aggaagctga gttggctgct gccaccgctg
5881 agcaataact agcataaccc attggggcct ctaaacgggt cttgaggggt tttttgctga
5941 aaggaggaac tatatccgga tatccacagg acgggtgtgg tcgccatgat cgcgtagtcg
6001 atagtggctc caagtagcga agcgagcagg actgggcggc ggccaaagcg gtcggacagt
6061 gctccgagaa cgggtgcgca tagaaattgc atcaacgcat atagcgctag cagcacgcca
6121 tagtgactgg cgatgctgtc ggaatggacg atatcccgca agaggcccgg cagtaccggc
6181 ataaccaagc ctatgcctac agcatccagg gtgacggtgc cgaggatgac gatgagcgca
6241 ttgttagatt tcatacacgg tgcctgactg cgttagcaat ttaactgtga taaactaccg
6301 cattaaagct tatcgatgat aagctgtcaa acatgagaat tcttgaagac gaaagggcct
6361 cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg
6421 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
6481 aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataac attgaaaaag
6541 gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg
6601 ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt
6661 gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt
6721 tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt
6781 attatcccgt gttgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa
6841 tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag
6901 agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac
6961 aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac
7021 tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac
7081 cacgatgcct gcagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac
7141 tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact
7201 tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg
7261 tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt
7321 tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat
7381 aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta
7441 gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa
7501 tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
7561 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
7621 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
7681 tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc
7741 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
7801 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
7861 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
7921 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
7981 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
8041 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
8101 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
8161 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
8221 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
8281 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga
8341 agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg
8401 caatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gtatacactc
8461 cgctatcgct acgtgactgg gtcatggctg cgccccgaca cccgccaaca cccgctgacg
8521 cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg
8581 ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt
8641 aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca
8701 gctcgttgag tttctccaga agcgttaatg tctggcttct gataaagcgg gccatgttaa
8761 gggcggtttt ttcctgtttg gtcactgatg cctccgtgta agggggattt ctgttcatgg
8821 gggtaatgat accgatgaaa cgagagagga tgctcacgat acgggttact gatgatgaac
8881 atgcccggtt actggaacgt tgtgagggta aacaactggc ggtatggatg cggcgggacc
8941 agagaaaaat cactcagggt caatgccagc gcttcgttaa tacagatgta ggtgttccac
9001 agggtagcca gcagcatcct gcgatgcaga tccggaacat aatggtgcag ggcgctgact
9061 tccgcgtttc cagactttac gaaacacgga aaccgaagac cattcatgtt gttgctcagg
9121 tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt gattcattct
9181 gctaaccagt aaggcaaccc cgccagccta gccgggtcct caacgacagg agcacgatca
9241 tgcgcacccg tggccaggac ccaacgctgc ccgagatgcg ccgcgtgcgg ctgctggaga
9301 tggcggacgc gatggatatg ttctgccaag ggttggtttg cgcattcaca gttctccgca
9361 agaattgatt ggctccaatt cttggagtgg tgaatccgtt agcgaggtgc cgccggcttc
9421 cattcaggtc gaggtggccc ggctccatgc accgcgacgc aacgcgggga ggcagacaag
9481 gtatagggcg gcgcctacaa tccatgccaa cccgttccat gtgctcgccg aggcggcata
9541 aatcgccgtg acgatcagcg gtccaatgat cgaagttagg ctggtaagag ccgcgagcga
9601 tccttgaagc tgtccctgat ggtcgtcatc tacctgcctg gacagcatgg cctgcaacgc
9661 gggcatcccg atgccgccgg aagcgagaag aatcataatg gggaaggcca tccagcctcg
9721 cgtcgcgaac gccagcaaga cgtagcccag cgcgtcggcc gccatgccgg cgataatggc
9781 ctgcttctcg ccgaaacgtt tggtggcggg accagtgacg aaggcttgag cgagggcgtg
9841 caagattccg aataccgcaa gcgacaggcc gatcatcgtc gcgctccagc gaaagcggtc
9901 ctcgccgaaa atgacccaga gcgctgccgg cacctgtcct acgagttgca tgataaagaa
9961 gacagtcata agtgcggcga cgatagtcat gccccgcgcc caccggaagg agctgactgg
10021 gttgaaggct ctcaagggca tcggtcgacg ctctccctta tgcgactcct gca
//
