| 质粒名称: | pJSC115 - Bacterial expression plasmid for SpCas9-HF1, HNH FRET variant |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 101210 |
| 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/S355C/S867C/N497A/R661A/Q695A/Q926A |
| 物种来源: | Streptococcus pyogenes |
| 插入大小: | -- |
| 基因突变: | C80S, C574S, S355C, S867C, 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: | pJSC115 - Bacterial expression plasmid for SpCas9-HF1, HNH FRET variant was a gift from Jennifer Doudna & Keith Joung (Addgene plasmid # 101210 ; http://n2t.net/addgene:101210 ; RRID:Addgene_101210) |
| 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 |
pJSC115 - Bacterial expression plasmid for SpCas9-HF1, HNH F
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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 pJSC115 - Bacterial expression plasmid for SpCas9-HF1, HNH FRET variant 10073 bp ds-DNA circular SYN 13-MAY-2021
DEFINITION Bacterial expression plasmid for SpCas9-HF1, HNH FRET variant.
ACCESSION .
VERSION .
KEYWORDS pJSC115 - Bacterial expression plasmid for SpCas9-HF1, HNH FRE...
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 1016..1039
/label=MBP-F
/note="Maltose binding protein, forward primer"
CDS 1097..1117
/codon_start=1
/product="tobacco etch virus (TEV) protease recognition and
cleavage site"
/label=TEV site
/translation="ENLYFQS"
CDS 1124..5230
/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
QQLPEKYKEIFFDQCKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTAFDKNLPNEKVLPKHSLLYEY
FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIESFD
SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
LKTYAHLFDDKVMKQLKRRRYTGWGALSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
ALIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKCDNVP
SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRAITKH
VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
DATLIHQSITGLYETRIDLSQLGGD"
primer_bind complement(5356..5374)
/label=T7 Term
/note="T7 terminator, reverse primer"
terminator 5370..5417
/label=T7 terminator
/note="transcription terminator for bacteriophage T7 RNA
polymerase"
promoter complement(5783..5811)
/label=tet promoter
/note="E. coli promoter for tetracycline efflux protein
gene"
primer_bind complement(5838..5856)
/label=pBRforEco
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
promoter 5924..6028
/gene="bla"
/label=AmpR promoter
CDS 6029..6889
/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(6247..6266)
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
rep_origin 7060..7648
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 7549..7568
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
primer_bind 7802..7819
/label=L4440
/note="L4440 vector, forward primer"
misc_feature 7834..7973
/label=bom
/note="basis of mobility region from pBR322"
primer_bind complement(7899..7918)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 8059..8081
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
CDS complement(8075..8266)
/codon_start=1
/gene="rop"
/product="Rop protein, which maintains plasmids at low copy
number"
/label=rop
/translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
DELYRSCLARFGDDGENL"
primer_bind 9724..9743
/label=pBRrevBam
/note="pBR322 vectors, tet region, downstream of BamHI,
reverse primer"
primer_bind 9827..9846
/label=T7
/note="T7 promoter, forward primer"
promoter 9827..9845
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
RBS 9934..9956
/note="efficient ribosome binding site from bacteriophage
T7 gene 10 (Olins and Rangwala, 1989)"
CDS 9976..10005
/codon_start=1
/product="10xHis affinity tag"
/label=10xHis
/translation="HHHHHHHHHH"
CDS 10015..1042
/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"
ORIGIN
1 tctcgctgaa gtcggtaaga aattcgagaa agataccgga attaaagtca ccgttgagca
61 tccggataaa ctggaagaga aattcccaca ggttgcggca actggcgatg gccctgacat
121 tatcttctgg gcacacgacc gctttggtgg ctacgctcaa tctggcctgt tggctgaaat
181 caccccggac aaagcgttcc aggacaagct gtatccgttt acctgggatg ccgtacgtta
241 caacggcaag ctgattgctt acccgatcgc tgttgaagcg ttatcgctga tttataacaa
301 agatctgctg ccgaacccgc caaaaacctg ggaagagatc ccggcgctgg ataaagaact
361 gaaagcgaaa ggtaagagcg cgctgatgtt caacctgcaa gaaccgtact tcacctggcc
421 gctgattgct gctgacgggg gttatgcgtt caagtatgaa aacggcaagt acgacattaa
481 agacgtgggc gtggataacg ctggcgcaaa agcgggtctg accttcctgg ttgacctgat
541 taaaaacaaa cacatgaatg cagacaccga ttactccatc gcagaagctg cctttaataa
601 aggcgaaaca gcgatgacca tcaacggccc gtgggcatgg tccaacatcg acaccagcaa
661 agtgaattat ggtgtaacgg tactgccgac cttcaagggt caaccatcca aaccgttcgt
721 tggcgtgctg agcgcaggta ttaacgccgc cagtccgaac aaagagctgg caaaagagtt
781 cctcgaaaac tatctgctga ctgatgaagg tctggaagcg gttaataaag acaaaccgct
841 gggtgccgta gcgctgaagt cttacgagga agagttggcg aaagatccac gtattgccgc
901 cactatggaa aacgcccaga aaggtgaaat catgccgaac atcccgcaga tgtccgcttt
961 ctggtatgcc gtgcgtactg cggtgatcaa cgccgccagc ggtcgtcaga ctgtcgatga
1021 agccctgaaa gacgcgcaga ctaattcgag ctcgaacaac aacaacaata acaataacaa
1081 caacctcggg atcgaggaaa acctgtactt ccaatccaat gcaatggata agaaatactc
1141 aataggctta gatatcggca caaatagcgt cggatgggcg gtgatcactg atgaatataa
1201 ggttccgtct aaaaagttca aggttctggg aaatacagac cgccacagta tcaaaaaaaa
1261 tcttataggg gctcttttat ttgacagtgg agagacagcg gaagcgactc gtctcaaacg
1321 gacagctcgt agaaggtata cacgtcggaa gaatcgtatt tcttatctac aggagatttt
1381 ttcaaatgag atggcgaaag tagatgatag tttctttcat cgacttgaag agtctttttt
1441 ggtggaagaa gacaagaagc atgaacgtca tcctattttt ggaaatatag tagatgaagt
1501 tgcttatcat gagaaatatc caactatcta tcatctgcga aaaaaattgg tagattctac
1561 tgataaagcg gatttgcgct taatctattt ggccttagcg catatgatta agtttcgtgg
1621 tcattttttg attgagggag atttaaatcc tgataatagt gatgtggaca aactatttat
1681 ccagttggta caaacctaca atcaattatt tgaagaaaac cctattaacg caagtggagt
1741 agatgctaaa gcgattcttt ctgcacgatt gagtaaatca agacgattag aaaatctcat
1801 tgctcagctc cccggtgaga agaaaaatgg cttatttggg aatctcattg ctttgtcatt
1861 gggtttgacc cctaatttta aatcaaattt tgatttggca gaagatgcta aattacagct
1921 ttcaaaagat acttacgatg atgatttaga taatttattg gcgcaaattg gagatcaata
1981 tgctgatttg tttttggcag ctaagaattt atcagatgct attttacttt cagatatcct
2041 aagagtaaat actgaaataa ctaaggctcc cctatcagct tcaatgatta aacgctacga
2101 tgaacatcat caagacttga ctcttttaaa agctttagtt cgacaacaac ttccagaaaa
2161 gtataaagaa atcttttttg atcaatgtaa aaacggatat gcaggttata ttgatggggg
2221 agctagccaa gaagaatttt ataaatttat caaaccaatt ttagaaaaaa tggatggtac
2281 tgaggaatta ttggtgaaac taaatcgtga agatttgctg cgcaagcaac ggacctttga
2341 caacggctct attccccatc aaattcactt gggtgagctg catgctattt tgagaagaca
2401 agaagacttt tatccatttt taaaagacaa tcgtgagaag attgaaaaaa tcttgacttt
2461 tcgaattcct tattatgttg gtccattggc gcgtggcaat agtcgttttg catggatgac
2521 tcggaagtct gaagaaacaa ttaccccatg gaattttgaa gaagttgtcg ataaaggtgc
2581 ttcagctcaa tcatttattg aacgcatgac agcgtttgat aaaaatcttc caaatgaaaa
2641 agtactacca aaacatagtt tgctttatga gtattttacg gtttataacg aattgacaaa
2701 ggtcaaatat gttactgaag gaatgcgaaa accagcattt ctttcaggtg aacagaagaa
2761 agccattgtt gatttactct tcaaaacaaa tcgaaaagta accgttaagc aattaaaaga
2821 agattatttc aaaaaaatag aatcttttga tagtgttgaa atttcaggag ttgaagatag
2881 atttaatgct tcattaggta cctaccatga tttgctaaaa attattaaag ataaagattt
2941 tttggataat gaagaaaatg aagatatctt agaggatatt gttttaacat tgaccttatt
3001 tgaagatagg gagatgattg aggaaagact taaaacatat gctcacctct ttgatgataa
3061 ggtgatgaaa cagcttaaac gtcgccgtta tactggttgg ggagcgttgt ctcgaaaatt
3121 gattaatggt attagggata agcaatctgg caaaacaata ttagattttt tgaaatcaga
3181 tggttttgcc aatcgcaatt ttatggccct gatccatgat gatagtttga catttaaaga
3241 agacattcaa aaagcacaag tgtctggaca aggcgatagt ttacatgaac atattgcaaa
3301 tttagctggt agccctgcta ttaaaaaagg tattttacag actgtaaaag ttgttgatga
3361 attggtcaaa gtaatggggc ggcataagcc agaaaatatc gttattgaaa tggcacgtga
3421 aaatcagaca actcaaaagg gccagaaaaa ttcgcgagag cgtatgaaac gaatcgaaga
3481 aggtatcaaa gaattaggaa gtcagattct taaagagcat cctgttgaaa atactcaatt
3541 gcaaaatgaa aagctctatc tctattatct ccaaaatgga agagacatgt atgtggacca
3601 agaattagat attaatcgtt taagtgatta tgatgtcgat cacattgttc cacaaagttt
3661 ccttaaagac gattcaatag acaataaggt cttaacgcgt tctgataaaa atcgtggtaa
3721 atgcgataac gttccaagtg aagaagtagt caaaaagatg aaaaactatt ggagacaact
3781 tctaaacgcc aagttaatca ctcaacgtaa gtttgataat ttaacgaaag ctgaacgtgg
3841 aggtttgagt gaacttgata aagctggttt tatcaaacgc caattggttg aaactcgcgc
3901 catcactaag catgtggcac aaattttgga tagtcgcatg aatactaaat acgatgaaaa
3961 tgataaactt attcgagagg ttaaagtgat taccttaaaa tctaaattag tttctgactt
4021 ccgaaaagat ttccaattct ataaagtacg tgagattaac aattaccatc atgcccatga
4081 tgcgtatcta aatgccgtcg ttggaactgc tttgattaag aaatatccaa aacttgaatc
4141 ggagtttgtc tatggtgatt ataaagttta tgatgttcgt aaaatgattg ctaagtctga
4201 gcaagaaata ggcaaagcaa ccgcaaaata tttcttttac tctaatatca tgaacttctt
4261 caaaacagaa attacacttg caaatggaga gattcgcaaa cgccctctaa tcgaaactaa
4321 tggggaaact ggagaaattg tctgggataa agggcgagat tttgccacag tgcgcaaagt
4381 attgtccatg ccccaagtca atattgtcaa gaaaacagaa gtacagacag gcggattctc
4441 caaggagtca attttaccaa aaagaaattc ggacaagctt attgctcgta aaaaagactg
4501 ggatccaaaa aaatatggtg gttttgatag tccaacggta gcttattcag tcctagtggt
4561 tgctaaggtg gaaaaaggga aatcgaagaa gttaaaatcc gttaaagagt tactagggat
4621 cacaattatg gaaagaagtt cctttgaaaa aaatccgatt gactttttag aagctaaagg
4681 atataaggaa gttaaaaaag acttaatcat taaactacct aaatatagtc tttttgagtt
4741 agaaaacggt cgtaaacgga tgctggctag tgccggagaa ttacaaaaag gaaatgagct
4801 ggctctgcca agcaaatatg tgaatttttt atatttagct agtcattatg aaaagttgaa
4861 gggtagtcca gaagataacg aacaaaaaca attgtttgtg gagcagcata agcattattt
4921 agatgagatt attgagcaaa tcagtgaatt ttctaagcgt gttattttag cagatgccaa
4981 tttagataaa gttcttagtg catataacaa acatagagac aaaccaatac gtgaacaagc
5041 agaaaatatt attcatttat ttacgttgac gaatcttgga gctcccgctg cttttaaata
5101 ttttgataca acaattgatc gtaaacgata tacgtctaca aaagaagttt tagatgccac
5161 tcttatccat caatccatca ctggtcttta tgaaacacgc attgatttga gtcagctagg
5221 aggtgactaa taacattgga agtggataac ggatccgcga tcgcggcgcg ccacctggtg
5281 gccggccggt accacgcgtg cgctctgatc cggctgctaa caaagcccga aaggaagctg
5341 agttggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc tctaaacggg
5401 tcttgagggg ttttttgctg aaaggaggaa ctatatccgg atatccacag gacgggtgtg
5461 gtcgccatga tcgcgtagtc gatagtggct ccaagtagcg aagcgagcag gactgggcgg
5521 cggccaaagc ggtcggacag tgctccgaga acgggtgcgc atagaaattg catcaacgca
5581 tatagcgcta gcagcacgcc atagtgactg gcgatgctgt cggaatggac gatatcccgc
5641 aagaggcccg gcagtaccgg cataaccaag cctatgccta cagcatccag ggtgacggtg
5701 ccgaggatga cgatgagcgc attgttagat ttcatacacg gtgcctgact gcgttagcaa
5761 tttaactgtg ataaactacc gcattaaagc ttatcgatga taagctgtca aacatgagaa
5821 ttcttgaaga cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat
5881 aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
5941 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
6001 gcttcaataa cattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat
6061 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt
6121 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag
6181 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa
6241 agttctgcta tgtggcgcgg tattatcccg tgttgacgcc gggcaagagc aactcggtcg
6301 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct
6361 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac
6421 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca
6481 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
6541 accaaacgac gagcgtgaca ccacgatgcc tgcagcaatg gcaacaacgt tgcgcaaact
6601 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc
6661 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga
6721 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg
6781 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
6841 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca
6901 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
6961 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
7021 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
7081 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
7141 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
7201 tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
7261 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
7321 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
7381 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
7441 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
7501 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
7561 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
7621 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
7681 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
7741 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg
7801 cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca
7861 tctgtgcggt atttcacacc gcaatggtgc actctcagta caatctgctc tgatgccgca
7921 tagttaagcc agtatacact ccgctatcgc tacgtgactg ggtcatggct gcgccccgac
7981 acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca
8041 gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga
8101 aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat tcacagatgt
8161 ctgcctgttc atccgcgtcc agctcgttga gtttctccag aagcgttaat gtctggcttc
8221 tgataaagcg ggccatgtta agggcggttt tttcctgttt ggtcactgat gcctccgtgt
8281 aagggggatt tctgttcatg ggggtaatga taccgatgaa acgagagagg atgctcacga
8341 tacgggttac tgatgatgaa catgcccggt tactggaacg ttgtgagggt aaacaactgg
8401 cggtatggat gcggcgggac cagagaaaaa tcactcaggg tcaatgccag cgcttcgtta
8461 atacagatgt aggtgttcca cagggtagcc agcagcatcc tgcgatgcag atccggaaca
8521 taatggtgca gggcgctgac ttccgcgttt ccagacttta cgaaacacgg aaaccgaaga
8581 ccattcatgt tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt cacgttcgct
8641 cgcgtatcgg tgattcattc tgctaaccag taaggcaacc ccgccagcct agccgggtcc
8701 tcaacgacag gagcacgatc atgcgcaccc gtggccagga cccaacgctg cccgagatgc
8761 gccgcgtgcg gctgctggag atggcggacg cgatggatat gttctgccaa gggttggttt
8821 gcgcattcac agttctccgc aagaattgat tggctccaat tcttggagtg gtgaatccgt
8881 tagcgaggtg ccgccggctt ccattcaggt cgaggtggcc cggctccatg caccgcgacg
8941 caacgcgggg aggcagacaa ggtatagggc ggcgcctaca atccatgcca acccgttcca
9001 tgtgctcgcc gaggcggcat aaatcgccgt gacgatcagc ggtccaatga tcgaagttag
9061 gctggtaaga gccgcgagcg atccttgaag ctgtccctga tggtcgtcat ctacctgcct
9121 ggacagcatg gcctgcaacg cgggcatccc gatgccgccg gaagcgagaa gaatcataat
9181 ggggaaggcc atccagcctc gcgtcgcgaa cgccagcaag acgtagccca gcgcgtcggc
9241 cgccatgccg gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg gaccagtgac
9301 gaaggcttga gcgagggcgt gcaagattcc gaataccgca agcgacaggc cgatcatcgt
9361 cgcgctccag cgaaagcggt cctcgccgaa aatgacccag agcgctgccg gcacctgtcc
9421 tacgagttgc atgataaaga agacagtcat aagtgcggcg acgatagtca tgccccgcgc
9481 ccaccggaag gagctgactg ggttgaaggc tctcaagggc atcggtcgac gctctccctt
9541 atgcgactcc tgcattagga agcagcccag tagtaggttg aggccgttga gcaccgccgc
9601 cgcaaggaat ggtgcatgca aggagatggc gcccaacagt cccccggcca cggggcctgc
9661 caccataccc acgccgaaac aagcgctcat gagcccgaag tggcgagccc gatcttcccc
9721 atcggtgatg tcggcgatat aggcgccagc aaccgcacct gtggcgccgg tgatgccggc
9781 cacgatgcgt ccggcgtaga ggatcgagat ctcgatcccg cgaaattaat acgactcact
9841 atagggagac cacaacggtt tccctctagt gccggctccg gagagctctt taattaagcg
9901 gccgccctgc aggactcgag ttctagaaat aattttgttt aactttaaga aggagatata
9961 catatgaaat cttctcacca tcaccatcac catcaccatc accatggttc ttctatgaaa
10021 atcgaagaag gtaaactggt aatctggatt aacggcgata aaggctataa cgg
//
