| 质粒名称: | pSHS306 - Bacterial expression plasmid for SpCas9, HNH FRET variant |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 87350 |
| 存储实验室: | Jennifer Doudna |
|
载体骨架基本信息 |
|
| 载体名称: | pCT10 |
| 载体出品公司: | -- |
| 空载体大小: | 6000 bp |
| 完整质粒大小: | 10000 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: | Cas9 |
| 物种来源: | S. pyogenes |
| 插入大小: | 4100 bp |
| 基因突变: | C80S/S355C/C574S/S867C |
| GenBank编号 | -- |
| 启动子: | T7 |
| 标签/融合蛋白: | His-MBP (N terminal on backbone) |
| 克隆方法: | -- |
| 5’克隆位点: | -- |
| 3’克隆位点: | -- |
| 5’测序引物 | CGCAGACTAATTCGAGCTCG |
| 3’测序引物 | GAAAGGAAGCTGAGTTGGCTG |
|
文献引用方法 |
|
| 材料和方法部分: | pSHS306 - Bacterial expression plasmid for SpCas9, HNH FRET variant was a gift from Jennifer Doudna (Addgene plasmid # 87350 ; http://n2t.net/addgene:87350 ; RRID:Addgene_87350) |
| 参考文献部分: | Conformational control of DNA target cleavage by CRISPR-Cas9. Sternberg SH, LaFrance B, Kaplan M, Doudna JA. Nature. 2015 Nov 5;527(7576):110-3. doi: 10.1038/nature15544. Epub 2015 Oct 28. 10.1038/nature15544 PubMed 26524520 |
pSHS306 - Bacterial expression plasmid for SpCas9, HNH FRET
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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 pSHS306 - Bacterial expression plasmid for SpCas9, HNH FRET variant 10073 bp ds-DNA circular SYN 12-MAY-2021
DEFINITION S867C/S355C in cysteine-free (C80S/C574S) S. pyogenes Cas9
expression plasmid, HNH-1 FRET construct.
ACCESSION .
VERSION .
KEYWORDS pSHS306 - Bacterial expression plasmid for SpCas9, HNH FRET va...
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 10073)
AUTHORS Sternberg SH, LaFrance B, Kaplan M, Doudna JA
TITLE Conformational control of DNA target cleavage by CRISPR-Cas9.
JOURNAL Nature. 2015 Nov 5;527(7576):110-3. doi: 10.1038/nature15544. Epub
2015 Oct 28.
PUBMED 26524520
REFERENCE 2 (bases 1 to 10073)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 12, 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 63..82
/label=pBRrevBam
/note="pBR322 vectors, tet region, downstream of BamHI,
reverse primer"
primer_bind 166..185
/label=T7
/note="T7 promoter, forward primer"
promoter 166..184
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
RBS 273..295
/note="efficient ribosome binding site from bacteriophage
T7 gene 10 (Olins and Rangwala, 1989)"
CDS 315..344
/codon_start=1
/product="10xHis affinity tag"
/label=10xHis
/translation="HHHHHHHHHH"
CDS 354..1454
/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 1428..1451
/label=MBP-F
/note="Maltose binding protein, forward primer"
CDS 1509..1529
/codon_start=1
/product="tobacco etch virus (TEV) protease recognition and
cleavage site"
/label=TEV site
/translation="ENLYFQS"
CDS 1536..5642
/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
NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIESFD
SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKCDNVP
SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
DATLIHQSITGLYETRIDLSQLGGD"
primer_bind complement(5768..5786)
/label=T7 Term
/note="T7 terminator, reverse primer"
terminator 5782..5829
/label=T7 terminator
/note="transcription terminator for bacteriophage T7 RNA
polymerase"
promoter complement(6195..6223)
/label=tet promoter
/note="E. coli promoter for tetracycline efflux protein
gene"
primer_bind complement(6250..6268)
/label=pBRforEco
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
promoter 6336..6440
/gene="bla"
/label=AmpR promoter
CDS 6441..7301
/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(6659..6678)
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
rep_origin 7472..8060
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 7961..7980
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
primer_bind 8214..8231
/label=L4440
/note="L4440 vector, forward primer"
misc_feature 8246..8385
/label=bom
/note="basis of mobility region from pBR322"
primer_bind complement(8311..8330)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 8471..8493
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
CDS complement(8487..8678)
/codon_start=1
/gene="rop"
/product="Rop protein, which maintains plasmids at low copy
number"
/label=rop
/translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
DELYRSCLARFGDDGENL"
ORIGIN
1 accataccca cgccgaaaca agcgctcatg agcccgaagt ggcgagcccg atcttcccca
61 tcggtgatgt cggcgatata ggcgccagca accgcacctg tggcgccggt gatgccggcc
121 acgatgcgtc cggcgtagag gatcgagatc tcgatcccgc gaaattaata cgactcacta
181 tagggagacc acaacggttt ccctctagtg ccggctccgg agagctcttt aattaagcgg
241 ccgccctgca ggactcgagt tctagaaata attttgttta actttaagaa ggagatatac
301 atatgaaatc ttctcaccat caccatcacc atcaccatca ccatggttct tctatgaaaa
361 tcgaagaagg taaactggta atctggatta acggcgataa aggctataac ggtctcgctg
421 aagtcggtaa gaaattcgag aaagataccg gaattaaagt caccgttgag catccggata
481 aactggaaga gaaattccca caggttgcgg caactggcga tggccctgac attatcttct
541 gggcacacga ccgctttggt ggctacgctc aatctggcct gttggctgaa atcaccccgg
601 acaaagcgtt ccaggacaag ctgtatccgt ttacctggga tgccgtacgt tacaacggca
661 agctgattgc ttacccgatc gctgttgaag cgttatcgct gatttataac aaagatctgc
721 tgccgaaccc gccaaaaacc tgggaagaga tcccggcgct ggataaagaa ctgaaagcga
781 aaggtaagag cgcgctgatg ttcaacctgc aagaaccgta cttcacctgg ccgctgattg
841 ctgctgacgg gggttatgcg ttcaagtatg aaaacggcaa gtacgacatt aaagacgtgg
901 gcgtggataa cgctggcgca aaagcgggtc tgaccttcct ggttgacctg attaaaaaca
961 aacacatgaa tgcagacacc gattactcca tcgcagaagc tgcctttaat aaaggcgaaa
1021 cagcgatgac catcaacggc ccgtgggcat ggtccaacat cgacaccagc aaagtgaatt
1081 atggtgtaac ggtactgccg accttcaagg gtcaaccatc caaaccgttc gttggcgtgc
1141 tgagcgcagg tattaacgcc gccagtccga acaaagagct ggcaaaagag ttcctcgaaa
1201 actatctgct gactgatgaa ggtctggaag cggttaataa agacaaaccg ctgggtgccg
1261 tagcgctgaa gtcttacgag gaagagttgg cgaaagatcc acgtattgcc gccactatgg
1321 aaaacgccca gaaaggtgaa atcatgccga acatcccgca gatgtccgct ttctggtatg
1381 ccgtgcgtac tgcggtgatc aacgccgcca gcggtcgtca gactgtcgat gaagccctga
1441 aagacgcgca gactaattcg agctcgaaca acaacaacaa taacaataac aacaacctcg
1501 ggatcgagga aaacctgtac ttccaatcca atgcaatgga taagaaatac tcaataggct
1561 tagatatcgg cacaaatagc gtcggatggg cggtgatcac tgatgaatat aaggttccgt
1621 ctaaaaagtt caaggttctg ggaaatacag accgccacag tatcaaaaaa aatcttatag
1681 gggctctttt atttgacagt ggagagacag cggaagcgac tcgtctcaaa cggacagctc
1741 gtagaaggta tacacgtcgg aagaatcgta tttcttatct acaggagatt ttttcaaatg
1801 agatggcgaa agtagatgat agtttctttc atcgacttga agagtctttt ttggtggaag
1861 aagacaagaa gcatgaacgt catcctattt ttggaaatat agtagatgaa gttgcttatc
1921 atgagaaata tccaactatc tatcatctgc gaaaaaaatt ggtagattct actgataaag
1981 cggatttgcg cttaatctat ttggccttag cgcatatgat taagtttcgt ggtcattttt
2041 tgattgaggg agatttaaat cctgataata gtgatgtgga caaactattt atccagttgg
2101 tacaaaccta caatcaatta tttgaagaaa accctattaa cgcaagtgga gtagatgcta
2161 aagcgattct ttctgcacga ttgagtaaat caagacgatt agaaaatctc attgctcagc
2221 tccccggtga gaagaaaaat ggcttatttg ggaatctcat tgctttgtca ttgggtttga
2281 cccctaattt taaatcaaat tttgatttgg cagaagatgc taaattacag ctttcaaaag
2341 atacttacga tgatgattta gataatttat tggcgcaaat tggagatcaa tatgctgatt
2401 tgtttttggc agctaagaat ttatcagatg ctattttact ttcagatatc ctaagagtaa
2461 atactgaaat aactaaggct cccctatcag cttcaatgat taaacgctac gatgaacatc
2521 atcaagactt gactctttta aaagctttag ttcgacaaca acttccagaa aagtataaag
2581 aaatcttttt tgatcaatgt aaaaacggat atgcaggtta tattgatggg ggagctagcc
2641 aagaagaatt ttataaattt atcaaaccaa ttttagaaaa aatggatggt actgaggaat
2701 tattggtgaa actaaatcgt gaagatttgc tgcgcaagca acggaccttt gacaacggct
2761 ctattcccca tcaaattcac ttgggtgagc tgcatgctat tttgagaaga caagaagact
2821 tttatccatt tttaaaagac aatcgtgaga agattgaaaa aatcttgact tttcgaattc
2881 cttattatgt tggtccattg gcgcgtggca atagtcgttt tgcatggatg actcggaagt
2941 ctgaagaaac aattacccca tggaattttg aagaagttgt cgataaaggt gcttcagctc
3001 aatcatttat tgaacgcatg acaaactttg ataaaaatct tccaaatgaa aaagtactac
3061 caaaacatag tttgctttat gagtatttta cggtttataa cgaattgaca aaggtcaaat
3121 atgttactga aggaatgcga aaaccagcat ttctttcagg tgaacagaag aaagccattg
3181 ttgatttact cttcaaaaca aatcgaaaag taaccgttaa gcaattaaaa gaagattatt
3241 tcaaaaaaat agaatctttt gatagtgttg aaatttcagg agttgaagat agatttaatg
3301 cttcattagg tacctaccat gatttgctaa aaattattaa agataaagat tttttggata
3361 atgaagaaaa tgaagatatc ttagaggata ttgttttaac attgacctta tttgaagata
3421 gggagatgat tgaggaaaga cttaaaacat atgctcacct ctttgatgat aaggtgatga
3481 aacagcttaa acgtcgccgt tatactggtt ggggacgttt gtctcgaaaa ttgattaatg
3541 gtattaggga taagcaatct ggcaaaacaa tattagattt tttgaaatca gatggttttg
3601 ccaatcgcaa ttttatgcag ctgatccatg atgatagttt gacatttaaa gaagacattc
3661 aaaaagcaca agtgtctgga caaggcgata gtttacatga acatattgca aatttagctg
3721 gtagccctgc tattaaaaaa ggtattttac agactgtaaa agttgttgat gaattggtca
3781 aagtaatggg gcggcataag ccagaaaata tcgttattga aatggcacgt gaaaatcaga
3841 caactcaaaa gggccagaaa aattcgcgag agcgtatgaa acgaatcgaa gaaggtatca
3901 aagaattagg aagtcagatt cttaaagagc atcctgttga aaatactcaa ttgcaaaatg
3961 aaaagctcta tctctattat ctccaaaatg gaagagacat gtatgtggac caagaattag
4021 atattaatcg tttaagtgat tatgatgtcg atcacattgt tccacaaagt ttccttaaag
4081 acgattcaat agacaataag gtcttaacgc gttctgataa aaatcgtggt aaatgcgata
4141 acgttccaag tgaagaagta gtcaaaaaga tgaaaaacta ttggagacaa cttctaaacg
4201 ccaagttaat cactcaacgt aagtttgata atttaacgaa agctgaacgt ggaggtttga
4261 gtgaacttga taaagctggt tttatcaaac gccaattggt tgaaactcgc caaatcacta
4321 agcatgtggc acaaattttg gatagtcgca tgaatactaa atacgatgaa aatgataaac
4381 ttattcgaga ggttaaagtg attaccttaa aatctaaatt agtttctgac ttccgaaaag
4441 atttccaatt ctataaagta cgtgagatta acaattacca tcatgcccat gatgcgtatc
4501 taaatgccgt cgttggaact gctttgatta agaaatatcc aaaacttgaa tcggagtttg
4561 tctatggtga ttataaagtt tatgatgttc gtaaaatgat tgctaagtct gagcaagaaa
4621 taggcaaagc aaccgcaaaa tatttctttt actctaatat catgaacttc ttcaaaacag
4681 aaattacact tgcaaatgga gagattcgca aacgccctct aatcgaaact aatggggaaa
4741 ctggagaaat tgtctgggat aaagggcgag attttgccac agtgcgcaaa gtattgtcca
4801 tgccccaagt caatattgtc aagaaaacag aagtacagac aggcggattc tccaaggagt
4861 caattttacc aaaaagaaat tcggacaagc ttattgctcg taaaaaagac tgggatccaa
4921 aaaaatatgg tggttttgat agtccaacgg tagcttattc agtcctagtg gttgctaagg
4981 tggaaaaagg gaaatcgaag aagttaaaat ccgttaaaga gttactaggg atcacaatta
5041 tggaaagaag ttcctttgaa aaaaatccga ttgacttttt agaagctaaa ggatataagg
5101 aagttaaaaa agacttaatc attaaactac ctaaatatag tctttttgag ttagaaaacg
5161 gtcgtaaacg gatgctggct agtgccggag aattacaaaa aggaaatgag ctggctctgc
5221 caagcaaata tgtgaatttt ttatatttag ctagtcatta tgaaaagttg aagggtagtc
5281 cagaagataa cgaacaaaaa caattgtttg tggagcagca taagcattat ttagatgaga
5341 ttattgagca aatcagtgaa ttttctaagc gtgttatttt agcagatgcc aatttagata
5401 aagttcttag tgcatataac aaacatagag acaaaccaat acgtgaacaa gcagaaaata
5461 ttattcattt atttacgttg acgaatcttg gagctcccgc tgcttttaaa tattttgata
5521 caacaattga tcgtaaacga tatacgtcta caaaagaagt tttagatgcc actcttatcc
5581 atcaatccat cactggtctt tatgaaacac gcattgattt gagtcagcta ggaggtgact
5641 aataacattg gaagtggata acggatccgc gatcgcggcg cgccacctgg tggccggccg
5701 gtaccacgcg tgcgctctga tccggctgct aacaaagccc gaaaggaagc tgagttggct
5761 gctgccaccg ctgagcaata actagcataa ccccttgggg cctctaaacg ggtcttgagg
5821 ggttttttgc tgaaaggagg aactatatcc ggatatccac aggacgggtg tggtcgccat
5881 gatcgcgtag tcgatagtgg ctccaagtag cgaagcgagc aggactgggc ggcggccaaa
5941 gcggtcggac agtgctccga gaacgggtgc gcatagaaat tgcatcaacg catatagcgc
6001 tagcagcacg ccatagtgac tggcgatgct gtcggaatgg acgatatccc gcaagaggcc
6061 cggcagtacc ggcataacca agcctatgcc tacagcatcc agggtgacgg tgccgaggat
6121 gacgatgagc gcattgttag atttcataca cggtgcctga ctgcgttagc aatttaactg
6181 tgataaacta ccgcattaaa gcttatcgat gataagctgt caaacatgag aattcttgaa
6241 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
6301 cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
6361 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
6421 aacattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
6481 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
6541 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
6601 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
6661 tatgtggcgc ggtattatcc cgtgttgacg ccgggcaaga gcaactcggt cgccgcatac
6721 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
6781 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
6841 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
6901 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
6961 acgagcgtga caccacgatg cctgcagcaa tggcaacaac gttgcgcaaa ctattaactg
7021 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
7081 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
7141 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct
7201 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
7261 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
7321 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
7381 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
7441 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
7501 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
7561 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc
7621 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
7681 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
7741 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
7801 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
7861 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
7921 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
7981 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
8041 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
8101 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
8161 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
8221 cagtgagcga ggaagcggaa gagcgcctga tgcggtattt tctccttacg catctgtgcg
8281 gtatttcaca ccgcaatggt gcactctcag tacaatctgc tctgatgccg catagttaag
8341 ccagtataca ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg acacccgcca
8401 acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct
8461 gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg
8521 aggcagctgc ggtaaagctc atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt
8581 tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta atgtctggct tctgataaag
8641 cgggccatgt taagggcggt tttttcctgt ttggtcactg atgcctccgt gtaaggggga
8701 tttctgttca tgggggtaat gataccgatg aaacgagaga ggatgctcac gatacgggtt
8761 actgatgatg aacatgcccg gttactggaa cgttgtgagg gtaaacaact ggcggtatgg
8821 atgcggcggg accagagaaa aatcactcag ggtcaatgcc agcgcttcgt taatacagat
8881 gtaggtgttc cacagggtag ccagcagcat cctgcgatgc agatccggaa cataatggtg
8941 cagggcgctg acttccgcgt ttccagactt tacgaaacac ggaaaccgaa gaccattcat
9001 gttgttgctc aggtcgcaga cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc
9061 ggtgattcat tctgctaacc agtaaggcaa ccccgccagc ctagccgggt cctcaacgac
9121 aggagcacga tcatgcgcac ccgtggccag gacccaacgc tgcccgagat gcgccgcgtg
9181 cggctgctgg agatggcgga cgcgatggat atgttctgcc aagggttggt ttgcgcattc
9241 acagttctcc gcaagaattg attggctcca attcttggag tggtgaatcc gttagcgagg
9301 tgccgccggc ttccattcag gtcgaggtgg cccggctcca tgcaccgcga cgcaacgcgg
9361 ggaggcagac aaggtatagg gcggcgccta caatccatgc caacccgttc catgtgctcg
9421 ccgaggcggc ataaatcgcc gtgacgatca gcggtccaat gatcgaagtt aggctggtaa
9481 gagccgcgag cgatccttga agctgtccct gatggtcgtc atctacctgc ctggacagca
9541 tggcctgcaa cgcgggcatc ccgatgccgc cggaagcgag aagaatcata atggggaagg
9601 ccatccagcc tcgcgtcgcg aacgccagca agacgtagcc cagcgcgtcg gccgccatgc
9661 cggcgataat ggcctgcttc tcgccgaaac gtttggtggc gggaccagtg acgaaggctt
9721 gagcgagggc gtgcaagatt ccgaataccg caagcgacag gccgatcatc gtcgcgctcc
9781 agcgaaagcg gtcctcgccg aaaatgaccc agagcgctgc cggcacctgt cctacgagtt
9841 gcatgataaa gaagacagtc ataagtgcgg cgacgatagt catgccccgc gcccaccgga
9901 aggagctgac tgggttgaag gctctcaagg gcatcggtcg acgctctccc ttatgcgact
9961 cctgcattag gaagcagccc agtagtaggt tgaggccgtt gagcaccgcc gccgcaagga
10021 atggtgcatg caaggagatg gcgcccaaca gtcccccggc cacggggcct gcc
//
