| 质粒名称: | pNICKclos1.0 |
|---|---|
| 出品公司: | 爱迪基因 |
| 目录编号: | 73639 |
| 存储实验室: | Sheng Yang |
|
载体骨架基本信息 |
|
| 载体名称: | pIMP1-ptb |
| 载体出品公司: | -- |
| 载体抗性: | 氨苄青霉素 |
| 空载体大小: | 4806 bp |
| 完整质粒大小: | -- |
| 载体修饰: | -- |
| 载体类型: | E、 大肠杆菌梭状芽孢杆菌穿梭载体 |
| 筛选标记: | 梭状芽孢杆菌红霉素 |
| 高拷贝/低拷贝: | 低拷贝 |
| 生长条件: | 37 ℃ |
|
插入基因信息 |
|
| 插入基因名称: | Cas9 nickase |
| 物种来源: | S. pygogenes |
| 插入大小: | -- |
| 基因突变: | D10A |
| GenBank编号 | -- |
| 启动子 | ptb |
| 标签/融合蛋白: | -- |
| 克隆方法: | -- |
| 5' 克隆位点: | -- |
| 3' 克隆位点: | -- |
| 5' 测序引物 | SpCas9N-R TACGAGCTGTCCGTTTGAGA |
| 3' 测序引物 | -- |
|
文献引用方法 |
|
| 材料和方法部分: | pNICKclos1.0 was a gift from Sheng Yang (Addgene plasmid # 73639 ; http://n2t.net/addgene:73639 ; RRID:Addgene_73639) |
| 参考文献部分: | CRISPR-based genome editing and expression control systems in Clostridium acetobutylicum and Clostridium beijerinckii. Li Q, Chen J, Minton NP, Zhang Y, Wen Z, Liu J, Yang H, Zeng Z, Ren X, Yang J, Gu Y, Jiang W, Jiang Y, Yang S. Biotechnol J. 2016 May 23. doi: 10.1002/biot.201600053. 10.1002/biot.201600053 PubMed 27213844 |
买家导航
pNICKclos1.0载体质粒基本信息
pNICKclos1.0质粒图谱载体图谱和pNICKclos1.0载体序列质粒序列多克隆位点信息
pNICKclos1.0质粒载体简介
pNICKclos1.0质粒序列载体序列
LOCUS pNICKclos1.0 11082 bp ds-DNA circular SYN 06-AUG-2018
DEFINITION Genome editing for gene pyrE (CAC-002) in Clostridium acetobutylicum
ATCC 824.
ACCESSION .
VERSION .
KEYWORDS pNICKclos1.0
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 11082)
AUTHORS Li Q, Chen J, Minton NP, Zhang Y, Wen Z, Liu J, Yang H, Zeng Z, Ren
X, Yang J, Gu Y, Jiang W, Jiang Y, Yang S
TITLE CRISPR-based genome editing and expression control systems in
Clostridium acetobutylicum and Clostridium beijerinckii.
JOURNAL Biotechnol J. 2016 May 23. doi: 10.1002/biot.201600053.
PUBMED 27213844
REFERENCE 2 (bases 1 to 11082)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Aug 6, 2018 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..11082
/organism="synthetic DNA construct"
/mol_type="other DNA"
promoter 14..48
/label=J23119(SpeI) promoter
/note="bacterial promoter (Registry of Standard Biological
Parts BBa_J23119) modified to end with an SpeI site"
misc_RNA 69..144
/label=gRNA scaffold
/note="guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system"
CDS 2304..6410
/codon_start=1
/product="nickase mutant of the Cas9 endonuclease from the
Streptococcus pyogenes Type II CRISPR/Cas system"
/label=Cas9(D10A)
/note="generates RNA-guided single strand nicks in DNA due
to the D10A mutation in the RuvC catalytic domain"
/translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
DATLIHQSITGLYETRIDLSQLGGD"
primer_bind complement(6422..6439)
/label=M13 Forward
/note="In lacZ gene. Also called M13-F20 or M13 (-21)
Forward"
primer_bind complement(6422..6438)
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(6431..6453)
/label=M13/pUC Forward
/note="In lacZ gene"
primer_bind complement(6647..6666)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 6766..6788
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
primer_bind complement(6826..6844)
/label=pBRforEco
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
promoter 6912..7016
/gene="bla"
/label=AmpR promoter
CDS 7017..7877
/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(7235..7254)
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
rep_origin 8048..8636
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 8537..8556
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
primer_bind 8790..8807
/label=L4440
/note="L4440 vector, forward primer"
protein_bind 8924..8945
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 8960..8990
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 8998..9014
/label=lac operator
/bound_moiety="lac repressor encoded by lacI"
/note="The lac repressor binds to the lac operator to
inhibit transcription in E. coli. This inhibition can be
relieved by adding lactose or
isopropyl-beta-D-thiogalactopyranoside (IPTG)."
primer_bind 9003..9025
/label=M13/pUC Reverse
/note="In lacZ gene"
primer_bind 9022..9038
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind 9022..9038
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
ORIGIN
1 agcttggctg cagttgacag ctagctcagt cctaggtata atactagtta tacttaaatc
61 acaggctggt tttagagcta gaaatagcaa gttaaaataa ggctagtccg ttatcaactt
121 gaaaaagtgg caccgagtcg gtgctttttt gcggccgcat tcttatgaaa attcaagtgc
181 tctaactgta atttttccag gaataaatta tggtcatgaa aagcctcttc tgtattatgc
241 aaggaaagca gctgcacaga ataatataga tattctatgc atcaactata agaataagat
301 ttcttgggag gacataggaa aacaaagtat tgaagatgta gctattgaaa taaagggcat
361 tataaaaaat gttattgaaa aaaagtatga taacatttat tttttaggga aaagtgttgg
421 aacagaaata gctggatgta catcttctaa attaaatata gataatataa agtttcttta
481 cttaacacct attgaagaga ctttaaagta tatagttgac agaaagtgca ttgtggtgtc
541 aggcactaag gatgaatttt ttaaaagtga atgtataaat aaaatcagag aaaataaaaa
601 tgttaaattg atgttgtttg agggagctaa tcattctctt gagattaagg atgatattat
661 aagaagttta agaattttag ataacgtagc acaattgtat ttggacttct ttaaataaaa
721 acatggaggg ctaaaaatgg aacaatacaa acaagaattt atagaattta tggtggagag
781 taatgtactt acctttgggg atttcataac taaaagcggc agaagaacac cattttttat
841 aaatacaggt aactacaaga caggtaatca attaaataag ttggctaagt tttatgctaa
901 agcaatatat gataattttg gagatgattt tgatatttta tttgggcctg catataaagg
961 aataccttta agtgtttcag tagctatggc acttgataat atttatggaa ttaatgcagc
1021 ttattgttca aatagaaaag aagttaaaga tcacggtgat aagggaatac ttcttggagc
1081 aaagcttgaa gaaggagaca gagttataat tgtagaagat gtcacaacag ctggtacatc
1141 agtatacgaa acaatgccga tatcaggttg atgtaaaggg aatcataata tcagtggata
1201 gaatggaaag aggtaaggga gataagagtg ccttaactga acttaaagaa aagtttggat
1261 ttaaaacatg ttctattgtt actatggaag aggtagtaga atatttgtat aagaaaaata
1321 tcaatggcaa agtaatcata gatgataaaa tgaaagatag aattaatgag tactataaag
1381 agtatggagt aaaatagtaa aataaatgtg cctcaactta gatgttaagg cacatttatt
1441 ttatatatta ttcatgtttt gaaacatttt tatcttttgt gtattttacg tgtagtaatt
1501 tgtgagcaag tccttcacct ggttttccaa agtagctatc atacatttta ataatagctg
1561 gattatcatg tgactttctc tttgaaagaa catttttatc ttggttgtat aatactgatg
1621 ctcttagttt tctgtaatca acattttctc tatcaagagc atttacgtga ggttgacctc
1681 caccatttat acatccacca gggcaagcca ttacttctat aaagtgatat tgtttttcgt
1741 tcatttttcc agatttcata aactcgaaga agttagaagc accatttata acagcaacgt
1801 ttagtttatt tccagcaatt tcaacttccg cttcttttat gcctttaaag cctcttactt
1861 cagtgtaatc aacattttca agttctttat tttcagcaaa gtctttagct gatcttattg
1921 cagcttccat aacgccaccg gttgcaccaa agatagctcc agcaccactg taagtaccca
1981 tagcaggatc aacttcacca tcttcaagat ctgcaaattt aatttttgca tctttaatca
2041 tttttgcaag ctctcttgta gttaaggatg catcaatatc tcttaagctg ttagtttcca
2101 tgaaaggaat atctgcttca tattttttat cattacaagg catgatagta actgtataaa
2161 catcctcgag ctgcagtata aaatataaat aattttctaa aaaacttaac ttcatgtgaa
2221 aagtttgtta aaatataaat gagcacgtta atcatttaac atagataatt aaatagtaaa
2281 agggagtgtc gaggatcccc gggatggata agaaatactc aataggctta gctatcggca
2341 caaatagcgt cggatgggcg gtgatcactg atgaatataa ggttccgtct aaaaagttca
2401 aggttctggg aaatacagac cgccacagta tcaaaaaaaa tcttataggg gctcttttat
2461 ttgacagtgg agagacagcg gaagcgactc gtctcaaacg gacagctcgt agaaggtata
2521 cacgtcggaa gaatcgtatt tgttatctac aggagatttt ttcaaatgag atggcgaaag
2581 tagatgatag tttctttcat cgacttgaag agtctttttt ggtggaagaa gacaagaagc
2641 atgaacgtca tcctattttt ggaaatatag tagatgaagt tgcttatcat gagaaatatc
2701 caactatcta tcatctgcga aaaaaattgg tagattctac tgataaagcg gatttgcgct
2761 taatctattt ggccttagcg catatgatta agtttcgtgg tcattttttg attgagggag
2821 atttaaatcc tgataatagt gatgtggaca aactatttat ccagttggta caaacctaca
2881 atcaattatt tgaagaaaac cctattaacg caagtggagt agatgctaaa gcgattcttt
2941 ctgcacgatt gagtaaatca agacgattag aaaatctcat tgctcagctc cccggtgaga
3001 agaaaaatgg cttatttggg aatctcattg ctttgtcatt gggtttgacc cctaatttta
3061 aatcaaattt tgatttggca gaagatgcta aattacagct ttcaaaagat acttacgatg
3121 atgatttaga taatttattg gcgcaaattg gagatcaata tgctgatttg tttttggcag
3181 ctaagaattt atcagatgct attttacttt cagatatcct aagagtaaat actgaaataa
3241 ctaaggctcc cctatcagct tcaatgatta aacgctacga tgaacatcat caagacttga
3301 ctcttttaaa agctttagtt cgacaacaac ttccagaaaa gtataaagaa atcttttttg
3361 atcaatcaaa aaacggatat gcaggttata ttgatggggg agctagccaa gaagaatttt
3421 ataaatttat caaaccaatt ttagaaaaaa tggatggtac tgaggaatta ttggtgaaac
3481 taaatcgtga agatttgctg cgcaagcaac ggacctttga caacggctct attccccatc
3541 aaattcactt gggtgagctg catgctattt tgagaagaca agaagacttt tatccatttt
3601 taaaagacaa tcgtgagaag attgaaaaaa tcttgacttt tcgaattcct tattatgttg
3661 gtccattggc gcgtggcaat agtcgttttg catggatgac tcggaagtct gaagaaacaa
3721 ttaccccatg gaattttgaa gaagttgtcg ataaaggtgc ttcagctcaa tcatttattg
3781 aacgcatgac aaactttgat aaaaatcttc caaatgaaaa agtactacca aaacatagtt
3841 tgctttatga gtattttacg gtttataacg aattgacaaa ggtcaaatat gttactgaag
3901 gaatgcgaaa accagcattt ctttcaggtg aacagaagaa agccattgtt gatttactct
3961 tcaaaacaaa tcgaaaagta accgttaagc aattaaaaga agattatttc aaaaaaatag
4021 aatgttttga tagtgttgaa atttcaggag ttgaagatag atttaatgct tcattaggta
4081 cctaccatga tttgctaaaa attattaaag ataaagattt tttggataat gaagaaaatg
4141 aagatatctt agaggatatt gttttaacat tgaccttatt tgaagatagg gagatgattg
4201 aggaaagact taaaacatat gctcacctct ttgatgataa ggtgatgaaa cagcttaaac
4261 gtcgccgtta tactggttgg ggacgtttgt ctcgaaaatt gattaatggt attagggata
4321 agcaatctgg caaaacaata ttagattttt tgaaatcaga tggttttgcc aatcgcaatt
4381 ttatgcagct gatccatgat gatagtttga catttaaaga agacattcaa aaagcacaag
4441 tgtctggaca aggcgatagt ttacatgaac atattgcaaa tttagctggt agccctgcta
4501 ttaaaaaagg tattttacag actgtaaaag ttgttgatga attggtcaaa gtaatggggc
4561 ggcataagcc agaaaatatc gttattgaaa tggcacgtga aaatcagaca actcaaaagg
4621 gccagaaaaa ttcgcgagag cgtatgaaac gaatcgaaga aggtatcaaa gaattaggaa
4681 gtcagattct taaagagcat cctgttgaaa atactcaatt gcaaaatgaa aagctctatc
4741 tctattatct ccaaaatgga agagacatgt atgtggacca agaattagat attaatcgtt
4801 taagtgatta tgatgtcgat cacattgttc cacaaagttt ccttaaagac gattcaatag
4861 acaataaggt cttaacgcgt tctgataaaa atcgtggtaa atcggataac gttccaagtg
4921 aagaagtagt caaaaagatg aaaaactatt ggagacaact tctaaacgcc aagttaatca
4981 ctcaacgtaa gtttgataat ttaacgaaag ctgaacgtgg aggtttgagt gaacttgata
5041 aagctggttt tatcaaacgc caattggttg aaactcgcca aatcactaag catgtggcac
5101 aaattttgga tagtcgcatg aatactaaat acgatgaaaa tgataaactt attcgagagg
5161 ttaaagtgat taccttaaaa tctaaattag tttctgactt ccgaaaagat ttccaattct
5221 ataaagtacg tgagattaac aattaccatc atgcccatga tgcgtatcta aatgccgtcg
5281 ttggaactgc tttgattaag aaatatccaa aacttgaatc ggagtttgtc tatggtgatt
5341 ataaagttta tgatgttcgt aaaatgattg ctaagtctga gcaagaaata ggcaaagcaa
5401 ccgcaaaata tttcttttac tctaatatca tgaacttctt caaaacagaa attacacttg
5461 caaatggaga gattcgcaaa cgccctctaa tcgaaactaa tggggaaact ggagaaattg
5521 tctgggataa agggcgagat tttgccacag tgcgcaaagt attgtccatg ccccaagtca
5581 atattgtcaa gaaaacagaa gtacagacag gcggattctc caaggagtca attttaccaa
5641 aaagaaattc ggacaagctt attgctcgta aaaaagactg ggatccaaaa aaatatggtg
5701 gttttgatag tccaacggta gcttattcag tcctagtggt tgctaaggtg gaaaaaggga
5761 aatcgaagaa gttaaaatcc gttaaagagt tactagggat cacaattatg gaaagaagtt
5821 cctttgaaaa aaatccgatt gactttttag aagctaaagg atataaggaa gttaaaaaag
5881 acttaatcat taaactacct aaatatagtc tttttgagtt agaaaacggt cgtaaacgga
5941 tgctggctag tgccggagaa ttacaaaaag gaaatgagct ggctctgcca agcaaatatg
6001 tgaatttttt atatttagct agtcattatg aaaagttgaa gggtagtcca gaagataacg
6061 aacaaaaaca attgtttgtg gagcagcata agcattattt agatgagatt attgagcaaa
6121 tcagtgaatt ttctaagcgt gttattttag cagatgccaa tttagataaa gttcttagtg
6181 catataacaa acatagagac aaaccaatac gtgaacaagc agaaaatatt attcatttat
6241 ttacgttgac gaatcttgga gctcccgctg cttttaaata ttttgataca acaattgatc
6301 gtaaacgata tacgtctaca aaagaagttt tagatgccac tcttatccat caatccatca
6361 ctggtcttta tgaaacacgc attgatttga gtcagctagg aggtgactga cccgggaatt
6421 cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc caacttaatc
6481 gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc
6541 gcccttccca acagttgcgc agcctgaatg gcgaatggcg cctgatgcgg tattttctcc
6601 ttacgcatct gtgcggtatt tcacaccgca tatggtgcac tctcagtaca atctgctctg
6661 atgccgcata gttaagccag ccccgacacc cgccaacacc cgctgacgcg ccctgacggg
6721 cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt
6781 gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc
6841 tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc
6901 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc
6961 cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga
7021 gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt
7081 ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag
7141 tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag
7201 aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta
7261 ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg
7321 agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca
7381 gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag
7441 gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc
7501 gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg
7561 tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc
7621 ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg
7681 cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg
7741 gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga
7801 cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac
7861 tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa
7921 aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca
7981 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
8041 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
8101 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
8161 ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc
8221 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
8281 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
8341 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
8401 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
8461 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
8521 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
8581 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
8641 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct
8701 ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata
8761 ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc
8821 gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg
8881 acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg agttagctca
8941 ctcattaggc accccaggct ttacacttta tgcttccggc tcgtatgttg tgtggaattg
9001 tgagcggata acaatttcac acaggaaaca gctatgacca tgattacgcc aagctttggc
9061 taacacacac gccattccaa ccaatagttt tctcggcata aagccatgct ctgacgctta
9121 aatgcactaa tgccttaaaa aaacattaaa gtctaacaca ctagacttat ttacttcgta
9181 attaagtcgt taaaccgtgt gctctacgac caaaagtata aaacctttaa gaactttctt
9241 ttttcttgta aaaaaagaaa ctagataaat ctctcatatc ttttattcaa taatcgcatc
9301 agattgcagt ataaatttaa cgatcactca tcatgttcat atttatcaga gctcgtgcta
9361 taattatact aattttataa ggaggaaaaa ataaagaggg ttataatgaa cgagaaaaat
9421 ataaaacaca gtcaaaactt tattacttca aaacataata tagataaaat aatgacaaat
9481 ataagattaa atgaacatga taatatcttt gaaatcggct caggaaaagg gcattttacc
9541 cttgaattag tacagaggtg taatttcgta actgccattg aaatagacca taaattatgc
9601 aaaactacag aaaataaact tgttgatcac gataatttcc aagttttaaa caaggatata
9661 ttgcagttta aatttcctaa aaaccaatcc tataaaatat ttggtaatat accttataac
9721 ataagtacgg atataatacg caaaattgtt tttgatagta tagctgatga gatttattta
9781 atcgtggaat acgggtttgc taaaagatta ttaaatacaa aacgctcatt ggcattattt
9841 ttaatggcag aagttgatat ttctatatta agtatggttc caagagaata ttttcatcct
9901 aaacctaaag tgaatagctc acttatcaga ttaaatagaa aaaaatcaag aatatcacac
9961 aaagataaac agaagtataa ttatttcgtt atgaaatggg ttaacaaaga atacaagaaa
10021 atatttacaa aaaatcaatt taacaattcc ttaaaacatg caggaattga cgatttaaac
10081 aatattagct ttgaacaatt cttatctctt ttcaatagct ataaattatt taataagtaa
10141 gttaagggat gcataaactg catcccttaa cttgtttttc gtgtacctat tttttgtgaa
10201 tcgattatgt cttttgcgca ttcacttctt ttctatataa atatgagcga agcgaataag
10261 cgtcggaaaa gcagcaaaaa gtttcctttt tgctgttgga gcatgggggt tcagggggtg
10321 cagtatctga cgtcaatgcc gagcgaaagc gagccgaagg gtagcattta cgttagataa
10381 ccccctgata tgctccgacg ctttatatag aaaagaagat tcaactaggt aaaatcttaa
10441 tataggttga gatgataagg tttataagga atttgtttgt tctaattttt cactcatttt
10501 gttctaattt cttttaacaa atgttctttt ttttttagaa cagttatgat atagttagaa
10561 tagtttaaaa taaggagtga gaaaaagatg aaagaaagat atggaacagt ctataaaggc
10621 tctcagaggc tcatagacga agaaagtgga gaagtcatag aggtagacaa gttataccgt
10681 aaacaaacgt ctggtaactt cgtaaaggca tatatagtgc aattaataag tatgttagat
10741 atgattggcg gaaaaaaact taaaatcgtt aactatatcc tagataatgt ccacttaagt
10801 aacaatacaa tgatagctac aacaagagaa atagcaaaag ctacaggaac aagtctacaa
10861 acagtaataa caacacttaa aatcttagaa gaaggaaata ttataaaaag aaaaactgga
10921 gtattaatgt taaaccctga actactaatg agaggcgacg accaaaaaca aaaatacctc
10981 ttactcgaat ttgggaactt tgagcaagag gcaaatgaaa tagattgacc tcccaataac
11041 accacgtagt tattgggagg tcaatctatg aaaatgcgat ta
//
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