| 质粒名称: | pEcCas |
|---|---|
| 出品公司: | 爱迪基因 |
| 目录编号: | 73227 |
| 存储实验室: | Sheng Yang |
|
载体骨架基本信息 |
|
| 载体名称: | pWM119 |
| 载体出品公司: | -- |
| 载体抗性: | 卡那霉素 |
| 空载体大小: | -- |
| 完整质粒大小: | -- |
| 载体修饰: | 用Kan替换Amp |
| 载体类型: | 细菌表达，CRISPR；红色重组酶 |
| 筛选标记: | -- |
| 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,pCASsac |
| 物种来源: | S. pyogenes MGAS5005 |
| 插入大小: | -- |
| 基因突变: | -- |
| GenBank编号 | -- |
| 启动子 | native promoter |
| 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 | pENTR-R |
| 3' 测序引物 | AraC-F-New |
|
文献引用方法 |
|
| 材料和方法部分: | pEcCas was a gift from Sheng Yang (Addgene plasmid # 73227 ; http://n2t.net/addgene:73227 ; RRID:Addgene_73227) |
| 参考文献部分: | A modified pCas/pTargetF system for CRISPR-Cas9-assisted genome editing in Escherichia coli. Li , Q i, Sun, Bingbing, Chen, Jun, Zhang, Yiwen, Jiang, Y u Jiang, Yang, Sheng. Acta Biochimica et Biophysica Sinica, 2021 10.1093/abbs/gmab036 |
买家导航
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 pEcCas 14605 bp ds-DNA circular SYN 12-MAY-2021
DEFINITION Constitutive expression of cas9 and inducible expression of lambda
RED and sgRNA.
ACCESSION .
VERSION .
KEYWORDS pEcCas
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 14605)
AUTHORS Li , Q i, Sun, Bingbing, Chen, Jun, Zhang, Yiwen, Jiang, Y u Jiang,
Yang, Sheng
TITLE A modified pCas/pTargetF system for CRISPR-Cas9-assisted genome
editing in Escherichia coli
JOURNAL Acta Biochimica et Biophysica Sinica, 2021
REFERENCE 2 (bases 1 to 14605)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..14605
/organism="synthetic DNA construct"
/mol_type="other DNA"
CDS complement(107..1057)
/codon_start=1
/gene="rep101"
/product="RepA protein needed for replication with the
pSC101 origin"
/label=Rep101
/translation="MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRKE
RTVSFTYNQYAQMMNISRENAYGVLAKATRELMTRTVEIRNPLVKGFEIFQWTNYAKFS
SEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHKANI
EISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTLIFQV
ELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLRNGLRKTLHDALTAKIQLTSFEAKF
LSDMQSKYDLNGSFSWLTQKQRTTLENILAKYGRI"
rep_origin 1105..1327
/label=pSC101 ori
/note="low-copy replication origin that requires the Rep101
protein"
CDS complement(1832..2647)
/codon_start=1
/gene="aph(3')-Ia"
/product="aminoglycoside phosphotransferase"
/label=KanR
/note="confers resistance to kanamycin in bacteria or G418
(Geneticin(R)) in eukaryotes"
/translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
primer_bind 2555..2574
/label=Kan-R
/note="Kanamycin resistance gene, reverse primer"
primer_bind 2639..2658
/label=pENTR-R
/note="pENTR vectors, reverse primer"
CDS 2995..7101
/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="MDKKYSIGLDIGTNSVGWAVITDDYKVPSKKFKVLGNTDRHSIKK
NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
DATLIHQSITGLYETRIDLSQLGGD"
CDS complement(7231..8109)
/codon_start=1
/gene="araC"
/product="L-arabinose regulatory protein"
/label=araC
/translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
EKVNDVAVKLS"
promoter 8136..8420
/gene="araBAD"
/label=araBAD promoter
/note="promoter of the L-arabinose operon of E. coli; the
araC regulatory gene is transcribed in the opposite
direction (Guzman et al., 1995)"
primer_bind 8343..8362
/label=pBAD Forward
/note="For vectors with E. coli araBAD promoter, forward
primer"
RBS 8447..8455
/label=Shine-Dalgarno sequence
/note="full consensus sequence for ribosome-binding sites
upstream of start codons in E. coli; complementary to a
region in the 3' end of the 16S rRNA (Chen et al., 1994)"
CDS 8464..8880
/codon_start=1
/gene="gam"
/product="inhibitor of the host RecBCD nuclease in the
lambda Red system"
/label=Gam
/note="protects the double-stranded DNA substrate for
recombination"
/translation="MDINTETEIKQKHSLTPFPVFLISPAFRGRYFHSYFRSSAMNAYY
IQDRLEAQSWARHYQQLAREEKEAELADDMEKGLPQHLFESLCIDHLQRHGASKKSITR
AFDDDVEFQERMAEHIRYMVETIAHHQVDIDSEV"
CDS 8886..9671
/codon_start=1
/gene="bet"
/product="single-stranded DNA binding recombinase in the
lambda Red system"
/label=Beta
/note="mediates pairing of a single-stranded DNA with a
complementary target"
/translation="MSTALATLAGKLAERVGMDSVDPQELITTLRQTAFKGDASDAQFI
ALLIVANQYGLNPWTKEIYAFPDKQNGIVPVVGVDGWSRIINENQQFDGMDFEQDNESC
TCRIYRKDRNHPICVTEWMDECRREPFKTREGREITGPWQSHPKRMLRHKAMIQCARLA
FGFAGIYDKDEAERIVENTAYTAERQPERDITPVNDETMQEINTLLIALDKTWDDDLLP
LCSQIFRRDIRASSELTQAEAVKALGFLKQKAAEQKVAA"
CDS 9668..10348
/codon_start=1
/gene="exo"
/product="5' to 3' double-stranded DNA exonuclease in the
lambda Red system"
/label=Exo
/note="creates 3' single-stranded DNA tails"
/translation="MTPDIILQRTGIDVRAVEQGDDAWHKLRLGVITASEVHNVIAKPR
SGKKWPDMKMSYFHTLLAEVCTGVAPEVNAKALAWGKQYENDARTLFEFTSGVNVTESP
IIYRDESMRTACSPDGLCSDGNGLELKCPFTSRDFMKFRLGGFEAIKSAYMAQVQYSMW
VTRKNAWYFANYDPRMKREGLHYVVIERDEKYMASFDEIVPEFIEKMDEALAEIGFVFG
EQWR"
terminator 10349..10593
/label=-lambda tL3 terminator
/note="transcription terminator tL3 from phage lambda"
CDS 10930..12351
/codon_start=1
/gene="Bacillus subtilis sacB"
/product="secreted levansucrase that renders bacterial
growth sensitive to sucrose"
/label=SacB
/note="negative selection marker"
/translation="MNIKKFAKQATVLTFTTALLAGGATQAFAKETNQKPYKETYGISH
ITRHDMLQIPEQQKNEKYQVPEFDSSTIKNISSAKGLDVWDSWPLQNADGTVANYHGYH
IVFALAGDPKNADDTSIYMFYQKVGETSIDSWKNAGRVFKDSDKFDANDSILKDQTQEW
SGSATFTSDGKIRLFYTDFSGKHYGKQTLTTAQVNVSASDSSLNINGVEDYKSIFDGDG
KTYQNVQQFIDEGNYSSGDNHTLRDPHYVEDKGHKYLVFEANTGTEDGYQGEESLFNKA
YYGKSTSFFRQESQKLLQSDKKRTAELANGALGMIELNDDYTLKKVMKPLIASNTVTDE
IERANVFKMNGKWYLFTDSRGSKMTIDGITSNDIYMLGYVSNSLTGPYKPLNKTGLVLK
MDLDPNDVTFTYSHFAVPQAKGNNVVITSYMTNRGFYADKQSTFAPSFLLNIKGKKTSV
VKDSILEQGQLTVNK"
misc_RNA complement(12474..12549)
/label=gRNA scaffold
/note="guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system"
promoter complement(12571..12689)
/label=rhaB promoter
/note="promoter of the E. coli rhaBAD operon, conferring
tight induction with L-rhamnose and repression with
D-glucose in the presence of RhaR and RhaS (Giacalone et
al., 2006)"
CDS 12835..13671
/codon_start=1
/gene="E. coli rhaS"
/product="positive regulator of the rhaB promoter
"
/label=rhaS
/note="activated by L-rhamnose"
/translation="MTVLHSVDFFPSGNASVAIEPRLPQADFPEHHHDFHEIVIVEHGT
GIHVFNGQPYTITGGTVCFVRDHDRHLYEHTDNLCLTNVLYRSPDRFQFLAGLNQLLPQ
ELDGQYPSHWRVNHSVLQQVRQLVAQMEQQEGENDLPSTASREILFMQLLLLLRKSSLQ
ENLENSASRLNLLLAWLEDHFADEVNWDAVADQFSLSLRTLHRQLKQQTGLTPQRYLNR
LRLMKARHLLRHSEASVTDIAYRCGFSDSNHFSTLFRREFNWSPRDIRQGRDGFLQ"
CDS 13745..14593
/codon_start=1
/gene="E. coli rhaR"
/product="transcriptional activator of rhaR and rhaS"
/label=rhaR
/note="needed for rhamnose-dependent activation of the rhaB
promoter"
/translation="MAHQLKLLKDDFFASDQQAVAVADRYPQDVFAEHTHDFCELVIVW
RGNGLHVLNDRPYRITRGDLFYIHADDKHSYASVNDLVLQNIIYCPERLKLNLDWQGAI
PGFNASAGQPHWRLGSMGMAQARQVIGQLEHESSQHVPFANEMAELLFGQLVMLLNRHR
YTSDSLPPTSSETLLDKLITRLAASLKSPFALDKFCDEASCSERVLRQQFRQQTGMTIN
QYLRQVRVCHAQYLLQHSRLLISDISTECGFEDSNYFSVVFTRETGMTPSQWRHLNSQK
D"
ORIGIN
1 catgggtatg gacagttttc cctttgatat gtaacggtga acagttgttc tacttttgtt
61 tgttagtctt gatgcttcac tgatagatac aagagccata agaacctcag atccttccgt
121 atttagccag tatgttctct agtgtggttc gttgtttttg cgtgagccat gagaacgaac
181 cattgagatc atacttactt tgcatgtcac tcaaaaattt tgcctcaaaa ctggtgagct
241 gaatttttgc agttaaagca tcgtgtagtg tttttcttag tccgttacgt aggtaggaat
301 ctgatgtaat ggttgttggt attttgtcac cattcatttt tatctggttg ttctcaagtt
361 cggttacgag atccatttgt ctatctagtt caacttggaa aatcaacgta tcagtcgggc
421 ggcctcgctt atcaaccacc aatttcatat tgctgtaagt gtttaaatct ttacttattg
481 gtttcaaaac ccattggtta agccttttaa actcatggta gttattttca agcattaaca
541 tgaacttaaa ttcatcaagg ctaatctcta tatttgcctt gtgagttttc ttttgtgtta
601 gttcttttaa taaccactca taaatcctca tagagtattt gttttcaaaa gacttaacat
661 gttccagatt atattttatg aattttttta actggaaaag ataaggcaat atctcttcac
721 taaaaactaa ttctaatttt tcgcttgaga acttggcata gtttgtccac tggaaaatct
781 caaagccttt aaccaaagga ttcctgattt ccacagttct cgtcatcagc tctctggttg
841 ctttagctaa tacaccataa gcattttccc tactgatgtt catcatctga gcgtattggt
901 tataagtgaa cgataccgtc cgttctttcc ttgtagggtt ttcaatcgtg gggttgagta
961 gtgccacaca gcataaaatt agcttggttt catgctccgt taagtcatag cgactaatcg
1021 ctagttcatt tgctttgaaa acaactaatt cagacataca tctcaattgg tctaggtgat
1081 tttaatcact ataccaattg agatgggcta gtcaatgata attactagtc cttttccttt
1141 gagttgtggg tatctgtaaa ttctgctaga cctttgctgg aaaacttgta aattctgcta
1201 gaccctctgt aaattccgct agacctttgt gtgttttttt tgtttatatt caagtggtta
1261 taatttatag aataaagaaa gaataaaaaa agataaaaag aatagatccc agccctgtgt
1321 ataactcact actttagtca gttccgcagt attacaaaag gatgtcgcaa acgctgtttg
1381 ctcctctaca aaacagacct taaaacccta aaggcttaag tagcaccctc gcaagctcgg
1441 ttgcggccgc aatcgggcaa atcgctgaat attccttttg tctccgacca tcaggcacct
1501 gagtcgctgt ctttttcgtg acattcagtt cgctgcgctc acggctctgg cagtgaatgg
1561 gggtaaatgg cactacaggc gccttttatg gattcatgca aggaaactac ccataataca
1621 agaaaagccc gtcacgggct tctcagggcg ttttatggcg ggtctgctat gtggtgctat
1681 ctgacttttt gctgttcagc agttcctgcc ctctgatttt ccagtctgac cacttcggat
1741 tatcccgtga caggtcattc agactggcta atgcacccag taaggcagcg gtatcatcaa
1801 cccactgcag caaccaatta accaattctg attagaaaaa ctcatcgagc atcaaatgaa
1861 actgcaattt attcatatca ggattatcaa taccatattt ttgaaaaagc cgtttctgta
1921 atgaaggaga aaactcaccg aggcagttcc ataggatggc aagatcctgg tatcggtctg
1981 cgattccgac tcgtccaaca tcaatacaac ctattaattt cccctcgtca aaaataaggt
2041 tatcaagtga gaaatcacca tgagtgacga ctgaatccgg tgagaatggc aaaagcttat
2101 gcatttcttt ccagacttgt tcaacaggcc agccattacg ctcgtcatca aaatcactcg
2161 catcaaccaa accgttattc attcgtgatt gcgcctgagc gagacgaaat acgcgatcgc
2221 tgttaaaagg acaattacaa acaggaatcg aatgcaaccg gcgcaggaac actgccagcg
2281 catcaacaat attttcacct gaatcaggat attcttctaa tacctggaat gctgttttcc
2341 cggggatcgc agtggtgagt aaccatgcat catcaggagt acggataaaa tgcttgatgg
2401 tcggaagagg cataaattcc gtcagccagt ttagtctgac catctcatct gtaacatcat
2461 tggcaacgct acctttgcca tgtttcagaa acaactctgg cgcatcgggc ttcccataca
2521 atcgatagat tgtcgcacct gattgcccga cattatcgcg agcccattta tacccatata
2581 aatcagcatc catgttggaa tttaatcgcg gcctcgagca agacgtttcc cgttgaatat
2641 ggctcataac accccttgta ttactgttta tgtaagcaga cagttttatt gttcatgatg
2701 atatattttt atcttgtgca atgtaacatc agagattttg agacacaacg tggctttccc
2761 tgcagctgaa caatacttaa tactatagaa tgataacaaa ataaactact ttttaaaaga
2821 attttgtgtt ataatctatt tattattaag tattgggtaa tattttttga agagatattt
2881 tgaaaaagaa aaattaaagc atattaaact aatttcggag gtcattaaaa ctattattga
2941 aatcatcaaa ctcattatgg atttaattta aactttttat tttaggaggc aaaaatggat
3001 aagaaatact caataggctt agatatcggc acaaatagcg tcggatgggc ggtgatcact
3061 gatgattata aggttccgtc taaaaagttc aaggttctgg gaaatacaga ccgccacagt
3121 atcaaaaaaa atcttatagg ggctctttta tttgacagtg gagagacagc ggaagcgact
3181 cgtctcaaac ggacagctcg tagaaggtat acacgtcgga agaatcgtat ttgttatcta
3241 caggagattt tttcaaatga gatggcgaaa gtagatgata gtttctttca tcgacttgaa
3301 gagtcttttt tggtggaaga agacaagaag catgaacgtc atcctatttt tggaaatata
3361 gtagatgaag ttgcttatca tgagaaatat ccaactatct atcatctgcg aaaaaaattg
3421 gtagattcta ctgataaagc ggatttgcgc ttaatctatt tggccttagc gcatatgatt
3481 aagtttcgtg gtcatttttt gattgaggga gatttaaatc ctgataatag tgatgtggac
3541 aaactattta tccagttggt acaaacctac aatcaattat ttgaagaaaa ccctattaac
3601 gcaagtggag tagatgctaa agcgattctt tctgcacgat tgagtaaatc aagacgatta
3661 gaaaatctca ttgctcagct ccccggtgag aagaaaaatg gcttatttgg gaatctcatt
3721 gctttgtcat tgggtttgac ccctaatttt aaatcaaatt ttgatttggc agaagatgct
3781 aaattacagc tttcaaaaga tacttacgat gatgatttag ataatttatt ggcgcaaatt
3841 ggagatcaat atgctgattt gtttttggca gctaagaatt tatcagatgc tattttactt
3901 tcagatatcc taagagtaaa tactgaaata actaaggctc ccctatcagc ttcaatgatt
3961 aaacgctacg atgaacatca tcaagacttg actcttttaa aagctttagt tcgacaacaa
4021 cttccagaaa agtataaaga aatctttttt gatcaatcaa aaaacggata tgcaggttat
4081 attgatgggg gagctagcca agaagaattt tataaattta tcaaaccaat tttagaaaaa
4141 atggatggta ctgaggaatt attggtgaaa ctaaatcgtg aagatttgct gcgcaagcaa
4201 cggacctttg acaacggctc tattccccat caaattcact tgggtgagct gcatgctatt
4261 ttgagaagac aagaagactt ttatccattt ttaaaagaca atcgtgagaa gattgaaaaa
4321 atcttgactt ttcgaattcc ttattatgtt ggtccattgg cgcgtggcaa tagtcgtttt
4381 gcatggatga ctcggaagtc tgaagaaaca attaccccat ggaattttga agaagttgtc
4441 gataaaggtg cttcagctca atcatttatt gaacgcatga caaactttga taaaaatctt
4501 ccaaatgaaa aagtactacc aaaacatagt ttgctttatg agtattttac ggtttataac
4561 gaattgacaa aggtcaaata tgttactgaa ggaatgcgaa aaccagcatt tctttcaggt
4621 gaacagaaga aagccattgt tgatttactc ttcaaaacaa atcgaaaagt aaccgttaag
4681 caattaaaag aagattattt caaaaaaata gaatgttttg atagtgttga aatttcagga
4741 gttgaagata gatttaatgc ttcattaggt acctaccatg atttgctaaa aattattaaa
4801 gataaagatt ttttggataa tgaagaaaat gaagatatct tagaggatat tgttttaaca
4861 ttgaccttat ttgaagatag ggagatgatt gaggaaagac ttaaaacata tgctcacctc
4921 tttgatgata aggtgatgaa acagcttaaa cgtcgccgtt atactggttg gggacgtttg
4981 tctcgaaaat tgattaatgg tattagggat aagcaatctg gcaaaacaat attagatttt
5041 ttgaaatcag atggttttgc caatcgcaat tttatgcagc tgatccatga tgatagtttg
5101 acatttaaag aagacattca aaaagcacaa gtgtctggac aaggcgatag tttacatgaa
5161 catattgcaa atttagctgg tagccctgct attaaaaaag gtattttaca gactgtaaaa
5221 gttgttgatg aattggtcaa agtaatgggg cggcataagc cagaaaatat cgttattgaa
5281 atggcacgtg aaaatcagac aactcaaaag ggccagaaaa attcgcgaga gcgtatgaaa
5341 cgaatcgaag aaggtatcaa agaattagga agtcagattc ttaaagagca tcctgttgaa
5401 aatactcaat tgcaaaatga aaagctctat ctctattatc tccaaaatgg aagagacatg
5461 tatgtggacc aagaattaga tattaatcgt ttaagtgatt atgatgtcga tcacattgtt
5521 ccacaaagtt tccttaaaga cgattcaata gacaataagg tcttaacgcg ttctgataaa
5581 aatcgtggta aatcggataa cgttccaagt gaagaagtag tcaaaaagat gaaaaactat
5641 tggagacaac ttctaaacgc caagttaatc actcaacgta agtttgataa tttaacgaaa
5701 gctgaacgtg gaggtttgag tgaacttgat aaagctggtt ttatcaaacg ccaattggtt
5761 gaaactcgcc aaatcactaa gcatgtggca caaattttgg atagtcgcat gaatactaaa
5821 tacgatgaaa atgataaact tattcgagag gttaaagtga ttaccttaaa atctaaatta
5881 gtttctgact tccgaaaaga tttccaattc tataaagtac gtgagattaa caattaccat
5941 catgcccatg atgcgtatct aaatgccgtc gttggaactg ctttgattaa gaaatatcca
6001 aaacttgaat cggagtttgt ctatggtgat tataaagttt atgatgttcg taaaatgatt
6061 gctaagtctg agcaagaaat aggcaaagca accgcaaaat atttctttta ctctaatatc
6121 atgaacttct tcaaaacaga aattacactt gcaaatggag agattcgcaa acgccctcta
6181 atcgaaacta atggggaaac tggagaaatt gtctgggata aagggcgaga ttttgccaca
6241 gtgcgcaaag tattgtccat gccccaagtc aatattgtca agaaaacaga agtacagaca
6301 ggcggattct ccaaggagtc aattttacca aaaagaaatt cggacaagct tattgctcgt
6361 aaaaaagact gggatccaaa aaaatatggt ggttttgata gtccaacggt agcttattca
6421 gtcctagtgg ttgctaaggt ggaaaaaggg aaatcgaaga agttaaaatc cgttaaagag
6481 ttactaggga tcacaattat ggaaagaagt tcctttgaaa aaaatccgat tgacttttta
6541 gaagctaaag gatataagga agttaaaaaa gacttaatca ttaaactacc taaatatagt
6601 ctttttgagt tagaaaacgg tcgtaaacgg atgctggcta gtgccggaga attacaaaaa
6661 ggaaatgagc tggctctgcc aagcaaatat gtgaattttt tatatttagc tagtcattat
6721 gaaaagttga agggtagtcc agaagataac gaacaaaaac aattgtttgt ggagcagcat
6781 aagcattatt tagatgagat tattgagcaa atcagtgaat tttctaagcg tgttatttta
6841 gcagatgcca atttagataa agttcttagt gcatataaca aacatagaga caaaccaata
6901 cgtgaacaag cagaaaatat tattcattta tttacgttga cgaatcttgg agctcccgct
6961 gcttttaaat attttgatac aacaattgat cgtaaacgat atacgtctac aaaagaagtt
7021 ttagatgcca ctcttatcca tcaatccatc actggtcttt atgaaacacg cattgatttg
7081 agtcagctag gaggtgactg aagtatattt tagatgaaga ttatttctta atctagacat
7141 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt
7201 tccccgaaaa gtgccacctg catcgattta ttatgacaac ttgacggcta catcattcac
7261 tttttcttca caaccggcac ggaactcgct cgggctggcc ccggtgcatt ttttaaatac
7321 ccgcgagaaa tagagttgat cgtcaaaacc aacattgcga ccgacggtgg cgataggcat
7381 ccgggtggtg ctcaaaagca gcttcgcctg gctgatacgt tggtcctcgc gccagcttaa
7441 gacgctaatc cctaactgct ggcggaaaag atgtgacaga cgcgacggcg acaagcaaac
7501 atgctgtgcg acgctggcga tatcaaaatt gctgtctgcc aggtgatcgc tgatgtactg
7561 acaagcctcg cgtacccgat tatccatcgg tggatggagc gactcgttaa tcgcttccat
7621 gcgccgcagt aacaattgct caagcagatt tatcgccagc agctccgaat agcgcccttc
7681 cccttgcccg gcgttaatga tttgcccaaa caggtcgctg aaatgcggct ggtgcgcttc
7741 atccgggcga aagaaccccg tattggcaaa tattgacggc cagttaagcc attcatgcca
7801 gtaggcgcgc ggacgaaagt aaacccactg gtgataccat tcgcgagcct ccggatgacg
7861 accgtagtga tgaatctctc ctggcgggaa cagcaaaata tcacccggtc ggcaaacaaa
7921 ttctcgtccc tgatttttca ccaccccctg accgcgaatg gtgagattga gaatataacc
7981 tttcattccc agcggtcggt cgataaaaaa atcgagataa ccgttggcct caatcggcgt
8041 taaacccgcc accagatggg cattaaacga gtatcccggc agcaggggat cattttgcgc
8101 ttcagccata cttttcatac tcccgccatt cagagaagaa accaattgtc catattgcat
8161 cagacattgc cgtcactgcg tcttttactg gctcttctcg ctaaccaaac cggtaacccc
8221 gcttattaaa agcattctgt aacaaagcgg gaccaaagcc atgacaaaaa cgcgtaacaa
8281 aagtgtctat aatcacggca gaaaagtcca cattgattat ttgcacggcg tcacactttg
8341 ctatgccata gcatttttat ccataagatt agcggatcct acctgacgct ttttatcgca
8401 actctctact gtttctccat acccgttttt ttgggaattc gagctctaag gaggttataa
8461 aaaatggata ttaatactga aactgagatc aagcaaaagc attcactaac cccctttcct
8521 gttttcctaa tcagcccggc atttcgcggg cgatattttc acagctattt caggagttca
8581 gccatgaacg cttattacat tcaggatcgt cttgaggctc agagctgggc gcgtcactac
8641 cagcagctcg cccgtgaaga gaaagaggca gaactggcag acgacatgga aaaaggcctg
8701 ccccagcacc tgtttgaatc gctatgcatc gatcatttgc aacgccacgg ggccagcaaa
8761 aaatccatta cccgtgcgtt tgatgacgat gttgagtttc aggagcgcat ggcagaacac
8821 atccggtaca tggttgaaac cattgctcac caccaggttg atattgattc agaggtataa
8881 aacgaatgag tactgcactc gcaacgctgg ctgggaagct ggctgaacgt gtcggcatgg
8941 attctgtcga cccacaggaa ctgatcacca ctcttcgcca gacggcattt aaaggtgatg
9001 ccagcgatgc gcagttcatc gcattactga tcgttgccaa ccagtacggc cttaatccgt
9061 ggacgaaaga aatttacgcc tttcctgata agcagaatgg catcgttccg gtggtgggcg
9121 ttgatggctg gtcccgcatc atcaatgaaa accagcagtt tgatggcatg gactttgagc
9181 aggacaatga atcctgtaca tgccggattt accgcaagga ccgtaatcat ccgatctgcg
9241 ttaccgaatg gatggatgaa tgccgccgcg aaccattcaa aactcgcgaa ggcagagaaa
9301 tcacggggcc gtggcagtcg catcccaaac ggatgttacg tcataaagcc atgattcagt
9361 gtgcccgtct ggccttcgga tttgctggta tctatgacaa ggatgaagcc gagcgcattg
9421 tcgaaaatac tgcatacact gcagaacgtc agccggaacg cgacatcact ccggttaacg
9481 atgaaaccat gcaggagatt aacactctgc tgatcgccct ggataaaaca tgggatgacg
9541 acttattgcc gctctgttcc cagatatttc gccgcgacat tcgtgcatcg tcagaactga
9601 cacaggccga agcagtaaaa gctcttggat tcctgaaaca gaaagccgca gagcagaagg
9661 tggcagcatg acaccggaca ttatcctgca gcgtaccggg atcgatgtga gagctgtcga
9721 acagggggat gatgcgtggc acaaattacg gctcggcgtc atcaccgctt cagaagttca
9781 caacgtgata gcaaaacccc gctccggaaa gaagtggcct gacatgaaaa tgtcctactt
9841 ccacaccctg cttgctgagg tttgcaccgg tgtggctccg gaagttaacg ctaaagcact
9901 ggcctgggga aaacagtacg agaacgacgc cagaaccctg tttgaattca cttccggcgt
9961 gaatgttact gaatccccga tcatctatcg cgacgaaagt atgcgtaccg cctgctctcc
10021 cgatggttta tgcagtgacg gcaacggcct tgaactgaaa tgcccgttta cctcccggga
10081 tttcatgaag ttccggctcg gtggtttcga ggccataaag tcagcttaca tggcccaggt
10141 gcagtacagc atgtgggtga cgcgaaaaaa tgcctggtac tttgccaact atgacccgcg
10201 tatgaagcgt gaaggcctgc attatgtcgt gattgagcgg gatgaaaagt acatggcgag
10261 ttttgacgag atcgtgccgg agttcatcga aaaaatggac gaggcactgg ctgaaattgg
10321 ttttgtattt ggggagcaat ggcgatgacg catcctcacg ataatatccg ggtaggcgca
10381 atcactttcg tctactccgt tacaaagcga ggctgggtat ttcccggcct ttctgttatc
10441 cgaaatccac tgaaagcaca gcggctggct gaggagataa ataataaacg aggggctgta
10501 tgcacaaagc atcttctgtt gagttaagaa cgagtatcga gatggcacat agccttgctc
10561 aaattggaat caggtttgtg ccaataccag tagaaacaga cgaagaatcc atgggtatgg
10621 acagatcctc tttaggcccg tagtctgcaa atccttttat gattttctat caaacaaaag
10681 aggaaaatag accagttgca atccaaacga gagtctaata gaatgaggtc gaaaagtaaa
10741 tcgcgcgggt ttgttactga taaagcaggc aagacctaaa atgtgtaaag ggcaaagtgt
10801 atactttggc gtcacccctt acatatttta ggtctttttt tattgtgcgt aactaacttg
10861 ccatcttcaa acaggagggc tggaagaagc agaccgctaa cacagtacat aaaaaaggag
10921 acatgaacga tgaacatcaa aaagtttgca aaacaagcaa cagtattaac ctttactacc
10981 gcactgctgg caggaggcgc aactcaagcg tttgcgaaag aaacgaacca aaagccatat
11041 aaggaaacat acggcatttc ccatattaca cgccatgata tgctgcaaat ccctgaacag
11101 caaaaaaatg aaaaatatca agttcctgaa ttcgattcgt ccacaattaa aaatatctct
11161 tctgcaaaag gcctggacgt ttgggacagc tggccattac aaaacgctga cggcactgtc
11221 gcaaactatc acggctacca catcgtcttt gcattagccg gagatcctaa aaatgcggat
11281 gacacatcga tttacatgtt ctatcaaaaa gtcggcgaaa cttctattga cagctggaaa
11341 aacgctggcc gcgtctttaa agacagcgac aaattcgatg caaatgattc tatcctaaaa
11401 gaccaaacac aagaatggtc aggttcagcc acatttacat ctgacggaaa aatccgttta
11461 ttctacactg atttctccgg taaacattac ggcaaacaaa cactgacaac tgcacaagtt
11521 aacgtatcag catcagacag ctctttgaac atcaacggtg tagaggatta taaatcaatc
11581 tttgacggtg acggaaaaac gtatcaaaat gtacagcagt tcatcgatga aggcaactac
11641 agctcaggcg acaaccatac gctgagagat cctcactacg tagaagataa aggccacaaa
11701 tacttagtat ttgaagcaaa cactggaact gaagatggct accaaggcga agaatcttta
11761 tttaacaaag catactatgg caaaagcaca tcattcttcc gtcaagaaag tcaaaaactt
11821 ctgcaaagcg ataaaaaacg cacggctgag ttagcaaacg gcgctctcgg tatgattgag
11881 ctaaacgatg attacacact gaaaaaagtg atgaaaccgc tgattgcatc taacacagta
11941 acagatgaaa ttgaacgcgc gaacgtcttt aaaatgaacg gcaaatggta cctgttcact
12001 gactcccgcg gatcaaaaat gacgattgac ggcattacgt ctaacgatat ttacatgctt
12061 ggttatgttt ctaattcttt aactggccca tacaagccgc tgaacaaaac tggccttgtg
12121 ttaaaaatgg atcttgatcc taacgatgta acctttactt actcacactt cgctgtacct
12181 caagcgaaag gaaacaatgt cgtgattaca agctatatga caaacagagg attctacgca
12241 gacaaacaat caacgtttgc gccaagcttc ctgctgaaca tcaaaggcaa gaaaacatct
12301 gttgtcaaag acagcatcct tgaacaagga caattaacag ttaacaaata aaaacgcaaa
12361 agaaaatgcc gatatcctat tggcattttc ttttatttct tatcaacata aaggtgaatc
12421 ccatatgaac tatataaaag caggcaaatg gctaaccgta ttcctcaaaa aaagcaccga
12481 ctcggtgcca ctttttcaag ttgataacgg actagcctta ttttaacttg ctatttctag
12541 ctctaaaacg gttggactca agacgatagt tacgaccagt ctaaaaagcg cctgaattcg
12601 cgaccttctc gttactgaca ggaaaatggg ccattggcaa ccagggaaag atgaacgtga
12661 tgatgttcac aatttgctga attgtggtga tgtgatgctc accgcatttc ctgaaaattc
12721 acgctgtatc ttgaaaaatc gacgtttttt acgtggtttt ccgtcgaaaa tttaaggtaa
12781 gaacctgacc tcgtgattac tatttcgccg tgttgacgac atcaggaggc cagtatgacc
12841 gtattacata gtgtggattt ttttccgtct ggtaacgcgt ccgtggcgat agaaccccgg
12901 ctcccgcagg cggattttcc tgaacatcat catgattttc atgaaattgt gattgtcgaa
12961 catggcacgg gtattcatgt gtttaatggg cagccctata ccatcaccgg tggcacggtc
13021 tgtttcgtac gcgatcatga tcggcatctg tatgaacata ccgataatct gtgtctgacc
13081 aatgtgctgt atcgctcgcc ggatcgattt cagtttctcg ccgggctgaa tcagttgctg
13141 ccacaagagc tggatgggca gtatccgtct cactggcgcg ttaaccacag cgtattgcag
13201 caggtgcgac agctggttgc acagatggaa cagcaggaag gggaaaatga tttaccctcg
13261 accgccagtc gcgagatctt gtttatgcaa ttactgctct tgctgcgtaa aagcagtttg
13321 caggagaacc tggaaaacag cgcatcacgt ctcaacttgc ttctggcctg gctggaggac
13381 cattttgccg atgaggtgaa ttgggatgcc gtggcggatc aattttctct ttcactgcgt
13441 acgctacatc ggcagcttaa gcagcaaacg ggactgacgc ctcagcgata cctgaaccgc
13501 ctgcgactga tgaaagcccg acatctgcta cgccacagcg aggccagcgt tactgacatc
13561 gcctatcgct gtggattcag cgacagtaac cacttttcga cgctttttcg ccgagagttt
13621 aactggtcac cgcgtgatat tcgccaggga cgggatggct ttctgcaata acgcgaatct
13681 tctcaacgta tttgtacgcc atattgcgaa taatcaactt cgttctctgg ccgaggtagc
13741 cacggtggcg catcagttaa aacttctcaa agatgatttt tttgccagcg accagcaggc
13801 agtcgctgtg gctgaccgtt atccgcaaga tgtctttgct gaacatacac atgatttttg
13861 tgagctggtg attgtctggc gcggtaatgg cctgcatgta ctcaacgatc gcccttatcg
13921 cattacccgt ggcgatctct tttacattca tgctgacgat aaacactcct acgcttccgt
13981 taacgatctg gttttgcaga atattattta ttgcccggag cgtctgaagc tgaatcttga
14041 ctggcagggg gcgattccgg gatttaacgc cagcgcaggg caaccacact ggcgcttagg
14101 tagcatgggg atggcgcagg cgcggcaggt tatcggtcag cttgagcatg aaagtagtca
14161 gcatgtgccg tttgctaacg aaatggctga gttgctgttc gggcagttgg tgatgttgct
14221 gaatcgccat cgttacacca gtgattcgtt gccgccaaca tccagcgaaa cgttgctgga
14281 taagctgatt acccggctgg cggctagcct gaaaagtccc tttgcgctgg ataaattttg
14341 tgatgaggca tcgtgcagtg agcgcgtttt gcgtcagcaa tttcgccagc agactggaat
14401 gaccatcaat caatatctgc gacaggtcag agtgtgtcat gcgcaatatc ttctccagca
14461 tagccgcctg ttaatcagtg atatttcgac cgaatgtggc tttgaagata gtaactattt
14521 ttcggtggtg tttacccggg aaaccgggat gacgcccagc cagtggcgtc atctcaattc
14581 gcagaaagat taattcgcca tgccc
//
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