| 质粒名称: | pmGFP-P2A-K(AAG)20-P2A-RFP |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 190697 |
| 存储实验室: | Sheng Yang |
|
载体骨架基本信息 |
|
| 载体名称: | p15A |
| 载体出品公司: | Clontech |
| 完整质粒大小: | 10550 bp |
| 载体类型: | 细菌表达 |
| 载体抗性: | Chloramphenicol, 25 μg/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 and Lamda Red system、CRISPR-associated protein Csn1 |
| 物种来源: | Streptococcus pyogenes |
| 克隆方法: | Gibson Cloning |
| 5’测序引物 | n/a |
|
文献引用方法 |
|
| 材料和方法部分: | pEcCas-2.0 was a gift from Sheng Yang (Addgene plasmid # 190697 ; http://n2t.net/addgene:190697 ; RRID:Addgene_190697) |
| 参考文献部分: | Improving the Editing Efficiency of CRISPR-Cas9 by Reducing the Generation of Escapers Based on the Surviving Mechanism. Li Q, Sun M, Lv L, Zuo Y, Zhang S, Zhang Y, Yang S. ACS Synth Biol. 2023 Mar 3. doi: 10.1021/acssynbio.2c00619. 10.1021/acssynbio.2c00619 PubMed 36867054 |
买家导航
pEcCas-2.0载体质粒基本信息
pEcCas-2.0质粒图谱载体图谱和pEcCas-2.0载体序列质粒序列多克隆位点信息
pEcCas-2.0质粒载体简介
pEcCas-2.0质粒序列载体序列
LOCUS pEcCas-2.0 10550 bp DNA circular SYN 25-AUG-2024
DEFINITION Constitutive expressing of Cas9 and inducible expression of lambda
Red.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 10550)
AUTHORS Li Q, Sun M, Lv L, Zuo Y, Zhang S, Zhang Y, Yang S
TITLE Improving the Editing Efficiency of CRISPR-Cas9 by Reducing the
Generation of Escapers Based on the Surviving Mechanism.
JOURNAL ACS Synth Biol. 2023 Mar 3. doi: 10.1021/acssynbio.2c00619.
PUBMED 36867054
REFERENCE 2 (bases 1 to 10550)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Aug 25, 2024 from SnapGene Server 7.0.3
https://www.biofeng.com
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "ACS Synth
Biol. 2023 Mar 3. doi: 10.1021/acssynbio.2c00619."
FEATURES Location/Qualifiers
source 1..10550
/mol_type="other DNA"
/organism="synthetic DNA construct"
primer_bind complement(288..305)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(422..967)
/direction=LEFT
/label=p15A ori
/note="Plasmids containing the medium-copy-number p15A
origin of replication can be propagated in E. coli cells
that contain a second plasmid with the ColE1 origin.
"
primer_bind complement(502..521)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS 1540..5646
/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 1540..5643
/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(5776..6654)
/codon_start=1
/gene="araC"
/product="L-arabinose regulatory protein"
/label=araC
/translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
EKVNDVAVKLS"
promoter 6681..6965
/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 6888..6907
/label=pBAD Forward
/note="For vectors with E. coli araBAD promoter, forward
primer"
RBS 6992..7000
/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 7009..7425
/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 7431..8216
/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 8213..8893
/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 8894..9138
/label=lambda tL3 terminator
/note="transcription terminator tL3 from phage lambda"
primer_bind complement(9234..9253)
/label=pBRrevBam
/note="pBR322 vectors, tet region, downstream of BamHI,
reverse primer"
CDS complement(9684..10343)
/codon_start=1
/gene="cat"
/product="chloramphenicol acetyltransferase"
/label=CmR
/note="confers resistance to chloramphenicol"
/translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
primer_bind 10258..10277
/label=CAT-R
/note="Chloramphenicol resistance gene, reverse primer"
promoter complement(10344..10446)
/label=cat promoter
/note="promoter of the E. coli cat gene encoding
chloramphenicol acetyltransferase"
ORIGIN
1 ggcgcaaagt gcgtcgggtg atgctgccaa cttactgatt tagtgtatga tggtgttttt
61 gaggtgctcc agtggcttct gtttctatca gctgtccctc ctgttcagct actgacgggg
121 tggtgcgtaa cggcaaaagc accgccggac atcagcgcta gcggagtgta tactggctta
181 ctatgttggc actgatgagg gtgtcagtga agtgcttcat gtggcaggag aaaaaaggct
241 gcaccggtgc gtcagcagaa tatgtgatac aggatatatt ccgcttcctc gctcactgac
301 tcgctacgct cggtcgttcg actgcggcga gcggaaatgg cttacgaacg gggcggagat
361 ttcctggaag atgccaggaa gatacttaac agggaagtga gagggccgcg gcaaagccgt
421 ttttccatag gctccgcccc cctgacaagc atcacgaaat ctgacgctca aatcagtggt
481 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggcggc tccctcgtgc
541 gctctcctgt tcctgccttt cggtttaccg gtgtcattcc gctgttatgg ccgcgtttgt
601 ctcattccac gcctgacact cagttccggg taggcagttc gctccaagct ggactgtatg
661 cacgaacccc ccgttcagtc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
721 aacccggaaa gacatgcaaa agcaccactg gcagcagcca ctggtaattg atttagagga
781 gttagtcttg aagtcatgcg ccggttaagg ctaaactgaa aggacaagtt ttggtgactg
841 cgctcctcca agccagttac ctcggttcaa agagttggta gctcagagaa ccttcgaaaa
901 accgccctgc aaggcggttt tttcgttttc agagcaagag attacgcgca gaccaaaacg
961 atctcaagaa gatcatctta ttaatatacg tagtatctac gatacgtagt atctacgata
1021 cgtaggaggt tgagaagtca tttaataagg ccactgttaa acgtagatat atctacgcgt
1081 agatatatct acgatacgta gcggccgacg cgctgggcta cgtcttaatg ggcagcgccc
1141 acatacgcag cgatttccaa cagcttgagg cggaagcttt aatgcgggtc ctaacacccc
1201 ttgtattact gtttatgtaa gcagacagtt ttattgttca tgatgatata tttttatctt
1261 gtgcaatgta acatcagaga ttttgagaca caacgtggct ttccctgcag ctgaacaata
1321 cttaatacta tagaatgata acaaaataaa ctacttttta aaagaatttt gtgttataat
1381 ctatttatta ttaagtattg ggtaatattt tttgaagaga tattttgaaa aagaaaaatt
1441 aaagcatatt aaactaattt cggaggtcat taaaactatt attgaaatca tcaaactcat
1501 tatggattta atttaaactt tttattttag gaggcaaaaa tggataagaa atactcaata
1561 ggcttagata tcggcacaaa tagcgtcgga tgggcggtga tcactgatga ttataaggtt
1621 ccgtctaaaa agttcaaggt tctgggaaat acagaccgcc acagtatcaa aaaaaatctt
1681 ataggggctc ttttatttga cagtggagag acagcggaag cgactcgtct caaacggaca
1741 gctcgtagaa ggtatacacg tcggaagaat cgtatttgtt atctacagga gattttttca
1801 aatgagatgg cgaaagtaga tgatagtttc tttcatcgac ttgaagagtc ttttttggtg
1861 gaagaagaca agaagcatga acgtcatcct atttttggaa atatagtaga tgaagttgct
1921 tatcatgaga aatatccaac tatctatcat ctgcgaaaaa aattggtaga ttctactgat
1981 aaagcggatt tgcgcttaat ctatttggcc ttagcgcata tgattaagtt tcgtggtcat
2041 tttttgattg agggagattt aaatcctgat aatagtgatg tggacaaact atttatccag
2101 ttggtacaaa cctacaatca attatttgaa gaaaacccta ttaacgcaag tggagtagat
2161 gctaaagcga ttctttctgc acgattgagt aaatcaagac gattagaaaa tctcattgct
2221 cagctccccg gtgagaagaa aaatggctta tttgggaatc tcattgcttt gtcattgggt
2281 ttgaccccta attttaaatc aaattttgat ttggcagaag atgctaaatt acagctttca
2341 aaagatactt acgatgatga tttagataat ttattggcgc aaattggaga tcaatatgct
2401 gatttgtttt tggcagctaa gaatttatca gatgctattt tactttcaga tatcctaaga
2461 gtaaatactg aaataactaa ggctccccta tcagcttcaa tgattaaacg ctacgatgaa
2521 catcatcaag acttgactct tttaaaagct ttagttcgac aacaacttcc agaaaagtat
2581 aaagaaatct tttttgatca atcaaaaaac ggatatgcag gttatattga tgggggagct
2641 agccaagaag aattttataa atttatcaaa ccaattttag aaaaaatgga tggtactgag
2701 gaattattgg tgaaactaaa tcgtgaagat ttgctgcgca agcaacggac ctttgacaac
2761 ggctctattc cccatcaaat tcacttgggt gagctgcatg ctattttgag aagacaagaa
2821 gacttttatc catttttaaa agacaatcgt gagaagattg aaaaaatctt gacttttcga
2881 attccttatt atgttggtcc attggcgcgt ggcaatagtc gttttgcatg gatgactcgg
2941 aagtctgaag aaacaattac cccatggaat tttgaagaag ttgtcgataa aggtgcttca
3001 gctcaatcat ttattgaacg catgacaaac tttgataaaa atcttccaaa tgaaaaagta
3061 ctaccaaaac atagtttgct ttatgagtat tttacggttt ataacgaatt gacaaaggtc
3121 aaatatgtta ctgaaggaat gcgaaaacca gcatttcttt caggtgaaca gaagaaagcc
3181 attgttgatt tactcttcaa aacaaatcga aaagtaaccg ttaagcaatt aaaagaagat
3241 tatttcaaaa aaatagaatg ttttgatagt gttgaaattt caggagttga agatagattt
3301 aatgcttcat taggtaccta ccatgatttg ctaaaaatta ttaaagataa agattttttg
3361 gataatgaag aaaatgaaga tatcttagag gatattgttt taacattgac cttatttgaa
3421 gatagggaga tgattgagga aagacttaaa acatatgctc acctctttga tgataaggtg
3481 atgaaacagc ttaaacgtcg ccgttatact ggttggggac gtttgtctcg aaaattgatt
3541 aatggtatta gggataagca atctggcaaa acaatattag attttttgaa atcagatggt
3601 tttgccaatc gcaattttat gcagctgatc catgatgata gtttgacatt taaagaagac
3661 attcaaaaag cacaagtgtc tggacaaggc gatagtttac atgaacatat tgcaaattta
3721 gctggtagcc ctgctattaa aaaaggtatt ttacagactg taaaagttgt tgatgaattg
3781 gtcaaagtaa tggggcggca taagccagaa aatatcgtta ttgaaatggc acgtgaaaat
3841 cagacaactc aaaagggcca gaaaaattcg cgagagcgta tgaaacgaat cgaagaaggt
3901 atcaaagaat taggaagtca gattcttaaa gagcatcctg ttgaaaatac tcaattgcaa
3961 aatgaaaagc tctatctcta ttatctccaa aatggaagag acatgtatgt ggaccaagaa
4021 ttagatatta atcgtttaag tgattatgat gtcgatcaca ttgttccaca aagtttcctt
4081 aaagacgatt caatagacaa taaggtctta acgcgttctg ataaaaatcg tggtaaatcg
4141 gataacgttc caagtgaaga agtagtcaaa aagatgaaaa actattggag acaacttcta
4201 aacgccaagt taatcactca acgtaagttt gataatttaa cgaaagctga acgtggaggt
4261 ttgagtgaac ttgataaagc tggttttatc aaacgccaat tggttgaaac tcgccaaatc
4321 actaagcatg tggcacaaat tttggatagt cgcatgaata ctaaatacga tgaaaatgat
4381 aaacttattc gagaggttaa agtgattacc ttaaaatcta aattagtttc tgacttccga
4441 aaagatttcc aattctataa agtacgtgag attaacaatt accatcatgc ccatgatgcg
4501 tatctaaatg ccgtcgttgg aactgctttg attaagaaat atccaaaact tgaatcggag
4561 tttgtctatg gtgattataa agtttatgat gttcgtaaaa tgattgctaa gtctgagcaa
4621 gaaataggca aagcaaccgc aaaatatttc ttttactcta atatcatgaa cttcttcaaa
4681 acagaaatta cacttgcaaa tggagagatt cgcaaacgcc ctctaatcga aactaatggg
4741 gaaactggag aaattgtctg ggataaaggg cgagattttg ccacagtgcg caaagtattg
4801 tccatgcccc aagtcaatat tgtcaagaaa acagaagtac agacaggcgg attctccaag
4861 gagtcaattt taccaaaaag aaattcggac aagcttattg ctcgtaaaaa agactgggat
4921 ccaaaaaaat atggtggttt tgatagtcca acggtagctt attcagtcct agtggttgct
4981 aaggtggaaa aagggaaatc gaagaagtta aaatccgtta aagagttact agggatcaca
5041 attatggaaa gaagttcctt tgaaaaaaat ccgattgact ttttagaagc taaaggatat
5101 aaggaagtta aaaaagactt aatcattaaa ctacctaaat atagtctttt tgagttagaa
5161 aacggtcgta aacggatgct ggctagtgcc ggagaattac aaaaaggaaa tgagctggct
5221 ctgccaagca aatatgtgaa ttttttatat ttagctagtc attatgaaaa gttgaagggt
5281 agtccagaag ataacgaaca aaaacaattg tttgtggagc agcataagca ttatttagat
5341 gagattattg agcaaatcag tgaattttct aagcgtgtta ttttagcaga tgccaattta
5401 gataaagttc ttagtgcata taacaaacat agagacaaac caatacgtga acaagcagaa
5461 aatattattc atttatttac gttgacgaat cttggagctc ccgctgcttt taaatatttt
5521 gatacaacaa ttgatcgtaa acgatatacg tctacaaaag aagttttaga tgccactctt
5581 atccatcaat ccatcactgg tctttatgaa acacgcattg atttgagtca gctaggaggt
5641 gactgaagta tattttagat gaagattatt tcttaatcta gacatgagcg gatacatatt
5701 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc
5761 acctgcatcg atttattatg acaacttgac ggctacatca ttcacttttt cttcacaacc
5821 ggcacggaac tcgctcgggc tggccccggt gcatttttta aatacccgcg agaagtagag
5881 ttgatcgtca aaaccaacat tgcgaccgac ggtggcgata ggcatccggg tggtgctcaa
5941 aagcagcttc gcctggctga tacgttggtc ctcgcgccag cttaagacgc taatccctaa
6001 ctgctggcgg aaaagatgtg acagacgcga cggcgacaag caaacatgct gtgcgacgct
6061 ggcgatatca aaattgctgt ctgccaggtg atcgctgatg tactgacaag cctcgcgtac
6121 ccgattatcc atcggtggat ggagcgactc gttaatcgct tccatgcgcc gcagtaacaa
6181 ttgctcaagc agatttatcg ccagcagctc cgaatagcgc ccttcccctt gcccggcgtt
6241 aatgatttgc ccaaacaggt cgctgaaatg cggctggtgc gcttcatccg ggcgaaagaa
6301 ccccgtattg gcaaatattg acggccagtt aagccattca tgccagtagg cgcgcggacg
6361 aaagtaaacc cactggtgat accattcgcg agcctccgga tgacgaccgt agtgatgaat
6421 ctctcctggc gggaacagca aaatatcacc cggtcggcaa acaaattctc gtccctgatt
6481 tttcaccacc ccctgaccgc gaatggtgag attgagaata taacctttca ttcccagcgg
6541 tcggtcgata aaaaaatcga gataaccgtt ggcctcaatc ggcgttaaac ccgccaccag
6601 atgggcatta aacgagtatc ccggcagcag gggatcattt tgcgcttcag ccatactttt
6661 catactcccg ccattcagag aagaaaccaa ttgtccatat tgcatcagac attgccgtca
6721 ctgcgtcttt tactggctct tctcgctaac caaaccggta accccgctta ttaaaagcat
6781 tctgtaacaa agcgggacca aagccatgac aaaaacgcgt aacaaaagtg tctataatca
6841 cggcagaaaa gtccacattg attatttgca cggcgtcaca ctttgctatg ccatagcatt
6901 tttatccata agattagcgg atcctacctg acgcttttta tcgcaactct ctactgtttc
6961 tccatacccg tttttttggg aattcgagct ctaaggaggt tataaaaaat ggatattaat
7021 actgaaactg agatcaagca aaagcattca ctaaccccct ttcctgtttt cctaatcagc
7081 ccggcatttc gcgggcgata ttttcacagc tatttcagga gttcagccat gaacgcttat
7141 tacattcagg atcgtcttga ggctcagagc tgggcgcgtc actaccagca gctcgcccgt
7201 gaagagaaag aggcagaact ggcagacgac atggaaaaag gcctgcccca gcacctgttt
7261 gaatcgctat gcatcgatca tttgcaacgc cacggggcca gcaaaaaatc cattacccgt
7321 gcgtttgatg acgatgttga gtttcaggag cgcatggcag aacacatccg gtacatggtt
7381 gaaaccattg ctcaccacca ggttgatatt gattcagagg tataaaacga atgagtactg
7441 cactcgcaac gctggctggg aagctggctg aacgtgtcgg catggattct gtcgacccac
7501 aggaactgat caccactctt cgccagacgg catttaaagg tgatgccagc gatgcgcagt
7561 tcatcgcatt actgatcgtt gccaaccagt acggccttaa tccgtggacg aaagaaattt
7621 acgcctttcc tgataagcag aatggcatcg ttccggtggt gggcgttgat ggctggtccc
7681 gcatcatcaa tgaaaaccag cagtttgatg gcatggactt tgagcaggac aatgaatcct
7741 gtacatgccg gatttaccgc aaggaccgta atcatccgat ctgcgttacc gaatggatgg
7801 atgaatgccg ccgcgaacca ttcaaaactc gcgaaggcag agaaatcacg gggccgtggc
7861 agtcgcatcc caaacggatg ttacgtcata aagccatgat tcagtgtgcc cgtctggcct
7921 tcggatttgc tggtatctat gacaaggatg aagccgagcg cattgtcgaa aatactgcat
7981 acactgcaga acgtcagccg gaacgcgaca tcactccggt taacgatgaa accatgcagg
8041 agattaacac tctgctgatc gccctggata aaacatggga tgacgactta ttgccgctct
8101 gttcccagat atttcgccgc gacattcgtg catcgtcaga actgacacag gccgaagcag
8161 taaaagctct tggattcctg aaacagaaag ccgcagagca gaaggtggca gcatgacacc
8221 ggacattatc ctgcagcgta ccgggatcga tgtgagagct gtcgaacagg gggatgatgc
8281 gtggcacaaa ttacggctcg gcgtcatcac cgcttcagaa gttcacaacg tgatagcaaa
8341 accccgctcc ggaaagaagt ggcctgacat gaaaatgtcc tacttccaca ccctgcttgc
8401 tgaggtttgc accggtgtgg ctccggaagt taacgctaaa gcactggcct ggggaaaaca
8461 gtacgagaac gacgccagaa ccctgtttga attcacttcc ggcgtgaatg ttactgaatc
8521 cccgatcatc tatcgcgacg aaagtatgcg taccgcctgc tctcccgatg gtttatgcag
8581 tgacggcaac ggccttgaac tgaaatgccc gtttacctcc cgggatttca tgaagttccg
8641 gctcggtggt ttcgaggcca taaagtcagc ttacatggcc caggtgcagt acagcatgtg
8701 ggtgacgcga aaaaatgcct ggtactttgc caactatgac ccgcgtatga agcgtgaagg
8761 cctgcattat gtcgtgattg agcgggatga aaagtacatg gcgagttttg acgagatcgt
8821 gccggagttc atcgaaaaaa tggacgaggc actggctgaa attggttttg tatttgggga
8881 gcaatggcga tgacgcatcc tcacgataat atccgggtag gcgcaatcac tttcgtctac
8941 tccgttacaa agcgaggctg ggtatttccc ggcctttctg ttatccgaaa tccactgaaa
9001 gcacagcggc tggctgagga gataaataat aaacgagggg ctgtatgcac aaagcatctt
9061 ctgttgagtt aagaacgagt atcgagatgg cacatagcct tgctcaaatt ggaatcaggt
9121 ttgtgccaat accagtagaa acagacgaag aatccatggg tatggacagt ggatcctcta
9181 cgccggacgc atcgtggccg gcatcaccgg cgccacaggt gcggttgctg gcgcctatat
9241 cgccgacatc accgatgggg aagatcgggc ctgccaccat acccacgccg aaacaagcgc
9301 cctgcaccat tatgttccgg atctgcatcg caggatgctg ctggctaccc tgtggaacac
9361 ctacatctgt attaacgaag cgctaaccgt ttttatcagg ctctgggagg cagaataaat
9421 gatcatatcg tcaattatta cctccacggg gagagcctga gcaaactggc ctcaggcatt
9481 tgagaagcac acggtcacac tgcttccggt agtcaataaa ccggtaaacc agcaatagac
9541 ataagcggct atttaacgac cctgccctga accgacgacc gggtcgaatt tgctttcgaa
9601 tttctgccat tcatccgctt attatcactt attcaggcgt agcaaccagg cgtttaaggg
9661 caccaataac tgccttaaaa aaattacgcc ccgccctgcc actcatcgca gtactgttgt
9721 aattcattaa gcattctgcc gacatggaag ccatcacaaa cggcatgatg aacctgaatc
9781 gccagcggca tcagcacctt gtcgccttgc gtataatatt tgcccatggt gaaaacgggg
9841 gcgaagaagt tgtccatatt ggccacgttt aaatcaaaac tggtgaaact cacccaggga
9901 ttggctgaga cgaaaaacat attctcaata aaccctttag ggaaataggc caggttttca
9961 ccgtaacacg ccacatcttg cgaatatatg tgtagaaact gccggaaatc gtcgtggtat
10021 tcactccaga gcgatgaaaa cgtttcagtt tgctcatgga aaacggtgta acaagggtga
10081 acactatccc atatcaccag ctcaccgtct ttcattgcca tacggaattc cggatgagca
10141 ttcatcaggc gggcaagaat gtgaataaag gccggataaa acttgtgctt atttttcttt
10201 acggtcttta aaaaggccgt aatatccagc tgaacggtct ggttataggt acattgagca
10261 actgactgaa atgcctcaaa atgttcttta cgatgccatt gggatatatc aacggtggta
10321 tatccagtga tttttttctc cattttagct tccttagctc ctgaaaatct cgataactca
10381 aaaaatacgc ccggtagtga tcttatttca ttatggtgaa agttggaacc tcttacgtgc
10441 cgatcaacgt ctcattttcg ccaaaagttg gcccagggct tcccggtatc aacagggaca
10501 ccaggattta tttattctgc gaagtgatct tccgtcacag gtatttattc
//
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