| 质粒名称: | pcDNA3.1_pCMV-nCas-PmCDA1-ugi pH1-gRNA(HPRT) |
|---|---|
| 目录编号: | 79620 |
| 存储实验室: | Akihiko Kondo |
|
载体骨架基本信息 |
|
| 载体名称: | pcDNA3.1 |
| 载体出品公司: | Invitrogen |
| 空载体大小: | 5428bp |
| 完整质粒大小: | 10414 bp |
| 筛选标记: | Neomycin (select with G418) |
| 载体抗性: | Ampicillin, 100 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: | SpCas9 |
| 物种来源: | Streptococcus pyogenes |
| 基因突变: | D10A for nickase Cas9 |
| 启动子: | CMV |
| 标签/融合蛋白: |
PmCDA1 (C terminal on insert)
UGI (C terminal on insert)
|
| 克隆方法: | Gibson Cloning |
| 5’测序引物 | ttcgctattacgccagctg |
| 3’测序引物 | GACAGGTCGGTCTTGACA |
|
文献引用方法 |
|
| 材料和方法部分: | pcDNA3.1_pCMV-nCas-PmCDA1-ugi pH1-gRNA(HPRT) was a gift from Akihiko Kondo (Addgene plasmid # 79620 ; http://n2t.net/addgene:79620 ; RRID:Addgene_79620) |
| 参考文献部分: | Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems. Nishida K, Arazoe T, Yachie N, Banno S, Kakimoto M, Tabata M, Mochizuki M, Miyabe A, Araki M, Hara KY, Shimatani Z, Kondo A. Science. 2016 Aug 4. pii: aaf8729. 10.1126/science.aaf8729 PubMed 2749247 |
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pcDNA3.1_pCMV-nCas-PmCDA1-ugi pH1-gRNA(HPRT)载体质粒基本信息
pcDNA3.1_pCMV-nCas-PmCDA1-ugi pH1-gRNA(HPRT)质粒图谱载体图谱和pcDNA3.1_pCMV-nCas-PmCDA1-ugi pH1-gRNA(HPRT)载体序列质粒序列多克隆位点信息
79620质粒图谱.png)
pcDNA3.1_pCMV-nCas-PmCDA1-ugi pH1-gRNA(HPRT)质粒载体简介
pcDNA3.1_pCMV-nCas-PmCDA1-ugi pH1-gRNA(HPRT)质粒序列载体序列
LOCUS pcDNA3.1_pCMV-nCas-PmCDA1-ugi pH1-gRNA(HPRT) 10419 bp ds-DNA circular SYN 12-MAY-2021
DEFINITION Expresses nCas9-PmCDA1-UGI and gRNA(HPRT) in mammalian cells.
ACCESSION .
VERSION .
KEYWORDS pcDNA3.1_pCMV-nCas-PmCDA1-ugi pH1-gRNA(HPRT)
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 10419)
AUTHORS Nishida K, Arazoe T, Yachie N, Banno S, Kakimoto M, Tabata M,
Mochizuki M, Miyabe A, Araki M, Hara KY, Shimatani Z, Kondo A
TITLE Targeted nucleotide editing using hybrid prokaryotic and vertebrate
adaptive immune systems.
JOURNAL Science. 2016 Aug 4. pii: aaf8729.
PUBMED 27492474
REFERENCE 2 (bases 1 to 10419)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58
http://www.biofeng.com
FEATURES Location/Qualifiers
source 1..10419
/organism="synthetic DNA construct"
/mol_type="other DNA"
primer_bind complement(35..55)
/label=T3
/note="T3 promoter, forward primer"
promoter complement(35..53)
/label=T3 promoter
/note="promoter for bacteriophage T3 RNA polymerase"
primer_bind complement(74..90)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(74..90)
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind complement(87..109)
/label=M13/pUC Reverse
/note="In lacZ gene"
protein_bind 98..114
/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)."
promoter complement(122..152)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 167..188
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(305..322)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(476..1064)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(556..575)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS complement(1235..2095)
/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 1858..1877
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
promoter complement(2096..2200)
/gene="bla"
/label=AmpR promoter
rep_origin complement(2226..2681)
/direction=LEFT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(2363..2384)
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind 2575..2594
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 2808..2830
/label=M13/pUC Forward
/note="In lacZ gene"
primer_bind 2822..2839
/label=M13 Forward
/note="In lacZ gene. Also called M13-F20 or M13 (-21)
Forward"
primer_bind 2823..2839
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
promoter 2849..2867
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
primer_bind 3027..3046
/label=H1
/note="Human H1 promoter, forward primer"
misc_RNA 3136..3211
/label=gRNA scaffold
/note="guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system"
enhancer 3226..3605
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 3606..3809
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
primer_bind 3760..3780
/label=CMV-F
/note="Human CMV immediate early promoter, forward primer"
promoter 3854..3872
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
CDS 3901..3921
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 3946..8049
/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"
CDS 8062..8082
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 8290..8355
/codon_start=1
/product="three tandem FLAG(R) epitope tags, followed by an
enterokinase cleavage site"
/label=3xFLAG
/translation="DYKDHDGDYKDHDIDYKDDDDK"
CDS 8989..9009
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 9016..9267
/codon_start=1
/product="uracil-DNA glycosylase inhibitor from a Bacillus
subtilis bacteriophage (Mol et al., 1995)"
/label=UGI
/translation="MTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHT
AYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
CDS 9268..9321
/codon_start=1
/product="2A peptide from Thosea asigna virus capsid
protein"
/label=T2A
/note="Eukaryotic ribosomes fail to insert a peptide bond
between the Gly and Pro residues, yielding separate
polypeptides."
/translation="EGRGSLLTCGDVEENPGP"
CDS 9328..10122
/codon_start=1
/gene="aph(3')-II (or nptII)"
/product="aminoglycoside phosphotransferase from Tn5"
/label=NeoR/KanR
/note="confers resistance to neomycin, kanamycin, and G418
(Geneticin(R))"
/translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
primer_bind complement(9382..9401)
/label=Neo-R
/note="Neomycin resistance gene, reverse primer"
primer_bind 9992..10011
/label=Neo-F
/note="Neomycin resistance gene, forward primer"
polyA_signal 10296..10417
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(10333..10352)
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
primer_bind 10387..10406
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
ORIGIN
1 ggccgccacc gcggtggagc tccagctttt gttcccttta gtgagggtta attgcgcgct
61 tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac
121 acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac
181 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
241 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg
301 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
361 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
421 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
481 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
541 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
601 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
661 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
721 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
781 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
841 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
901 acggctacac tagaaggaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
961 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
1021 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
1081 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
1141 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
1201 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
1261 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
1321 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
1381 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
1441 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
1501 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
1561 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
1621 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
1681 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
1741 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
1801 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
1861 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
1921 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
1981 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
2041 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
2101 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
2161 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
2221 cacctaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag
2281 ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac
2341 cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga
2401 ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc
2461 accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg
2521 gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa
2581 gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac
2641 caccacaccc gccgcgctta atgcgccgct acagggcgcg tcccattcgc cattcaggct
2701 gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa
2761 agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg
2821 ttgtaaaacg acggccagtg agcgcgcgta atacgactca ctatagggcg aattgggtac
2881 cgggccaatt cgaacgctga cgtcatcaac ccgctccaag gaatcgcggg cccagtgtca
2941 ctaggcggga acacccagcg cgcgtgcgcc ctggcaggaa gatggctgtg agggacaggg
3001 gagtggcgcc ctgcaatatt tgcatgtcgc tatgtgttct gggaaatcac cataaacgtg
3061 aaatgtcttt ggatttggga atcttataag ttctgtatga ggaccacaga tccccccgag
3121 atgtcatgaa agagagtttt agagctagaa atagcaagtt aaaataaggc tagtccgtta
3181 tcaacttgaa aaagtggcac cgagtcggtg ctttttttac gcgttgacat tgattattga
3241 ctagttatta atagtaatca attacggggt cattagttca tagcccatat atggagttcc
3301 gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat
3361 tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc
3421 aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc
3481 caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt
3541 acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta
3601 ccatggtgat gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg
3661 gatttccaag tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac
3721 gggactttcc aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg
3781 tacggtggga ggtctatata agcagagctc tctggctaac tagagaaccc actgcttact
3841 ggcttatcga aattaatacg actcactata gggagaccca agctggctag cgaaatggca
3901 ccgaagaaga agcgtaaagt cggaatccac ggagttcctg cggcaatgga caagaagtac
3961 tccattgggc tcgctatcgg cacaaacagc gtcggttggg ccgtcattac ggacgagtac
4021 aaggtgccga gcaaaaaatt caaagttctg ggcaataccg atcgccacag cataaagaag
4081 aacctcattg gcgccctcct gttcgactcc ggggagacgg ccgaagccac gcggctcaaa
4141 agaacagcac ggcgcagata tacccgcaga aagaatcgga tctgctacct gcaggagatc
4201 tttagtaatg agatggctaa ggtggatgac tctttcttcc ataggctgga ggagtccttt
4261 ttggtggagg aggataaaaa gcacgagcgc cacccaatct ttggcaatat cgtggacgag
4321 gtggcgtacc atgaaaagta cccaaccata tatcatctga ggaagaagct tgtagacagt
4381 actgataagg ctgacttgcg gttgatctat ctcgcgctgg cgcatatgat caaatttcgg
4441 ggacacttcc tcatcgaggg ggacctgaac ccagacaaca gcgatgtcga caaactcttt
4501 atccaactgg ttcagactta caatcagctt ttcgaagaga acccgatcaa cgcatccgga
4561 gttgacgcca aagcaatcct gagcgctagg ctgtccaaat cccggcggct cgaaaacctc
4621 atcgcacagc tccctgggga gaagaagaac ggcctgtttg gtaatcttat cgccctgtca
4681 ctcgggctga cccccaactt taaatctaac ttcgacctgg ccgaagatgc caagcttcaa
4741 ctgagcaaag acacctacga tgatgatctc gacaatctgc tggcccagat cggcgaccag
4801 tacgcagacc tttttttggc ggcaaagaac ctgtcagacg ccattctgct gagtgatatt
4861 ctgcgagtga acacggagat caccaaagct ccgctgagcg ctagtatgat caagcgctat
4921 gatgagcacc accaagactt gactttgctg aaggcccttg tcagacagca actgcctgag
4981 aagtacaagg aaattttctt cgatcagtct aaaaatggct acgccggata cattgacggc
5041 ggagcaagcc aggaggaatt ttacaaattt attaagccca tcttggaaaa aatggacggc
5101 accgaggagc tgctggtaaa gcttaacaga gaagatctgt tgcgcaaaca gcgcactttc
5161 gacaatggaa gcatccccca ccagattcac ctgggcgaac tgcacgctat cctcaggcgg
5221 caagaggatt tctacccctt tttgaaagat aacagggaaa agattgagaa aatcctcaca
5281 tttcggatac cctactatgt aggccccctc gcccggggaa attccagatt cgcgtggatg
5341 actcgcaaat cagaagagac catcactccc tggaacttcg aggaagtcgt ggataagggg
5401 gcctctgccc agtccttcat cgaaaggatg actaactttg ataaaaatct gcctaacgaa
5461 aaggtgcttc ctaaacactc tctgctgtac gagtacttca cagtttataa cgagctcacc
5521 aaggtcaaat acgtcacaga agggatgaga aagccagcat tcctgtctgg agagcagaag
5581 aaagctatcg tggacctcct cttcaagacg aaccggaaag ttaccgtgaa acagctcaaa
5641 gaagactatt tcaaaaagat tgaatgtttc gactctgttg aaatcagcgg agtggaggat
5701 cgcttcaacg catccctggg aacgtatcac gatctcctga aaatcattaa agacaaggac
5761 ttcctggaca atgaggagaa cgaggacatt cttgaggaca ttgtcctcac ccttacgttg
5821 tttgaagata gggagatgat tgaagaacgc ttgaaaactt acgctcatct cttcgacgac
5881 aaagtcatga aacagctcaa gaggcgccga tatacaggat gggggcggct gtcaagaaaa
5941 ctgatcaatg ggatccgaga caagcagagt ggaaagacaa tcctggattt tcttaagtcc
6001 gatggatttg ccaaccggaa cttcatgcag ttgatccatg atgactctct cacctttaag
6061 gaggacatcc agaaagcaca agtttctggc cagggggaca gtcttcacga gcacatcgct
6121 aatcttgcag gtagcccagc tatcaaaaag ggaatactgc agaccgttaa ggtcgtggat
6181 gaactcgtca aagtaatggg aaggcataag cccgagaata tcgttatcga gatggcccga
6241 gagaaccaaa ctacccagaa gggacagaag aacagtaggg aaaggatgaa gaggattgaa
6301 gagggtataa aagaactggg gtcccaaatc cttaaggaac acccagttga aaacacccag
6361 cttcagaatg agaagctcta cctgtactac ctgcagaacg gcagggacat gtacgtggat
6421 caggaactgg acatcaatcg gctctccgac tacgacgtgg atcatatcgt gccccagtct
6481 tttctcaaag atgattctat tgataataaa gtgttgacaa gatccgataa aaatagaggg
6541 aagagtgata acgtcccctc agaagaagtt gtcaagaaaa tgaaaaatta ttggcggcag
6601 ctgctgaacg ccaaactgat cacacaacgg aagttcgata atctgactaa ggctgaacga
6661 ggtggcctgt ctgagttgga taaagccggc ttcatcaaaa ggcagcttgt tgagacacgc
6721 cagatcacca agcacgtggc ccaaattctc gattcacgca tgaacaccaa gtacgatgaa
6781 aatgacaaac tgattcgaga ggtgaaagtt attactctga agtctaagct ggtctcagat
6841 ttcagaaagg actttcagtt ttataaggtg agagagatca acaattacca ccatgcgcat
6901 gatgcctacc tgaatgcagt ggtaggcact gcacttatca aaaaatatcc caagcttgaa
6961 tctgaatttg tttacggaga ctataaagtg tacgatgtta ggaaaatgat cgcaaagtct
7021 gagcaggaaa taggcaaggc caccgctaag tacttctttt acagcaatat tatgaatttt
7081 ttcaagaccg agattacact ggccaatgga gagattcgga agcgaccact tatcgaaaca
7141 aacggagaaa caggagaaat cgtgtgggac aagggtaggg atttcgcgac agtccggaag
7201 gtcctgtcca tgccgcaggt gaacatcgtt aaaaagaccg aagtacagac cggaggcttc
7261 tccaaggaaa gtatcctccc gaaaaggaac agcgacaagc tgatcgcacg caaaaaagat
7321 tgggacccca agaaatacgg cggattcgat tctcctacag tcgcttacag tgtactggtt
7381 gtggccaaag tggagaaagg gaagtctaaa aaactcaaaa gcgtcaagga actgctgggc
7441 atcacaatca tggagcgatc aagcttcgaa aaaaacccca tcgactttct cgaggcgaaa
7501 ggatataaag aggtcaaaaa agacctcatc attaagcttc ccaagtactc tctctttgag
7561 cttgaaaacg gccggaaacg aatgctcgct agtgcgggcg agctgcagaa aggtaacgag
7621 ctggcactgc cctctaaata cgttaatttc ttgtatctgg ccagccacta tgaaaagctc
7681 aaagggtctc ccgaagataa tgagcagaag cagctgttcg tggaacaaca caaacactac
7741 cttgatgaga tcatcgagca aataagcgaa ttctccaaaa gagtgatcct cgccgacgct
7801 aacctcgata aggtgctttc tgcttacaat aagcacaggg ataagcccat cagggagcag
7861 gcagaaaaca ttatccactt gtttactctg accaacttgg gcgcgcctgc agccttcaag
7921 tacttcgaca ccaccataga cagaaagcgg tacacctcta caaaggaggt cctggacgcc
7981 acactgattc atcagtcaat tacggggctc tatgaaacaa gaatcgacct ctctcagctc
8041 ggtggagaca gcagggctga ccccaagaag aagaggaagg tgggtggagg aggtaccggc
8101 ggtggaggct cagcagaata cgtacgagct ctgtttgact tcaatgggaa tgacgaggag
8161 gatctcccct ttaagaaggg cgatattctc cgcatcagag ataagcccga agaacaatgg
8221 tggaatgccg aggatagcga agggaaaagg ggcatgattc tggtgccata tgtggagaaa
8281 tattccggtg actacaaaga ccatgatggg gattacaaag accacgacat cgactacaaa
8341 gacgacgacg ataaatcagg gatgacagac gccgagtacg tgcgcattca tgagaaactg
8401 gatatttaca ccttcaagaa gcagttcttc aacaacaaga aatctgtgtc acaccgctgc
8461 tacgtgctgt ttgagttgaa gcgaaggggc gaaagaaggg cttgcttttg gggctatgcc
8521 gtcaacaagc cccaaagtgg caccgagaga ggaatacacg ctgagatatt cagtatccga
8581 aaggtggaag agtatcttcg ggataatcct gggcagttta cgatcaactg gtattccagc
8641 tggagtcctt gcgctgattg tgccgagaaa attctggaat ggtataatca ggaacttcgg
8701 ggaaacgggc acacattgaa aatctgggcc tgcaagctgt actacgagaa gaatgcccgg
8761 aaccagatag gactctggaa tctgagggac aatggtgtag gcctgaacgt gatggtttcc
8821 gagcactatc agtgttgtcg gaagattttc atccaaagct ctcataacca gctcaatgaa
8881 aaccgctggt tggagaaaac actgaaacgt gcggagaagt ggagatccga gctgagcatc
8941 atgatccagg tcaagattct gcataccact aagtctccag ccgttggtcc caagaagaaa
9001 agaaaagtcg gtaccatgac caacctttcc gacatcatag agaaggaaac aggcaaacag
9061 ttggtcatcc aagagtcgat actcatgctt cctgaagaag ttgaggaggt cattgggaat
9121 aagccggaaa gtgacattct cgtacacact gcgtatgatg agagcaccga tgagaacgtg
9181 atgctgctca cgtcagatgc cccagagtac aaaccctggg ctctggtgat tcaggactct
9241 aatggagaga acaagatcaa gatgctagaa ggcaggggaa gccttctgac ttgtggggat
9301 gtggaagaaa accctggtcc atctagaatg attgaacaag atggattgca cgcaggttct
9361 ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac aatcggctgc
9421 tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt tgtcaagacc
9481 gacctgtccg gtgccctgaa tgaactgcag gacgaggcag cgcggctatc gtggctggcc
9541 acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg aagggactgg
9601 ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag
9661 aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc
9721 ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat ggaagccggt
9781 cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc cgaactgttc
9841 gccaggctca aggcgcgcat gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc
9901 tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga ctgtggccgg
9961 ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat tgctgaagag
10021 cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg
10081 cagcgcatcg ccttctatcg ccttcttgac gagttcttct gagcgggact ctggggttcg
10141 aaatgaccga ccaagcgacg cccaacctgc catcacgaga tttcgattcc accgccgcct
10201 tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc
10261 gcggggatct catgctggag ttcttcgccc accccaactt gtttattgca gcttataatg
10321 gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt
10381 ctagttgtgg tttgtccaaa ctcatcaatg tatcttagc
//
