| 质粒名称: | pYLCRISPR/Cas9pUbi-N |
|---|---|
| 出品公司: | 爱迪基因 |
| 目录编号: | 106331 |
| 存储实验室: | Yao-Guang Liu |
|
载体骨架基本信息 |
|
| 载体名称: | pCAMBIA1300 |
| 载体出品公司: | CAMBIA |
| 载体抗性: | 卡那霉素 |
| 空载体大小: | -- |
| 完整质粒大小: | -- |
| 载体修饰: | -- |
| 载体类型: | 植物表达，CRISPR |
| 筛选标记: | -- |
| 高拷贝/低拷贝: | -- |
| 生长条件: | 37 ℃ |
|
插入基因信息 |
|
| 插入基因名称: | Cas9 |
| 物种来源: | Synthetic |
| 插入大小: | -- |
| 基因突变: | 植物密码子优化后，5'区（400bp）GC含量较高（62.5%），总GC含量为54.2% |
| GenBank编号 | MG719602 |
| 启动子 | ZmUbi |
| 标签/融合蛋白: | -- |
| 克隆方法: | 限制性内切酶 |
| 5' 克隆位点: | -- |
| 3' 克隆位点: | -- |
| 5' 测序引物 | -- |
| 3' 测序引物 | -- |
|
文献引用方法 |
|
| 材料和方法部分: | pYLCRISPR/Cas9pUbi-N was a gift from Yao-Guang Liu (Addgene plasmid # 106331 ; http://n2t.net/addgene:106331 ; RRID:Addgene_106331) |
| 参考文献部分: | A robust CRISPR/Cas9 system for convenient, high-efficiency multiplex genome editing in monocot and dicot plants. Ma X, Zhang Q, Zhu Q, Liu W, Chen Y, Qiu R, Wang B, Yang Z, Li H, Lin Y, Xie Y, Shen R, Chen S, Wang Z, Chen Y, Guo J, Chen L, Zhao X, Dong Z, Liu YG. Mol Plant. 2015 Apr 24. pii: S1674-2052(15)00204-X. doi: 10.1016/j.molp.2015.04.007. 10.1016/j.molp.2015.04.007 PubMed 25917172 |
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 pYLCRISPR/Cas9pUbi-N 17355 bp ds-DNA circular SYN 06-AUG-2018
DEFINITION Expression of Cas9 in plants, ZmUbi promoter, Kanamycin selection.
ACCESSION .
VERSION .
KEYWORDS pYLCRISPR/Cas9pUbi-N
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 17355)
AUTHORS Ma X, Zhang Q, Zhu Q, Liu W, Chen Y, Qiu R, Wang B, Yang Z, Li H,
Lin Y, Xie Y, Shen R, Chen S, Wang Z, Chen Y, Guo J, Chen L, Zhao X,
Dong Z, Liu YG
TITLE A robust CRISPR/Cas9 system for convenient, high-efficiency
multiplex genome editing in monocot and dicot plants.
JOURNAL Mol Plant. 2015 Apr 24. pii: S1674-2052(15)00204-X. doi:
10.1016/j.molp.2015.04.007.
PUBMED 25917172
REFERENCE 2 (bases 1 to 17355)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Aug 6, 2018 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..17355
/organism="synthetic DNA construct"
/mol_type="other DNA"
protein_bind 94..115
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 130..160
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 168..184
/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 173..195
/label=M13/pUC Reverse
/note="In lacZ gene"
primer_bind 192..208
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind 192..208
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
CDS 2316..2336
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 2361..6461
/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="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
ATLIHQSITGLYETRIDLSQLGGD"
RBS 3547..3555
/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 6462..6509
/codon_start=1
/product="bipartite nuclear localization signal from
nucleoplasmin"
/label=nucleoplasmin NLS
/translation="KRPAATKKAGQAKKKK"
terminator 6522..6774
/label=NOS terminator
/note="nopaline synthase terminator and poly(A) signal"
protein_bind 6892..6913
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 6928..6958
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 6966..6982
/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 7024..7042
/label=SP6 promoter
/note="promoter for bacteriophage SP6 RNA polymerase"
primer_bind 7024..7041
/label=SP6
/note="SP6 promoter, forward primer"
CDS 7116..7418
/codon_start=1
/gene="ccdB"
/product="CcdB, a bacterial toxin that poisons DNA gyrase"
/label=ccdB
/note="Plasmids containing the ccdB gene cannot be
propagated in standard E. coli strains."
/translation="QFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDKV
SRELYPVVHIGDESWRMMTTDMPSVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
primer_bind 7297..7316
/label=ccdB-fwd
/note="ccdB gene, forward primer"
protein_bind complement(7628..7661)
/label=loxP
/bound_moiety="Cre recombinase"
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (GCATACAT)."
primer_bind complement(7691..7708)
/label=M13 Forward
/note="In lacZ gene. Also called M13-F20 or M13 (-21)
Forward"
primer_bind complement(7691..7707)
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(7700..7722)
/label=M13/pUC Forward
/note="In lacZ gene"
misc_feature 7910..7934
/label=RB T-DNA repeat
/note="right border repeat from nopaline C58 T-DNA"
CDS 9235..9864
/codon_start=1
/product="stability protein from the Pseudomonas plasmid
pVS1 (Heeb et al., 2000)"
/label=pVS1 StaA
/translation="MKVIAVLNQKGGSGKTTIATHLARALQLAGADVLLVDSDPQGSAR
DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP
VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI
LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI"
CDS 10293..11366
/codon_start=1
/product="replication protein from the Pseudomonas plasmid
pVS1 (Heeb et al., 2000)"
/label=pVS1 RepA
/translation="MSGRKPSGPVQIGAALGDDLVEKLKAAQAAQRQRIEAEARPGESW
QAAADRIRKESRQPPAAGAPSIRKPPKGDEQPDFFVPMLYDVGTRDSRSIMDVAVFRLS
KRDRRAGEVIRYELPDGHVEVSAGPAGMASVWDYDLVLMAVSHLTESMNRYREGKGDKP
GRVFRPHVADVLKFCRRADGGKQKDDLVETCIRLNTTHVAMQRTKKAKNGRLVTVSEGE
ALISRYKIVKSETGRPEYIEIELADWMYREITEGKNPDVLTVHPDYFLIDPGIGRFLYR
LARRAAGKAEARWLFKTIYERSGSAGEFKKFCFTVRKLIGSNDLPEYDLKEEAGQAGPI
LVMRYRNLIEGEASAGS"
rep_origin 11432..11626
/label=pVS1 oriV
/note="origin of replication for the Pseudomonas plasmid
pVS1 (Heeb et al., 2000)"
primer_bind complement(11862..11884)
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
misc_feature 11970..12110
/label=bom
/note="basis of mobility region from pBR322"
primer_bind 12025..12044
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind complement(12125..12142)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(12296..12884)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(12376..12395)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS complement(12971..13765)
/codon_start=1
/gene="aphA-3"
/product="aminoglycoside phosphotransferase"
/label=KanR
/note="confers resistance to kanamycin"
/translation="MAKMRISPELKKLIEKYRCVKDTEGMSPAKVYKLVGENENLYLKM
TDSRYKGTTYDVEREKDMMLWLEGKLPVPKVLHFERHDGWSNLLMSEADGVLCSEEYED
EQSPEKIIELYAECIRLFHSIDISDCPYTNSLDSRLAELDYLLNNDLADVDCENWEEDT
PFKDPRELYDFLKTEKPEEELVFSHGDLGDSNIFVKDGKVSGFIDLGRSGRADKWYDIA
FCVRSIREDIGEEQYVELFFDLLGIKPDWEKIKYYILLDELF"
misc_feature 15389..15413
/label=LB T-DNA repeat
/note="left border repeat from nopaline C58 T-DNA"
polyA_signal 15491..15665
/label=CaMV poly(A) signal
/note="cauliflower mosaic virus polyadenylation signal"
CDS complement(15722..16519)
/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="MGIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGR
PVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLL
SSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEH
QGLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDI
ALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
primer_bind complement(15833..15852)
/label=Neo-F
/note="Neomycin resistance gene, forward primer"
primer_bind 16443..16462
/label=Neo-R
/note="Neomycin resistance gene, reverse primer"
promoter complement(16582..17258)
/label=CaMV 35S promoter (enhanced)
/note="cauliflower mosaic virus 35S promoter with a
duplicated enhancer region"
primer_bind complement(16625..16645)
/label=35S promoter
/note="CaMV 35S promoter, forward primer"
ORIGIN
1 caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc
61 cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg agttagctca ctcattaggc
121 accccaggct ttacacttta tgcttccggc tcgtatgttg tgtggaattg tgagcggata
181 acaatttcac acaggaaaca gctatgacat gattacgaat tcgagctcgg taccgctcgc
241 taccttaaga gaggatatcc ctccatccta taatgtaggc tataggaact agggcaaggc
301 cggccatgcg gccgcaagct gggtgcagcg tgacccggtc gtgcccctct ctagagataa
361 tgagcattgc atgtctaagt tataaaaaat taccacatat tttttttgtc acacttgttt
421 gaagtgcagt ttatctatct ttatacatat atttaaactt tactctacga ataatataat
481 ctatagtact acaataatat cagtgtttta gagaatcata taaatgaaca gttagacatg
541 gtctaaagga caattgagta ttttgacaac aggactctac agttttatct ttttagtgtg
601 catgtgttct cctttttttt tgcaaatagc ttcacctata taatacttca tccattttat
661 tagtacatcc atttagggtt tagggttaat ggtttttata gactaatttt tttagtacat
721 ctattttatt ctattttagc ctctaaatta agaaaactaa aactctattt tagttttttt
781 atttaataat ttagatataa aatagaataa aataaagtga ctaaaaatta aacaaatacc
841 ctttaagaaa ttaaaaaaac taaggaaaca tttttcttgt ttcgagtaga taatgccagc
901 ctgttaaacg ccgtcgacga gtctaacgga caccaaccag cgaaccagca gcgtcgcgtc
961 gggccaagcg aagcagacgg cacggcatct ctgtcgctgc ctctggaccc ctctcgagag
1021 ttccgctcca ccgttggact tgctccgctg tcggcatcca gaaattgcgt ggcggagcgg
1081 cagacgtgag ccggcacggc aggcggcctc ctcctcctct cacggcaccg gcagctacgg
1141 gggattcctt tcccaccgct ccttcgcttt cccttcctcg cccgccgtaa taaatagaca
1201 ccccctccac accctctttc cccaacctcg tgttgttcgg agcgcacaca cacacaacca
1261 gatctccccc aaatccaccc gtcggcacct ccgcttcaag gtacgccgct cgtcctnccc
1321 cccccccctc tctaccttct ctagatcggc gttccggtcc atggttaggg cccggtagtt
1381 ctacttctgt tcatgtttgt gttagatccg tgtttgtgtt agatccgtgc tgctagcgtt
1441 cgtacacgga tgcgacctgt acgtcagaca cgttctgatt gctaacttgc cagtgtttct
1501 ctttggggaa tcctgggatg gctctagccg ttccgcagac gggatcgatt tcatgatttt
1561 ttttgtttcg ttgcataggg tttggtttgc ccttttcctt tatttcaata tatgccgtgc
1621 acttgtttgt cgggtcatct tttcatgctt ttttttgtct tggttgtgat gatgtggtct
1681 ggttgggcgg tcgttctaga tcggagtaga attctgtttc aaactacctg gtggatttat
1741 taattttgga tctgtatgtg tgtgccatac atattcatag ttacgaattg aagatgatgg
1801 atggaaatat cgatctagga taggtataca tgttgatgcg ggttttactg atgcatatac
1861 agagatgctt tttgttcgct tggttgtgat gatgtggtgt ggttgggcgg tcgttcattc
1921 gttctagatc ggagtagaat actgtttcaa actacctggt gtatttatta attttggaac
1981 tgtatgtgtg tgtcatacat cttcatagtt acgagtttaa gatggatgga aatatcgatc
2041 taggataggt atacatgttg atgtgggttt tactgatgca tatacatgat ggcatatgca
2101 gcatctattc atatgctcta accttgagta cctatctatt ataataaaca agtatgtttt
2161 ataattattt tgatcttgat atacttggat gatggcatat gcagcagcta tatgtggatt
2221 tttttagccc tgccttcata cgctatttat ttgcttggta ctgtttcttt tgtcgatgct
2281 caccctgttg tttggtgtta cttctgcaga tggctcctaa gaagaagcgg aaggttggta
2341 ttcacggggt gcctgcggct gacaagaagt actccatcgg cctcgacatc ggcaccaaca
2401 gcgtcggctg ggcggtgatc accgacgagt acaaggtccc gtccaagaag ttcaaggtcc
2461 tgggcaacac cgaccgccac tccatcaaga agaacctcat cggcgccctc ctcttcgact
2521 ccggcgagac ggcggaggcg acccgcctca agcgcaccgc ccgccgccgc tacacccgcc
2581 gcaagaaccg catctgctac ctccaggaga tcttctccaa cgagatggcg aaggtcgacg
2641 actccttctt ccaccgcctc gaggagtcct tcctcgtgga ggaggacaag aagcacgagc
2701 gccaccccat cttcggcaac atcgtcgacg aggtcgccta ccacgagaag taccccacta
2761 tctaccacct tcgtaagaag cttgttgact ctactgataa ggctgatctt cgtctcatct
2821 accttgctct cgctcacatg atcaagttcc gtggtcactt ccttatcgag ggtgacctta
2881 accctgataa ctccgacgtg gacaagctct tcatccagct cgtccagacc tacaaccagc
2941 tcttcgagga gaaccctatc aacgcttccg gtgtcgacgc taaggcgatc ctttccgcta
3001 ggctctccaa gtccaggcgt ctcgagaacc tcatcgccca gctccctggt gagaagaaga
3061 acggtctttt cggtaacctc atcgctctct ccctcggtct gacccctaac ttcaagtcca
3121 acttcgacct cgctgaggac gctaagcttc agctctccaa ggatacctac gacgatgatc
3181 tcgacaacct cctcgctcag attggagatc agtacgctga tctcttcctt gctgctaaga
3241 acctctccga tgctatcctc ctttcggata tccttagggt taacactgag atcactaagg
3301 ctcctctttc tgcttccatg atcaagcgct acgacgagca ccaccaggac ctcaccctcc
3361 tcaaggctct tgttcgtcag cagctccccg agaagtacaa ggagatcttc ttcgaccagt
3421 ccaagaacgg ctacgccggt tacattgacg gtggagctag ccaggaggag ttctacaagt
3481 tcatcaagcc aatccttgag aagatggatg gtactgagga gcttctcgtt aagcttaacc
3541 gtgaggacct ccttaggaag cagaggactt tcgataacgg ctctatccct caccagatcc
3601 accttggtga gcttcacgcc atccttcgta ggcaggagga cttctaccct ttcctcaagg
3661 acaaccgtga gaagatcgag aagatcctta ctttccgtat tccttactac gttggtcctc
3721 ttgctcgtgg taactcccgt ttcgcttgga tgactaggaa gtccgaggag actatcaccc
3781 cttggaactt cgaggaggtt gttgacaagg gtgcttccgc ccagtccttc atcgagcgca
3841 tgaccaactt cgacaagaac ctccccaacg agaaggtcct ccccaagcac tccctcctct
3901 acgagtactt cacggtctac aacgagctca ccaaggtcaa gtacgtcacc gagggtatgc
3961 gcaagcctgc cttcctctcc ggcgagcaga agaaggctat cgttgacctc ctcttcaaga
4021 ccaaccgcaa ggtcaccgtc aagcagctca aggaggacta cttcaagaag atcgagtgct
4081 tcgactccgt cgagatcagc ggcgttgagg accgtttcaa cgcttctctc ggtacctacc
4141 acgatctcct caagatcatc aaggacaagg acttcctcga caacgaggag aacgaggaca
4201 tcctcgagga catcgtcctc actcttactc tcttcgagga tagggagatg atcgaggaga
4261 ggctcaagac ttacgctcat ctcttcgatg acaaggttat gaagcagctc aagcgtcgcc
4321 gttacaccgg ttggggtagg ctctcccgca agctcatcaa cggtatcagg gataagcaga
4381 gcggcaagac tatcctcgac ttcctcaagt ctgatggttt cgctaacagg aacttcatgc
4441 agctcatcca cgatgactct cttaccttca aggaggatat tcagaaggct caggtgtccg
4501 gtcagggcga ctctctccac gagcacattg ctaaccttgc tggttcccct gctatcaaga
4561 agggcatcct tcagactgtt aaggttgtcg atgagcttgt caaggttatg ggtcgtcaca
4621 agcctgagaa catcgtcatc gagatggctc gtgagaacca gactacccag aagggtcaga
4681 agaactcgag ggagcgcatg aagaggattg aggagggtat caaggagctt ggttctcaga
4741 tccttaagga gcaccctgtc gagaacaccc agctccagaa cgagaagctc tacctctact
4801 acctccagaa cggtagggat atgtacgttg accaggagct cgacatcaac aggctttctg
4861 actacgacgt cgaccacatt gttcctcagt ctttccttaa ggatgactcc atcgacaaca
4921 aggtcctcac gaggtccgac aagaacaggg gtaagtcgga caacgtccct tccgaggagg
4981 ttgtcaagaa gatgaagaac tactggaggc agcttctcaa cgctaagctc attacccaga
5041 ggaagttcga caacctcacg aaggctgaga ggggtggcct ttccgagctt gacaaggctg
5101 gtttcatcaa gaggcagctt gttgagacga ggcagattac caagcacgtt gctcagatcc
5161 tcgattctag gatgaacacc aagtacgacg agaacgacaa gctcatccgc gaggtcaagg
5221 tgatcaccct caagtccaag ctcgtctccg acttccgcaa ggacttccag ttctacaagg
5281 tccgcgagat caacaactac caccacgctc acgatgctta ccttaacgct gtcgttggta
5341 ccgctcttat caagaagtac cctaagcttg agtccgagtt cgtctacggt gactacaagg
5401 tctacgacgt tcgtaagatg atcgccaagt ccgagcagga gatcggcaag gccaccgcca
5461 agtacttctt ctactccaac atcatgaact tcttcaagac cgagatcacc ctcgccaacg
5521 gcgagatccg caagcgccct cttatcgaga cgaacggtga gactggtgag atcgtttggg
5581 acaagggtcg cgacttcgct actgttcgca aggtcctttc tatgcctcag gttaacatcg
5641 tcaagaagac cgaggtccag accggtggct tctccaagga gtctatcctt ccaaagagaa
5701 actcggacaa gctcatcgct aggaagaagg attgggaccc taagaagtac ggtggtttcg
5761 actcccctac tgtcgcctac tccgtcctcg tggtcgccaa ggtggagaag ggtaagtcga
5821 agaagctcaa gtccgtcaag gagctcctcg gcatcaccat catggagcgc tcctccttcg
5881 agaagaaccc gatcgacttc ctcgaggcca agggctacaa ggaggtcaag aaggacctca
5941 tcatcaagct ccccaagtac tctcttttcg agctcgagaa cggtcgtaag aggatgctgg
6001 cttccgctgg tgagctccag aagggtaacg agcttgctct tccttccaag tacgtgaact
6061 tcctctacct cgcctcccac tacgagaagc tcaagggttc ccctgaggat aacgagcaga
6121 agcagctctt cgtggagcag cacaagcact acctcgacga gatcatcgag cagatctccg
6181 agttctccaa gcgcgtcatc ctcgctgacg ctaacctcga caaggtcctc tccgcctaca
6241 acaagcaccg cgacaagccc atccgcgagc aggccgagaa catcatccac ctcttcacgc
6301 tcacgaacct cggcgcccct gctgctttca agtacttcga caccaccatc gacaggaagc
6361 gttacacgtc caccaaggag gttctcgacg ctactctcat ccaccagtcc atcaccggtc
6421 tttacgagac tcgtatcgac ctttcccagc ttggtggtga taagcgtcct gctgccacca
6481 aaaaggccgg acaggctaag aaaaagaagt aggatcctcc cgatcgttca aacatttggc
6541 aataaagttt cttaagattg aatcctgttg ccggtcttgc gatgattatc atataatttc
6601 tgttgaatta cgttaagcat gtaataatta acatgtaatg catgacgtta tttatgaggt
6661 gggtttttat gattagagtc ccgcaattat acatttaata cgcgatagaa aacaaaatat
6721 agcgcgcaaa ctaggataaa ttatcgcgcg cggtgtcatc tatgttacta gatcgggagc
6781 accggtaagg cgcgccgtag tgctcgagag acctctgaag tggccgattc attaatgcag
6841 ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag
6901 ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg
6961 tggaattgtg agcggataac aatttcacac aagaaacagc tatgaccatg attacgccaa
7021 gctatttagg tgacactata gaatactcaa gctatgcatc aagctcaatg ggtctagtct
7081 gtagataccc atcacactgg cgaccgctcg aacatcagtt taaggtttac acctataaaa
7141 gagagagccg ttatcgtctg tttgtggatg tacagagtga tattattgac acgccggggc
7201 gacggatggt gatccccctg gccagtgcac gtctgctgtc agataaagtc tcccgtgaac
7261 tttacccggt ggtgcatatc ggggatgaaa gctggcgcat gatgaccacc gatatgccca
7321 gtgtgcctgt ctccgttatc ggggaagaag tggctgatct cagccaccgc gaaaatgaca
7381 tcaaaaacgc cattaacctg atgttctggg gaatataaat gtcaggcctg aatggcgaat
7441 ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgag cgtgggtctc gcggtatcat
7501 tggcgcgcct ctcgagctag cggccgcatg catcgatctc ctacatcgta taaattagcc
7561 tatacgaagt tattgcatct atgtcgggtg cggagaaaga ggtaatgaaa tggcagtatt
7621 agatctgata acttcgtata atgtatgcta tacgaagtta tgactgcaag tcgacaccca
7681 taagcttggc actggccgtc gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc
7741 aacttaatcg ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc
7801 gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg cgaatgctag agcagcttga
7861 gcttggatca gattgtcgtt tcccgccttc agtttaaact atcagtgttt gacaggatat
7921 attggcgggt aaacctaaga gaaaagagcg tttattagaa taatcggata tttaaaaggg
7981 cgtgaaaagg tttatccgtt cgtccatttg tatgtgcatg ccaaccacag ggttcccctc
8041 gggatcaaag tactttgatc caacccctcc gctgctatag tgcagtcggc ttctgacgtt
8101 cagtgcagcc gtcttctgaa aacgacatgt cgcacaagtc ctaagttacg cgacaggctg
8161 ccgccctgcc cttttcctgg cgttttcttg tcgcgtgttt tagtcgcata aagtagaata
8221 cttgcgacta gaaccggaga cattacgcca tgaacaagag cgccgccgct ggcctgctgg
8281 gctatgcccg cgtcagcacc gacgaccagg acttgaccaa ccaacgggcc gaactgcacg
8341 cggccggctg caccaagctg ttttccgaga agatcaccgg caccaggcgc gaccgcccgg
8401 agctggccag gatgcttgac cacctacgcc ctggcgacgt tgtgacagtg accaggctag
8461 accgcctggc ccgcagcacc cgcgacctac tggacattgc cgagcgcatc caggaggccg
8521 gcgcgggcct gcgtagcctg gcagagccgt gggccgacac caccacgccg gccggccgca
8581 tggtgttgac cgtgttcgcc ggcattgccg agttcgagcg ttccctaatc atcgaccgca
8641 cccggagcgg gcgcgaggcc gccaaggccc gaggcgtgaa gtttggcccc cgccctaccc
8701 tcaccccggc acagatcgcg cacgcccgcg agctgatcga ccaggaaggc cgcaccgtga
8761 aagaggcggc tgcactgctt ggcgtgcatc gctcgaccct gtaccgcgca cttgagcgca
8821 gcgaggaagt gacgcccacc gaggccaggc ggcgcggtgc cttccgtgag gacgcattga
8881 ccgaggccga cgccctggcg gccgccgaga atgaacgcca agaggaacaa gcatgaaacc
8941 gcaccaggac ggccaggacg aaccgttttt cattaccgaa gagatcgagg cggagatgat
9001 cgcggccggg tacgtgttcg agccgcccgc gcacgtctca accgtgcggc tgcatgaaat
9061 cctggccggt ttgtctgatg ccaagctggc ggcctggccg gccagcttgg ccgctgaaga
9121 aaccgagcgc cgccgtctaa aaaggtgatg tgtatttgag taaaacagct tgcgtcatgc
9181 ggtcgctgcg tatatgatgc gatgagtaaa taaacaaata cgcaagggga acgcatgaag
9241 gttatcgctg tacttaacca gaaaggcggg tcaggcaaga cgaccatcgc aacccatcta
9301 gcccgcgccc tgcaactcgc cggggccgat gttctgttag tcgattccga tccccagggc
9361 agtgcccgcg attgggcggc cgtgcgggaa gatcaaccgc taaccgttgt cggcatcgac
9421 cgcccgacga ttgaccgcga cgtgaaggcc atcggccggc gcgacttcgt agtgatcgac
9481 ggagcgcccc aggcggcgga cttggctgtg tccgcgatca aggcagccga cttcgtgctg
9541 attccggtgc agccaagccc ttacgacata tgggccaccg ccgacctggt ggagctggtt
9601 aagcagcgca ttgaggtcac ggatggaagg ctacaagcgg cctttgtcgt gtcgcgggcg
9661 atcaaaggca cgcgcatcgg cggtgaggtt gccgaggcgc tggccgggta cgagctgccc
9721 attcttgagt cccgtatcac gcagcgcgtg agctacccag gcactgccgc cgccggcaca
9781 accgttcttg aatcagaacc cgagggcgac gctgcccgcg aggtccaggc gctggccgct
9841 gaaattaaat caaaactcat ttgagttaat gaggtaaaga gaaaatgagc aaaagcacaa
9901 acacgctaag tgccggccgt ccgagcgcac gcagcagcaa ggctgcaacg ttggccagcc
9961 tggcagacac gccagccatg aagcgggtca actttcagtt gccggcggag gatcacacca
10021 agctgaagat gtacgcggta cgccaaggca agaccattac cgagctgcta tctgaataca
10081 tcgcgcagct accagagtaa atgagcaaat gaataaatga gtagatgaat tttagcggct
10141 aaaggaggcg gcatggaaaa tcaagaacaa ccaggcaccg acgccgtgga atgccccatg
10201 tgtggaggaa cgggcggttg gccaggcgta agcggctggg ttgcctgccg gccctgcaat
10261 ggcactggaa cccccaagcc cgaggaatcg gcgtgagcgg tcgcaaacca tccggcccgg
10321 tacaaatcgg cgcggcgctg ggtgatgacc tggtggagaa gttgaaggcc gcgcaggccg
10381 cccagcggca acgcatcgag gcagaagcac gccccggtga atcgtggcaa gcggccgctg
10441 atcgaatccg caaagaatcc cggcaaccgc cggcagccgg tgcgccgtcg attaggaagc
10501 cgcccaaggg cgacgagcaa ccagattttt tcgttccgat gctctatgac gtgggcaccc
10561 gcgatagtcg cagcatcatg gacgtggccg ttttccgtct gtcgaagcgt gaccgacgag
10621 ctggcgaggt gatccgctac gagcttccag acgggcacgt agaggtttcc gcagggccgg
10681 ccggcatggc cagtgtgtgg gattacgacc tggtactgat ggcggtttcc catctaaccg
10741 aatccatgaa ccgataccgg gaagggaagg gagacaagcc cggccgcgtg ttccgtccac
10801 acgttgcgga cgtactcaag ttctgccggc gagccgatgg cggaaagcag aaagacgacc
10861 tggtagaaac ctgcattcgg ttaaacacca cgcacgttgc catgcagcgt acgaagaagg
10921 ccaagaacgg ccgcctggtg acggtatccg agggtgaagc cttgattagc cgctacaaga
10981 tcgtaaagag cgaaaccggg cggccggagt acatcgagat cgagctagct gattggatgt
11041 accgcgagat cacagaaggc aagaacccgg acgtgctgac ggttcacccc gattactttt
11101 tgatcgatcc cggcatcggc cgttttctct accgcctggc acgccgcgcc gcaggcaagg
11161 cagaagccag atggttgttc aagacgatct acgaacgcag tggcagcgcc ggagagttca
11221 agaagttctg tttcaccgtg cgcaagctga tcgggtcaaa tgacctgccg gagtacgatt
11281 tgaaggagga ggcggggcag gctggcccga tcctagtcat gcgctaccgc aacctgatcg
11341 agggcgaagc atccgccggt tcctaatgta cggagcagat gctagggcaa attgccctag
11401 caggggaaaa aggtcgaaaa gttctctttc ctgtggatag cacgtacatt gggaacccaa
11461 agccgtacat tgggaaccgg aacccgtaca ttgggaaccc aaagccgtac attgggaacc
11521 ggtcacacat gtaagtgact gatataaaag agaaaaaagg cgatttttcc gcctaaaact
11581 ctttaaaact tattaaaact cttaaaaccc gcctggcctg tgcataactg tctggccagc
11641 gcacagccga agagctgcaa aaagcgccta cccttcggtc gctgcgctcc ctacgccccg
11701 ccgcttcgcg tcggcctatc gcggccgctg gccgctcaaa aatggctggc ctacggccag
11761 gcaatctacc agggcgcgga caagccgcgc cgtcgccact cgaccgccgg cgcccacatc
11821 aaggcaccct gcctcgcgcg tttcggtgat gacggtgaaa acctctgaca catgcagctc
11881 ccggagacgg tcacagcttg tctgtaagcg gatgccggga gcagacaagc ccgtcagggc
11941 gcgtcagcgg gtgttggcgg gtgtcggggc gcagccatga cccagtcacg tagcgatagc
12001 ggagtgtata ctggcttaac tatgcggcat cagagcagat tgtactgaga gtgcaccata
12061 tgcggtgtga aataccgcac agatgcgtaa ggagaaaata ccgcatcagg cgctcttccg
12121 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
12181 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
12241 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
12301 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
12361 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
12421 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
12481 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
12541 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
12601 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
12661 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
12721 acggctacac tagaaggaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
12781 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
12841 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
12901 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatgc
12961 attctaggta ctaaaacaat tcatccagta aaatataata ttttattttc tcccaatcag
13021 gcttgatccc cagtaagtca aaaaatagct cgacatactg ttcttccccg atatcctccc
13081 tgatcgaccg gacgcagaag gcaatgtcat accacttgtc cgccctgccg cttctcccaa
13141 gatcaataaa gccacttact ttgccatctt tcacaaagat gttgctgtct cccaggtcgc
13201 cgtgggaaaa gacaagttcc tcttcgggct tttccgtctt taaaaaatca tacagctcgc
13261 gcggatcttt aaatggagtg tcttcttccc agttttcgca atccacatcg gccagatcgt
13321 tattcagtaa gtaatccaat tcggctaagc ggctgtctaa gctattcgta tagggacaat
13381 ccgatatgtc gatggagtga aagagcctga tgcactccgc atacagctcg ataatctttt
13441 cagggctttg ttcatcttca tactcttccg agcaaaggac gccatcggcc tcactcatga
13501 gcagattgct ccagccatca tgccgttcaa agtgcaggac ctttggaaca ggcagctttc
13561 cttccagcca tagcatcatg tccttttccc gttccacatc ataggtggtc cctttatacc
13621 ggctgtccgt catttttaaa tataggtttt cattttctcc caccagctta tataccttag
13681 caggagacat tccttccgta tcttttacgc agcggtattt ttcgatcagt tttttcaatt
13741 ccggtgatat tctcatttta gccatttatt atttccttcc tcttttctac agtatttaaa
13801 gataccccaa gaagctaagg aaggtgcgaa caagtccctg atatgagatc atgtttgtca
13861 tctggagcca tagaacaggg ttcatcatga gtcatcaact taccttcgcc gacagtgaat
13921 tcagcagtaa gcgccgtcag accagaaaag agattttctt gtcccgcatg gagcagattc
13981 tgccatggca aaacatggtg gaagtcatcg agccgtttta ccccaaggct ggtaatggcc
14041 ggcgacctta tccgctggaa accatgctac gcattcactg catgcagcat tggtacaacc
14101 tgagcgatgg cgcgatggaa gatgctctgt acgaaatcgc ctccatgcgt ctgtttgccc
14161 ggttatccct ggatagcgcc ttgccggacc gcaccaccat catgaatttc cgccacctgc
14221 tggagcagca tcaactggcc cgccaattgt tcaagaccat caatcgctgg ctggccgaag
14281 caggcgtcat gatgactcaa ggcaccttgg tcgatgccac catcattgag gcacccagct
14341 cgaccaagaa caaagagcag caacgcgatc cggagatgca tcagaccaag aaaggcaatc
14401 agtggcactt tggcatgaag gcccacattg gtgtcgatgc caagagtggc ctgacccaca
14461 gcctggtcac caccgcggcc aacgagcatg acctcaatca gctgggtaat ctgctgcatg
14521 gagaggagca atttgtctca gccgatgccg gctaccaagg ggcgccacag cgcgaggagc
14581 tggccgaggt ggatgtggac tggctgatcg ccgagcgccc cggcaaggta agaaccttga
14641 aacagcatcc acgcaagaac aaaacggcca tcaacatcga atacatgaaa gccagcatcc
14701 gggccagggt ggagcaccca tttcgcatca tcaagcgaca gttcggcttc gtgaaagcca
14761 gatacaaggg gttgctgaaa aacgataacc aactggcgat gttattcacg ctggccaacc
14821 tgtttcgggc ggaccaaatg atacgtcagt gggagagatc tcactaaaaa ctggggataa
14881 cgccttaaat ggcgaagaaa cggtctaaat aggctgattc aaggcattta cgggagaaaa
14941 aatcggctca aacatgaaga aatgaaatga ctgagtcagc cgagaagaat ttccccgctt
15001 attcgcacct tccctaatta taacaagacg aactccaatt cactgttcct tgcattctaa
15061 aaccttaaat accagaaaac agctttttca aagttgtttt caaagttggc gtataacata
15121 gtatcgacgg agccgatttt gaaaccgcgg tgatcacagg cagcaacgct ctgtcatcgt
15181 tacaatcaac atgctaccct ccgcgagatc atccgtgttt caaacccggc agcttagttg
15241 ccgttcttcc gaatagcatc ggtaacatga gcaaagtctg ccgccttaca acggctctcc
15301 cgctgacgcc gtcccggact gatgggctgc ctgtatcgag tggtgatttt gtgccgagct
15361 gccggtcggg gagctgttgg ctggctggtg gcaggatata ttgtggtgta aacaaattga
15421 cgcttagaca acttaataac acattgcgga cgtttttaat gtactgaatt aacgccgaat
15481 taattcgggg gatctggatt ttagtactgg attttggttt taggaattag aaattttatt
15541 gatagaagta ttttacaaat acaaatacat actaagggtt tcttatatgc tcaacacatg
15601 agcgaaaccc tataggaacc ctaattccct tatctgggaa ctactcacac attattatgg
15661 agaaactcga gcttgtcgat cgactctagc tagaggatcg atccgaaccc cagagtcccg
15721 ctcagaagaa ctcgtcaaga aggcgataga aggcgatgcg ctgcgaatcg ggagcggcga
15781 taccgtaaag cacgaggaag cggtcagccc attcgccgcc aagctcttca gcaatatcac
15841 gggtagccaa cgctatgtcc tgatagcggt ccgccacacc cagccggcca cagtcgatga
15901 atccagaaaa gcggccattt tccaccatga tattcggcaa gcaggcatcg ccatgtgtca
15961 cgacgagatc ctcgccgtcg ggcatgcgcg ccttgagcct ggcgaacagt tcggctggcg
16021 cgagcccctg atgctcttcg tccagatcat cctgatcgac aagaccggct tccatccgag
16081 tacgtgctcg ctcgatgcga tgtttcgctt ggtggtcgaa tgggcaggta gccggatcaa
16141 gcgtatgcag ccgccgcatt gcatcagcca tgatggatac tttctcggca ggagcaaggt
16201 gagatgacag gagatcctgc cccggcactt cgcccaatag cagccagtcc cttcccgctt
16261 cagtgacaac gtcgagcaca gctgcgcaag gaacgcccgt cgtggccagc cacgatagcc
16321 gcgctgcctc gtcctggagt tcattcaggg caccggacag gtcggtcttg acaaaaagaa
16381 ccgggcgccc ctgcgctgac agccgaaaca cggcggcatc agagcagccg attgtctgtt
16441 gtgcccagtc atagccgaat agcctctcca cccaagcggc cggagaacct gcgtgcaatc
16501 catcttgttc aatccccatg gtcgatcgac agatctgcga aagctcgaga gagatagatt
16561 tgtagagaga gactggtgat ttcagcgtgt cctctccaaa tgaaatgaac ttccttatat
16621 agaggaaggg tcttgcgaag gatagtggga ttgtgcgtca tcccttacgt cagtggagat
16681 atcacatcaa tccacttgct ttgaagacgt ggttggaacg tcttcttttt ccacgatgct
16741 cttcgtgggt gggggtccat ctttgggacc actgtcggca gaggcatctt gaacgatagc
16801 ctttccttta tcgcaatgat ggcatttgta ggtgccacct tccttttcta ctgtcctttt
16861 gatgaagtga cagatagctg ggcaatggaa tccgaggagg tttcccgata ttaccctttg
16921 ttgaaaagtc tcaatagccc tttggtcttc tgagactgta tctttgatat tcttggagta
16981 gacgagagtg tcgtgctcca ccatgttcac atcaatccac ttgctttgaa gacgtggttg
17041 gaacgtcttc tttttccacg atgctcctcg tgggtggggg tccatctttg ggaccactgt
17101 cggcagaggc atcttgaacg atagcctttc ctttatcgca atgatggcat ttgtaggtgc
17161 caccttcctt ttctactgtc cttttgatga agtgacagat agctgggcaa tggaatccga
17221 ggaggtttcc cgatattacc ctttgttgaa aagtctcaat agccctttgg tcttctgaga
17281 ctgtatcttt gatattcttg gagtagacga gagtgtcgtg ctccaccatg ttggcaagct
17341 gctctagcca atacg
//
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