| 质粒名称: | pYLCRISPR/Cas9P35S-H |
|---|---|
| 出品公司: | 爱迪基因 |
| 目录编号: | 66189 |
| 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 |
| 筛选标记: | 潮霉素 |
| 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: | S. pyogenes |
| 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 | -- |
| 启动子 | cauliflower mosaic virus 35S promoter (P35S) |
| 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 | M13pUC-Rev |
| 3' 测序引物 | NOSterm-R attgccaaatgtttgaacga |
|
文献引用方法 |
|
| 材料和方法部分: | pYLCRISPR/Cas9P35S-H was a gift from Yao-Guang Liu (Addgene plasmid # 66189 ; http://n2t.net/addgene:66189 ; RRID:Addgene_66189) |
| 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/Cas9P35S-H 16427 bp ds-DNA circular SYN 06-AUG-2018
DEFINITION expression of Cas9 in plants, cauliflower mosaic virus 35S promoter
(P35S), hygro selection.
ACCESSION .
VERSION .
KEYWORDS pYLCRISPR/Cas9P35S-H
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 16427)
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 16427)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Aug 6, 2018 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..16427
/organism="synthetic DNA construct"
/mol_type="other DNA"
protein_bind 349..370
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 385..415
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 423..439
/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 428..450
/label=M13/pUC Reverse
/note="In lacZ gene"
primer_bind 447..463
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind 447..463
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
CDS complement(804..851)
/codon_start=1
/product="bipartite nuclear localization signal from
nucleoplasmin"
/label=nucleoplasmin NLS
/translation="KRPAATKKAGQAKKKK"
CDS complement(852..4952)
/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 3758..3766
/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 complement(4977..4997)
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
promoter complement(5146..5490)
/label=CaMV 35S promoter
/note="strong constitutive promoter from cauliflower mosaic
virus"
protein_bind 6003..6024
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 6039..6069
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 6077..6093
/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 6135..6153
/label=SP6 promoter
/note="promoter for bacteriophage SP6 RNA polymerase"
primer_bind 6135..6152
/label=SP6
/note="SP6 promoter, forward primer"
CDS 6227..6529
/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
SRELYPVVHIGDESWRTMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
protein_bind complement(6739..6772)
/label=loxP
/bound_moiety="Cre recombinase"
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (GCATACAT)."
primer_bind complement(6802..6819)
/label=M13 Forward
/note="In lacZ gene. Also called M13-F20 or M13 (-21)
Forward"
primer_bind complement(6802..6818)
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(6811..6833)
/label=M13/pUC Forward
/note="In lacZ gene"
misc_feature 7021..7045
/label=RB T-DNA repeat
/note="right border repeat from nopaline C58 T-DNA"
CDS 8346..8975
/codon_start=1
/product="stability protein from the Pseudomonas plasmid
pVS1 (Heeb et al., 2000)"
/label=pVS1 StaA
/translation="MKVIAVLNQKGGSGKTTIATHLARALQLAGADVLLVDSDPQGSAR
DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP
VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI
LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI"
CDS 9404..10477
/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 10543..10737
/label=pVS1 oriV
/note="origin of replication for the Pseudomonas plasmid
pVS1 (Heeb et al., 2000)"
primer_bind complement(10973..10995)
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
misc_feature 11081..11221
/label=bom
/note="basis of mobility region from pBR322"
primer_bind 11136..11155
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind complement(11236..11253)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(11407..11995)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(11487..11506)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS complement(12082..12876)
/codon_start=1
/gene="aphA-3"
/product="aminoglycoside phosphotransferase"
/label=KanR
/note="confers resistance to kanamycin"
/translation="MAKMRISPELKKLIEKYRCVKDTEGMSPAKVYKLVGENENLYLKM
TDSRYKGTTYDVEREKDMMLWLEGKLPVPKVLHFERHDGWSNLLMSEADGVLCSEEYED
EQSPEKIIELYAECIRLFHSIDISDCPYTNSLDSRLAELDYLLNNDLADVDCENWEEDT
PFKDPRELYDFLKTEKPEEELVFSHGDLGDSNIFVKDGKVSGFIDLGRSGRADKWYDIA
FCVRSIREDIGEEQYVELFFDLLGIKPDWEKIKYYILLDELF"
misc_feature 14500..14524
/label=LB T-DNA repeat
/note="left border repeat from nopaline C58 T-DNA"
polyA_signal 14602..14776
/label=CaMV poly(A) signal
/note="cauliflower mosaic virus polyadenylation signal"
CDS complement(14817..15842)
/codon_start=1
/gene="aph(4)-Ia"
/product="aminoglycoside phosphotransferase from E. coli"
/label=HygR
/note="confers resistance to hygromycin"
/translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRSQGVTLQDLP
ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
K"
promoter complement(15909..158)
/label=CaMV 35S promoter (enhanced)
/note="cauliflower mosaic virus 35S promoter with a
duplicated enhancer region"
ORIGIN
1 cggcagaggc atcttgaacg atagcctttc ctttatcgca atgatggcat ttgtaggtgc
61 caccttcctt ttctactgtc cttttgatga agtgacagat agctgggcaa tggaatccga
121 ggaggtttcc cgatattacc ctttgttgaa aagtctcaat agccctttgg tcttctgaga
181 ctgtatcttt gatattcttg gagtagacga gagtgtcgtg ctccaccatg ttggcaagct
241 gctctagcca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg
301 gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta
361 gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg
421 aattgtgagc ggataacaat ttcacacagg aaacagctat gacatgatta cgaattcgag
481 ctcggtaccg ctcgctacct taagagagga tatccctcca tcctataatg taggctatag
541 gaactagggc aaggccggcc ttatgcggcc gcgtcactgg attttggttt taggaattag
601 aaattttatt gatagaagta ttttacaaat acaaatacat actaagggtt tcttatatgc
661 tcaacacatg agcgaaaccc tataagaacc ctaattccct tatctgggaa ctactcacac
721 attattctgg agaaaaatag agagagatag atttgtagag agagactggt gatttttgcg
781 gactctagat caggtggatc ctacttcttt ttcttagcct gtccggcctt tttggtggca
841 gcaggacgct tatcaccacc aagctgggaa aggtcgatac gagtctcgta aagaccggtg
901 atggactggt ggatgagagt agcgtcgaga acctccttgg tggacgtgta acgcttcctg
961 tcgatggtgg tgtcgaagta cttgaaagca gcaggggcgc cgaggttcgt gagcgtgaag
1021 aggtggatga tgttctcggc ctgctcgcgg atgggcttgt cgcggtgctt gttgtaggcg
1081 gagaggacct tgtcgaggtt agcgtcagcg aggatgacgc gcttggagaa ctcggagatc
1141 tgctcgatga tctcgtcgag gtagtgcttg tgctgctcca cgaagagctg cttctgctcg
1201 ttatcctcag gggaaccctt gagcttctcg tagtgggagg cgaggtagag gaagttcacg
1261 tacttggaag gaagagcaag ctcgttaccc ttctggagct caccagcgga agccagcatc
1321 ctcttacgac cgttctcgag ctcgaaaaga gagtacttgg ggagcttgat gatgaggtcc
1381 ttcttgacct ccttgtagcc cttggcctcg aggaagtcga tcgggttctt ctcgaaggag
1441 gagcgctcca tgatggtgat gccgaggagc tccttgacgg acttgagctt cttcgactta
1501 cccttctcca ccttggcgac cacgaggacg gagtaggcga cagtagggga gtcgaaacca
1561 ccgtacttct tagggtccca atccttcttc ctagcgatga gcttgtccga gtttctcttt
1621 ggaaggatag actccttgga gaagccaccg gtctggacct cggtcttctt gacgatgtta
1681 acctgaggca tagaaaggac cttgcgaaca gtagcgaagt cgcgaccctt gtcccaaacg
1741 atctcaccag tctcaccgtt cgtctcgata agagggcgct tgcggatctc gccgttggcg
1801 agggtgatct cggtcttgaa gaagttcatg atgttggagt agaagaagta cttggcggtg
1861 gccttgccga tctcctgctc ggacttggcg atcatcttac gaacgtcgta gaccttgtag
1921 tcaccgtaga cgaactcgga ctcaagctta gggtacttct tgataagagc ggtaccaacg
1981 acagcgttaa ggtaagcatc gtgagcgtgg tggtagttgt tgatctcgcg gaccttgtag
2041 aactggaagt ccttgcggaa gtcggagacg agcttggact tgagggtgat caccttgacc
2101 tcgcggatga gcttgtcgtt ctcgtcgtac ttggtgttca tcctagaatc gaggatctga
2161 gcaacgtgct tggtaatctg cctcgtctca acaagctgcc tcttgatgaa accagccttg
2221 tcaagctcgg aaaggccacc cctctcagcc ttcgtgaggt tgtcgaactt cctctgggta
2281 atgagcttag cgttgagaag ctgcctccag tagttcttca tcttcttgac aacctcctcg
2341 gaagggacgt tgtccgactt acccctgttc ttgtcggacc tcgtgaggac cttgttgtcg
2401 atggagtcat ccttaaggaa agactgagga acaatgtggt cgacgtcgta gtcagaaagc
2461 ctgttgatgt cgagctcctg gtcaacgtac atatccctac cgttctggag gtagtagagg
2521 tagagcttct cgttctggag ctgggtgttc tcgacagggt gctccttaag gatctgagaa
2581 ccaagctcct tgataccctc ctcaatcctc ttcatgcgct ccctcgagtt cttctgaccc
2641 ttctgggtag tctggttctc acgagccatc tcgatgacga tgttctcagg cttgtgacga
2701 cccataacct tgacaagctc atcgacaacc ttaacagtct gaaggatgcc cttcttgata
2761 gcaggggaac cagcaaggtt agcaatgtgc tcgtggagag agtcgccctg accggacacc
2821 tgagccttct gaatatcctc cttgaaggta agagagtcat cgtggatgag ctgcatgaag
2881 ttcctgttag cgaaaccatc agacttgagg aagtcgagga tagtcttgcc gctctgctta
2941 tccctgatac cgttgatgag cttgcgggag agcctacccc aaccggtgta acggcgacgc
3001 ttgagctgct tcataacctt gtcatcgaag agatgagcgt aagtcttgag cctctcctcg
3061 atcatctccc tatcctcgaa gagagtaaga gtgaggacga tgtcctcgag gatgtcctcg
3121 ttctcctcgt tgtcgaggaa gtccttgtcc ttgatgatct tgaggagatc gtggtaggta
3181 ccgagagaag cgttgaaacg gtcctcaacg ccgctgatct cgacggagtc gaagcactcg
3241 atcttcttga agtagtcctc cttgagctgc ttgacggtga ccttgcggtt ggtcttgaag
3301 aggaggtcaa cgatagcctt cttctgctcg ccggagagga aggcaggctt gcgcataccc
3361 tcggtgacgt acttgacctt ggtgagctcg ttgtagaccg tgaagtactc gtagaggagg
3421 gagtgcttgg ggaggacctt ctcgttgggg aggttcttgt cgaagttggt catgcgctcg
3481 atgaaggact gggcggaagc acccttgtca acaacctcct cgaagttcca aggggtgata
3541 gtctcctcgg acttcctagt catccaagcg aaacgggagt taccacgagc aagaggacca
3601 acgtagtaag gaatacggaa agtaaggatc ttctcgatct tctcacggtt gtccttgagg
3661 aaagggtaga agtcctcctg cctacgaagg atggcgtgaa gctcaccaag gtggatctgg
3721 tgagggatag agccgttatc gaaagtcctc tgcttcctaa ggaggtcctc acggttaagc
3781 ttaacgagaa gctcctcagt accatccatc ttctcaagga ttggcttgat gaacttgtag
3841 aactcctcct ggctagctcc accgtcaatg taaccggcgt agccgttctt ggactggtcg
3901 aagaagatct ccttgtactt ctcggggagc tgctgacgaa caagagcctt gaggagggtg
3961 aggtcctggt ggtgctcgtc gtagcgcttg atcatggaag cagaaagagg agccttagtg
4021 atctcagtgt taaccctaag gatatccgaa aggaggatag catcggagag gttcttagca
4081 gcaaggaaga gatcagcgta ctgatctcca atctgagcga ggaggttgtc gagatcatcg
4141 tcgtaggtat ccttggagag ctgaagctta gcgtcctcag cgaggtcgaa gttggacttg
4201 aagttagggg tcagaccgag ggagagagcg atgaggttac cgaaaagacc gttcttcttc
4261 tcaccaggga gctgggcgat gaggttctcg agacgcctgg acttggagag cctagcggaa
4321 aggatcgcct tagcgtcgac accggaagcg ttgatagggt tctcctcgaa gagctggttg
4381 taggtctgga cgagctggat gaagagcttg tccacgtcgg agttatcagg gttaaggtca
4441 ccctcgataa ggaagtgacc acggaacttg atcatgtgag cgagagcaag gtagatgaga
4501 cgaagatcag ccttatcagt agagtcaaca agcttcttac gaaggtggta gatagtgggg
4561 tacttctcgt ggtaggcgac ctcgtcgacg atgttgccga agatggggtg gcgctcgtgc
4621 ttcttgtcct cctccacgag gaaggactcc tcgaggcggt ggaagaagga gtcgtcgacc
4681 ttcgccatct cgttggagaa gatctcctgg aggtagcaga tgcggttctt gcggcgggtg
4741 tagcggcggc gggcggtgcg cttgaggcgg gtcgcctccg ccgtctcgcc ggagtcgaag
4801 aggagggcgc cgatgaggtt cttcttgatg gagtggcggt cggtgttgcc caggaccttg
4861 aacttcttgg acgggacctt gtactcgtcg gtgatcaccg cccagccgac gctgttggtg
4921 ccgatgtcga ggccgatgga gtacttcttg tcagccgcag gcaccccgtg aataccaacc
4981 ttccgcttct tcttaggagc catggctatc gttcgtaaat ggtgaaaatt ttcagaaaat
5041 tgcttttgct ttaaaagaaa tgatttaaat tgctgcaata gaagtagaat gcttgattgc
5101 ttgagattcg tttgttttgt atatgttgtg ttgagaactc tcgacgtcct ctccaaatga
5161 aatgaacttc cttatataga ggaagggtct tgcgaaggat agtgggattg tgcgtcatcc
5221 cttacgtcag tggagatatc acatcaatcc acttgctttg aagacgtggt tggaacgtct
5281 tctttttcca cgatgctcct cgtgggtggg ggtccatctt tgggaccact gtcggcagag
5341 gcatcttcaa cgatggcctt tcctttatcg caatgatggc atttgtagga gccaccttcc
5401 ttttccacta tcttcacaat aaagtgacag atagctgggc aatggaatcc gaggaggttt
5461 ccggatatta ccctttgttg aaaagtctca attgcccttt ggtcttctga gactgtatct
5521 ttgatatttt tggagtagac aagtgtgtcg tgctccacca tgttatcaca tcaatccact
5581 tgctttgaag acgtggttgg aacgtcttct ttttccacga tgctcctcgt gggtgggggt
5641 ccatctttgg gaccactgtc ggcagaggca tcttcaacga tggcctttcc tttatcgcaa
5701 tgatggcatt tgtaggagcc accttccttt tccactatct tcacaataaa gtgacagata
5761 gctgggcaat ggaatccgag gaggtttccg gatattaccc tttgttgaaa agtctcaatt
5821 gccctttggt cttctgagac tgtatctttg atatttttgg agtagacaag tgtgtcgtgc
5881 tccacatgtt gaccggtaag gcgcgccgta gtgctcgaga gacctctgaa gtggccgatt
5941 cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca
6001 attaatgtga gttagctcac tcattaggca ccccaggctt tacactttat gcttccggct
6061 cgtatgttgt gtggaattgt gagcggataa caatttcaca caagaaacag ctatgaccat
6121 gattacgcca agctatttag gtgacactat agaatactca agctatgcat caagctcaat
6181 gggtctagtc tgtagatacc catcacactg gcgaccgctc gaacatcagt ttaaggttta
6241 cacctataaa agagagagcc gttatcgtct gtttgtggat gtacagagtg atattattga
6301 cacgccgggg cgacggatgg tgatccccct ggccagtgca cgtctgctgt cagataaagt
6361 ctcccgtgaa ctttacccgg tggtgcatat cggggatgaa agctggcgca cgatgaccac
6421 cgatatggcc agtgtgcctg tctccgttat cggggaagaa gtggctgatc tcagccaccg
6481 cgaaaatgac atcaaaaacg ccattaacct gatgttctgg ggaatataaa tgtcaggcct
6541 gaatggcgaa tggacgcgcc ctgtagcggc gcattaagcg cggcgggtga gcgtgggtct
6601 cgcggtatca ttggcgcgcc tctcgagcta gcggccgcat gcatcgatct cctacatcgt
6661 ataaattagc ctatacgaag ttattgcatc tatgtcgggt gcggagaaag aggtaatgaa
6721 atggcagtat tagatctgat aacttcgtat aatgtatgct atacgaagtt atgactgcaa
6781 gtcgacaccc ataagcttgg cactggccgt cgttttacaa cgtcgtgact gggaaaaccc
6841 tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag
6901 cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatgcta
6961 gagcagcttg agcttggatc agattgtcgt ttcccgcctt cagtttaaac tatcagtgtt
7021 tgacaggata tattggcggg taaacctaag agaaaagagc gtttattaga ataatcggat
7081 atttaaaagg gcgtgaaaag gtttatccgt tcgtccattt gtatgtgcat gccaaccaca
7141 gggttcccct cgggatcaaa gtactttgat ccaacccctc cgctgctata gtgcagtcgg
7201 cttctgacgt tcagtgcagc cgtcttctga aaacgacatg tcgcacaagt cctaagttac
7261 gcgacaggct gccgccctgc ccttttcctg gcgttttctt gtcgcgtgtt ttagtcgcat
7321 aaagtagaat acttgcgact agaaccggag acattacgcc atgaacaaga gcgccgccgc
7381 tggcctgctg ggctatgccc gcgtcagcac cgacgaccag gacttgacca accaacgggc
7441 cgaactgcac gcggccggct gcaccaagct gttttccgag aagatcaccg gcaccaggcg
7501 cgaccgcccg gagctggcca ggatgcttga ccacctacgc cctggcgacg ttgtgacagt
7561 gaccaggcta gaccgcctgg cccgcagcac ccgcgaccta ctggacattg ccgagcgcat
7621 ccaggaggcc ggcgcgggcc tgcgtagcct ggcagagccg tgggccgaca ccaccacgcc
7681 ggccggccgc atggtgttga ccgtgttcgc cggcattgcc gagttcgagc gttccctaat
7741 catcgaccgc acccggagcg ggcgcgaggc cgccaaggcc cgaggcgtga agtttggccc
7801 ccgccctacc ctcaccccgg cacagatcgc gcacgcccgc gagctgatcg accaggaagg
7861 ccgcaccgtg aaagaggcgg ctgcactgct tggcgtgcat cgctcgaccc tgtaccgcgc
7921 acttgagcgc agcgaggaag tgacgcccac cgaggccagg cggcgcggtg ccttccgtga
7981 ggacgcattg accgaggccg acgccctggc ggccgccgag aatgaacgcc aagaggaaca
8041 agcatgaaac cgcaccagga cggccaggac gaaccgtttt tcattaccga agagatcgag
8101 gcggagatga tcgcggccgg gtacgtgttc gagccgcccg cgcacgtctc aaccgtgcgg
8161 ctgcatgaaa tcctggccgg tttgtctgat gccaagctgg cggcctggcc ggccagcttg
8221 gccgctgaag aaaccgagcg ccgccgtcta aaaaggtgat gtgtatttga gtaaaacagc
8281 ttgcgtcatg cggtcgctgc gtatatgatg cgatgagtaa ataaacaaat acgcaagggg
8341 aacgcatgaa ggttatcgct gtacttaacc agaaaggcgg gtcaggcaag acgaccatcg
8401 caacccatct agcccgcgcc ctgcaactcg ccggggccga tgttctgtta gtcgattccg
8461 atccccaggg cagtgcccgc gattgggcgg ccgtgcggga agatcaaccg ctaaccgttg
8521 tcggcatcga ccgcccgacg attgaccgcg acgtgaaggc catcggccgg cgcgacttcg
8581 tagtgatcga cggagcgccc caggcggcgg acttggctgt gtccgcgatc aaggcagccg
8641 acttcgtgct gattccggtg cagccaagcc cttacgacat atgggccacc gccgacctgg
8701 tggagctggt taagcagcgc attgaggtca cggatggaag gctacaagcg gcctttgtcg
8761 tgtcgcgggc gatcaaaggc acgcgcatcg gcggtgaggt tgccgaggcg ctggccgggt
8821 acgagctgcc cattcttgag tcccgtatca cgcagcgcgt gagctaccca ggcactgccg
8881 ccgccggcac aaccgttctt gaatcagaac ccgagggcga cgctgcccgc gaggtccagg
8941 cgctggccgc tgaaattaaa tcaaaactca tttgagttaa tgaggtaaag agaaaatgag
9001 caaaagcaca aacacgctaa gtgccggccg tccgagcgca cgcagcagca aggctgcaac
9061 gttggccagc ctggcagaca cgccagccat gaagcgggtc aactttcagt tgccggcgga
9121 ggatcacacc aagctgaaga tgtacgcggt acgccaaggc aagaccatta ccgagctgct
9181 atctgaatac atcgcgcagc taccagagta aatgagcaaa tgaataaatg agtagatgaa
9241 ttttagcggc taaaggaggc ggcatggaaa atcaagaaca accaggcacc gacgccgtgg
9301 aatgccccat gtgtggagga acgggcggtt ggccaggcgt aagcggctgg gttgcctgcc
9361 ggccctgcaa tggcactgga acccccaagc ccgaggaatc ggcgtgagcg gtcgcaaacc
9421 atccggcccg gtacaaatcg gcgcggcgct gggtgatgac ctggtggaga agttgaaggc
9481 cgcgcaggcc gcccagcggc aacgcatcga ggcagaagca cgccccggtg aatcgtggca
9541 agcggccgct gatcgaatcc gcaaagaatc ccggcaaccg ccggcagccg gtgcgccgtc
9601 gattaggaag ccgcccaagg gcgacgagca accagatttt ttcgttccga tgctctatga
9661 cgtgggcacc cgcgatagtc gcagcatcat ggacgtggcc gttttccgtc tgtcgaagcg
9721 tgaccgacga gctggcgagg tgatccgcta cgagcttcca gacgggcacg tagaggtttc
9781 cgcagggccg gccggcatgg ccagtgtgtg ggattacgac ctggtactga tggcggtttc
9841 ccatctaacc gaatccatga accgataccg ggaagggaag ggagacaagc ccggccgcgt
9901 gttccgtcca cacgttgcgg acgtactcaa gttctgccgg cgagccgatg gcggaaagca
9961 gaaagacgac ctggtagaaa cctgcattcg gttaaacacc acgcacgttg ccatgcagcg
10021 tacgaagaag gccaagaacg gccgcctggt gacggtatcc gagggtgaag ccttgattag
10081 ccgctacaag atcgtaaaga gcgaaaccgg gcggccggag tacatcgaga tcgagctagc
10141 tgattggatg taccgcgaga tcacagaagg caagaacccg gacgtgctga cggttcaccc
10201 cgattacttt ttgatcgatc ccggcatcgg ccgttttctc taccgcctgg cacgccgcgc
10261 cgcaggcaag gcagaagcca gatggttgtt caagacgatc tacgaacgca gtggcagcgc
10321 cggagagttc aagaagttct gtttcaccgt gcgcaagctg atcgggtcaa atgacctgcc
10381 ggagtacgat ttgaaggagg aggcggggca ggctggcccg atcctagtca tgcgctaccg
10441 caacctgatc gagggcgaag catccgccgg ttcctaatgt acggagcaga tgctagggca
10501 aattgcccta gcaggggaaa aaggtcgaaa agttctcttt cctgtggata gcacgtacat
10561 tgggaaccca aagccgtaca ttgggaaccg gaacccgtac attgggaacc caaagccgta
10621 cattgggaac cggtcacaca tgtaagtgac tgatataaaa gagaaaaaag gcgatttttc
10681 cgcctaaaac tctttaaaac ttattaaaac tcttaaaacc cgcctggcct gtgcataact
10741 gtctggccag cgcacagccg aagagctgca aaaagcgcct acccttcggt cgctgcgctc
10801 cctacgcccc gccgcttcgc gtcggcctat cgcggccgct ggccgctcaa aaatggctgg
10861 cctacggcca ggcaatctac cagggcgcgg acaagccgcg ccgtcgccac tcgaccgccg
10921 gcgcccacat caaggcaccc tgcctcgcgc gtttcggtga tgacggtgaa aacctctgac
10981 acatgcagct cccggagacg gtcacagctt gtctgtaagc ggatgccggg agcagacaag
11041 cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg cgcagccatg acccagtcac
11101 gtagcgatag cggagtgtat actggcttaa ctatgcggca tcagagcaga ttgtactgag
11161 agtgcaccat atgcggtgtg aaataccgca cagatgcgta aggagaaaat accgcatcag
11221 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
11281 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
11341 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
11401 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
11461 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
11521 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
11581 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
11641 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
11701 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
11761 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
11821 gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc
11881 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
11941 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
12001 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
12061 tttggtcatg cattctaggt actaaaacaa ttcatccagt aaaatataat attttatttt
12121 ctcccaatca ggcttgatcc ccagtaagtc aaaaaatagc tcgacatact gttcttcccc
12181 gatatcctcc ctgatcgacc ggacgcagaa ggcaatgtca taccacttgt ccgccctgcc
12241 gcttctccca agatcaataa agccacttac tttgccatct ttcacaaaga tgttgctgtc
12301 tcccaggtcg ccgtgggaaa agacaagttc ctcttcgggc ttttccgtct ttaaaaaatc
12361 atacagctcg cgcggatctt taaatggagt gtcttcttcc cagttttcgc aatccacatc
12421 ggccagatcg ttattcagta agtaatccaa ttcggctaag cggctgtcta agctattcgt
12481 atagggacaa tccgatatgt cgatggagtg aaagagcctg atgcactccg catacagctc
12541 gataatcttt tcagggcttt gttcatcttc atactcttcc gagcaaagga cgccatcggc
12601 ctcactcatg agcagattgc tccagccatc atgccgttca aagtgcagga cctttggaac
12661 aggcagcttt ccttccagcc atagcatcat gtccttttcc cgttccacat cataggtggt
12721 ccctttatac cggctgtccg tcatttttaa atataggttt tcattttctc ccaccagctt
12781 atatacctta gcaggagaca ttccttccgt atcttttacg cagcggtatt tttcgatcag
12841 ttttttcaat tccggtgata ttctcatttt agccatttat tatttccttc ctcttttcta
12901 cagtatttaa agatacccca agaagctaag gaaggtgcga acaagtccct gatatgagat
12961 catgtttgtc atctggagcc atagaacagg gttcatcatg agtcatcaac ttaccttcgc
13021 cgacagtgaa ttcagcagta agcgccgtca gaccagaaaa gagattttct tgtcccgcat
13081 ggagcagatt ctgccatggc aaaacatggt ggaagtcatc gagccgtttt accccaaggc
13141 tggtaatggc cggcgacctt atccgctgga aaccatgcta cgcattcact gcatgcagca
13201 ttggtacaac ctgagcgatg gcgcgatgga agatgctctg tacgaaatcg cctccatgcg
13261 tctgtttgcc cggttatccc tggatagcgc cttgccggac cgcaccacca tcatgaattt
13321 ccgccacctg ctggagcagc atcaactggc ccgccaattg ttcaagacca tcaatcgctg
13381 gctggccgaa gcaggcgtca tgatgactca aggcaccttg gtcgatgcca ccatcattga
13441 ggcacccagc tcgaccaaga acaaagagca gcaacgcgat ccggagatgc atcagaccaa
13501 gaaaggcaat cagtggcact ttggcatgaa ggcccacatt ggtgtcgatg ccaagagtgg
13561 cctgacccac agcctggtca ccaccgcggc caacgagcat gacctcaatc agctgggtaa
13621 tctgctgcat ggagaggagc aatttgtctc agccgatgcc ggctaccaag gggcgccaca
13681 gcgcgaggag ctggccgagg tggatgtgga ctggctgatc gccgagcgcc ccggcaaggt
13741 aagaaccttg aaacagcatc cacgcaagaa caaaacggcc atcaacatcg aatacatgaa
13801 agccagcatc cgggccaggg tggagcaccc atttcgcatc atcaagcgac agttcggctt
13861 cgtgaaagcc agatacaagg ggttgctgaa aaacgataac caactggcga tgttattcac
13921 gctggccaac ctgtttcggg cggaccaaat gatacgtcag tgggagagat ctcactaaaa
13981 actggggata acgccttaaa tggcgaagaa acggtctaaa taggctgatt caaggcattt
14041 acgggagaaa aaatcggctc aaacatgaag aaatgaaatg actgagtcag ccgagaagaa
14101 tttccccgct tattcgcacc ttccctaatt ataacaagac gaactccaat tcactgttcc
14161 ttgcattcta aaaccttaaa taccagaaaa cagctttttc aaagttgttt tcaaagttgg
14221 cgtataacat agtatcgacg gagccgattt tgaaaccgcg gtgatcacag gcagcaacgc
14281 tctgtcatcg ttacaatcaa catgctaccc tccgcgagat catccgtgtt tcaaacccgg
14341 cagcttagtt gccgttcttc cgaatagcat cggtaacatg agcaaagtct gccgccttac
14401 aacggctctc ccgctgacgc cgtcccggac tgatgggctg cctgtatcga gtggtgattt
14461 tgtgccgagc tgccggtcgg ggagctgttg gctggctggt ggcaggatat attgtggtgt
14521 aaacaaattg acgcttagac aacttaataa cacattgcgg acgtttttaa tgtactgaat
14581 taacgccgaa ttaattcggg ggatctggat tttagtactg gattttggtt ttaggaatta
14641 gaaattttat tgatagaagt attttacaaa tacaaataca tactaagggt ttcttatatg
14701 ctcaacacat gagcgaaacc ctataggaac cctaattccc ttatctggga actactcaca
14761 cattattatg gagaaactcg agcttgtcga tcgacagatc ccggtcggca tctactctat
14821 ttctttgccc tcggacgagt gctggggcgt cggtttccac tatcggcgag tacttctaca
14881 cagccatcgg tccagacggc cgcgcttctg cgggcgattt gtgtacgccc gacagtcccg
14941 gctccggatc ggacgattgc gtcgcatcga ccctgcgccc aagctgcatc atcgaaattg
15001 ccgtcaacca agctctgata gagttggtca agaccaatgc ggagcatata cgcccggagt
15061 cgtggcgatc ctgcaagctc cggatgcctc cgctcgaagt agcgcgtctg ctgctccata
15121 caagccaacc acggcctcca gaagaagatg ttggcgacct cgtattggga atccccgaac
15181 atcgcctcgc tccagtcaat gaccgctgtt atgcggccat tgtccgtcag gacattgttg
15241 gagccgaaat ccgcgtgcac gaggtgccgg acttcggggc agtcctcggc ccaaagcatc
15301 agctcatcga gagcctgcgc gacggacgca ctgacggtgt cgtccatcac agtttgccag
15361 tgatacacat ggggatcagc aatcgcgcat atgaaatcac gccatgtagt gtattgaccg
15421 attccttgcg gtccgaatgg gccgaacccg ctcgtctggc taagatcggc cgcagcgatc
15481 gcatccatag cctccgcgac cggttgtaga acagcgggca gttcggtttc aggcaggtct
15541 tgcaacgtga caccctgtga acggcgggag atgcaatagg tcaggctctc gctaaactcc
15601 ccaatgtcaa gcacttccgg aatcgggagc gcggccgatg caaagtgccg ataaacataa
15661 cgatctttgt agaaaccatc ggcgcagcta tttacccgca ggacatatcc acgccctcct
15721 acatcgaagc tgaaagcacg agattcttcg ccctccgaga gctgcatcag gtcggagacg
15781 ctgtcgaact tttcgatcag aaacttctcg acagacgtcg cggtgagttc aggctttttc
15841 atatctcatt gccccccgga tctgcgaaag ctcgagagag atagatttgt agagagagac
15901 tggtgatttc agcgtgtcct ctccaaatga aatgaacttc cttatataga ggaagggtct
15961 tgcgaaggat agtgggattg tgcgtcatcc cttacgtcag tggagatatc acatcaatcc
16021 acttgctttg aagacgtggt tggaacgtct tctttttcca cgatgctctt cgtgggtggg
16081 ggtccatctt tgggaccact gtcggcagag gcatcttgaa cgatagcctt tcctttatcg
16141 caatgatggc atttgtaggt gccaccttcc ttttctactg tccttttgat gaagtgacag
16201 atagctgggc aatggaatcc gaggaggttt cccgatatta ccctttgttg aaaagtctca
16261 atagcccttt ggtcttctga gactgtatct ttgatattct tggagtagac gagagtgtcg
16321 tgctccacca tgttcacatc aatccacttg ctttgaagac gtggttggaa cgtcttcttt
16381 ttccacgatg ctcctcgtgg gtgggggtcc atctttggga ccactgt
//
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