| 质粒名称: | pHEE401E |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 71287 |
| 存储实验室: | Qi-Jun Chen |
|
载体骨架基本信息 |
|
| 载体名称: | pCambia |
| 载体出品公司: | -- |
| 载体抗性: | 卡那霉素 |
| 空载体大小: | -- |
| 完整质粒大小: | -- |
| 载体修饰: | -- |
| 载体类型: | 植物表达，CRISPR；植物二元载体 |
| 筛选标记: | 潮霉素 |
| 高拷贝/低拷贝: | 高拷贝 |
| 生长条件: | 37 ℃ |
|
插入基因信息 |
|
| 插入基因名称: | gRNA scaffold |
| 物种来源: | Synthetic |
| 插入大小: | -- |
| 基因突变: | -- |
| GenBank编号 | -- |
| 启动子 | U6-26p Arabidopsis U6 gene promoter |
| 标签/融合蛋白: | -- |
| 克隆方法: | 限制性内切酶 |
| 5' 克隆位点: | BsaI(未破坏) |
| 3' 克隆位点: | BsaI(未破坏) |
| 5' 测序引物 | M13-fwd |
| 3' 测序引物 | -- |
|
文献引用方法 |
|
| 材料和方法部分: | pHEE401E was a gift from Qi-Jun Chen (Addgene plasmid # 71287 ; http://n2t.net/addgene:71287 ; RRID:Addgene_71287) |
| 参考文献部分: | Egg cell-specific promoter-controlled CRISPR/Cas9 efficiently generates homozygous mutants for multiple target genes in Arabidopsis in a single generation. Wang ZP, Xing HL, Dong L, Zhang HY, Han CY, Wang XC, Chen QJ. Genome Biol. 2015 Jul 21;16:144. doi: 10.1186/s13059-015-0715-0. 10.1186/s13059-015-0715-0 [pii] PubMed 26193878 |
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 pHEE401E 17112 bp ds-DNA circular SYN 12-DEC-2018
DEFINITION Egg cell-specific promoter-controlled expression of
3×FLAG-NLS-zCas9-NLS. Contains gRNA scaffold for insertion of target
sequence (U6-26 promoter), Hyg resistance.
ACCESSION .
VERSION .
KEYWORDS pHEE401E
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 17112)
AUTHORS Wang ZP, Xing HL, Dong L, Zhang HY, Han CY, Wang XC, Chen QJ
TITLE Egg cell-specific promoter-controlled CRISPR/Cas9 efficiently
generates homozygous mutants for multiple target genes in
Arabidopsis in a single generation.
JOURNAL Genome Biol. 2015 Jul 21;16:144. doi: 10.1186/s13059-015-0715-0.
PUBMED 26193878
REFERENCE 2 (bases 1 to 17112)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Dec 12, 2018 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..17112
/organism="synthetic DNA construct"
/mol_type="other DNA"
primer_bind 78..100
/label=M13/pUC Forward
/note="In lacZ gene"
primer_bind 92..109
/label=M13 Forward
/note="In lacZ gene. Also called M13-F20 or M13 (-21)
Forward"
primer_bind 93..109
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
CDS 822..1613
/codon_start=1
/gene="aadA"
/product="aminoglycoside adenylyltransferase (Murphy,
1985)"
/label=SmR
/note="confers resistance to spectinomycin and
streptomycin"
/translation="MGEAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
misc_RNA 1765..1840
/label=gRNA scaffold
/note="guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system"
CDS 3410..3475
/codon_start=1
/product="three tandem FLAG(R) epitope tags, followed by an
enterokinase cleavage site"
/label=3xFLAG
/translation="DYKDHDGDYKDHDIDYKDDDDK"
CDS 3482..3502
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 3527..7627
/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 7540..7548
/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 7628..7675
/codon_start=1
/product="bipartite nuclear localization signal from
nucleoplasmin"
/label=nucleoplasmin NLS
/translation="KRPAATKKAGQAKKKK"
primer_bind complement(8332..8348)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(8332..8348)
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind complement(8345..8367)
/label=M13/pUC Reverse
/note="In lacZ gene"
protein_bind 8356..8372
/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(8380..8410)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 8425..8446
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 8637..9313
/label=CaMV 35S promoter (enhanced)
/note="cauliflower mosaic virus 35S promoter with a
duplicated enhancer region"
primer_bind 9250..9270
/label=35S promoter
/note="CaMV 35S promoter, forward primer"
CDS 9380..10405
/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"
polyA_signal 10446..10620
/label=CaMV poly(A) signal
/note="cauliflower mosaic virus polyadenylation signal"
misc_feature 10698..10722
/label=LB T-DNA repeat
/note="left border repeat from nopaline C58 T-DNA"
CDS 11147..11941
/codon_start=1
/gene="aphA-3"
/product="aminoglycoside phosphotransferase"
/label=KanR
/note="confers resistance to kanamycin"
/translation="MAKMRISPELKKLIEKYRCVKDTEGMSPAKVYKLVGENENLYLKM
TDSRYKGTTYDVEREKDMMLWLEGKLPVPKVLHFERHDGWSNLLMSEADGVLCSEEYED
EQSPEKIIELYAECIRLFHSIDISDCPYTNSLDSRLAELDYLLNNDLADVDCENWEEDT
PFKDPRELYDFLKTEKPEEELVFSHGDLGDSNIFVKDGKVSGFIDLGRSGRADKWYDIA
FCVRSIREDIGEEQYVELFFDLLGIKPDWEKIKYYILLDELF"
rep_origin 12028..12616
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 12517..12536
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
primer_bind 12770..12787
/label=L4440
/note="L4440 vector, forward primer"
misc_feature 12802..12942
/label=bom
/note="basis of mobility region from pBR322"
primer_bind complement(12868..12887)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 13028..13050
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
rep_origin 13286..13480
/label=pVS1 oriV
/note="origin of replication for the Pseudomonas plasmid
pVS1 (Heeb et al., 2000)"
CDS complement(13546..14619)
/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"
CDS complement(15048..15677)
/codon_start=1
/product="stability protein from the Pseudomonas plasmid
pVS1 (Heeb et al., 2000)"
/label=pVS1 StaA
/translation="MKVIAVLNQKGGSGKTTIATHLARALQLAGADVLLVDSDPQGSAR
DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP
VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI
LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI"
misc_feature 16978..17002
/label=RB T-DNA repeat
/note="right border repeat from nopaline C58 T-DNA"
ORIGIN
1 ggcctcttcg ctattacgcc agctggcgaa agggggatgt gctgcaaggc gattaagttg
61 ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg acggccagtg ccaagcttcg
121 acttgccttc cgcacaatac atcatttctt cttagctttt tttcttcttc ttcgttcata
181 cagttttttt ttgtttatca gcttacattt tcttgaaccg tagctttcgt tttcttcttt
241 ttaactttcc attcggagtt tttgtatctt gtttcatagt ttgtcccagg attagaatga
301 ttaggcatcg aaccttcaag aatttgattg aataaaacat cttcattctt aagatatgaa
361 gataatcttc aaaaggcccc tgggaatctg aaagaagaga agcaggccca tttatatggg
421 aaagaacaat agtatttctt atataggccc atttaagttg aaaacaatct tcaaaagtcc
481 cacatcgctt agataagaaa acgaagctga gtttatatac agctagagtc gaagtagtga
541 ttgggagacc aacccagtgg acataagcct gttcggttcg taagctgtaa tgcaagtagc
601 gtatgcgctc acgcaactgg tccagaacct tgaccgaacg cagcggtggt aacggcgcag
661 tggcggtttt catggcttgt tatgactgtt tttttggggt acagtctatg cctcgggcat
721 ccaagcagca agcgcgttac gccgtgggtc gatgtttgat gttatggagc agcaacgatg
781 ttacgcagca gggcagtcgc cctaaaacaa agttaaacat catgggggaa gcggtgatcg
841 ccgaagtatc gactcaacta tcagaggtag ttggcgtcat cgagcgccat ctcgaaccga
901 cgttgctggc cgtacatttg tacggctccg cagtggatgg cggcctgaag ccacacagtg
961 atattgattt gctggttacg gtgaccgtaa ggcttgatga aacaacgcgg cgagctttga
1021 tcaacgacct tttggaaact tcggcttccc ctggagagag cgagattctc cgcgctgtag
1081 aagtcaccat tgttgtgcac gacgacatca ttccgtggcg ttatccagct aagcgcgaac
1141 tgcaatttgg agaatggcag cgcaatgaca ttcttgcagg tatcttcgag ccagccacga
1201 tcgacattga tctggctatc ttgctgacaa aagcaagaga acatagcgtt gccttggtag
1261 gtccagcggc ggaggaactc tttgatccgg ttcctgaaca ggatctattt gaggcgctaa
1321 atgaaacctt aacgctatgg aactcgccgc ccgactgggc tggcgatgag cgaaatgtag
1381 tgcttacgtt gtcccgcatt tggtacagcg cagtaaccgg caaaatcgcg ccgaaggatg
1441 tcgctgccga ctgggcaatg gagcgcctgc cggcccagta tcagcccgtc atacttgaag
1501 ctagacaggc ttatcttgga caagaagaag atcgcttggc ctcgcgcgca gatcagttgg
1561 aagaatttgt ccactacgtg aaaggcgaga tcaccaaggt agtcggcaaa taatgtctag
1621 ctagaaattc gttcaagccg acgccgcttc gcggcgcggc ttaactcaag cgttagatgc
1681 actaagcaca taattgctca cagccaaact atcaggtcaa gtctgctttt attattttta
1741 agcgtgcata ataagccggt ctcggtttta gagctagaaa tagcaagtta aaataaggct
1801 agtccgttat caacttgaaa aagtggcacc gagtcggtgc ttttttttgc aaaattttcc
1861 agatcgattt cttcttcctc tgttcttcgg cgttcaattt ctggggtttt ctcttcgttt
1921 tctgtaactg aaacctaaaa tttgacctaa aaaaaatctc aaataatatg attcagtggt
1981 tttgtacttt tcagttagtt gagttttgca gttccgatga gataaaccaa taccatggtt
2041 atactagtga ataaaagcat ttgcgtttgg tttatcattg cgtttataca aggacagaga
2101 tccactgagc tggaatagct taaaaccatt atcagaacaa aataaaccat tttttgttaa
2161 gaatcagagc atagtaaaca acagaaacaa cctaagagag gtaacttgtc caagaagata
2221 gctaattata tctattttat aaaagttatc atagtttgta agtcacaaaa gatgcaaata
2281 acagagaaac taggagactt gagaatatac attcttgtat atttgtattc gagattgtga
2341 aaatttgacc ataagtttaa attcttaaaa agatatatct gatctaggtg atggttatag
2401 actgtaattt taccacatgt ttaatgatgg atagtgacac acatgacaca tcgacaacac
2461 tatagcatct tatttagatt acaacatgaa atttttctgt aatacatgtc tttgtacata
2521 atttaaaagt aattcctaag aaatatattt atacaaggag tttaaagaaa acatagcata
2581 aagttcaatg agtagtaaaa accatataca gtatatagca taaagttcaa tgagtttatt
2641 acaaaagcat tggttcactt tctgtaacac gacgttaaac cttcgtctcc aataggagcg
2701 ctactgattc aacatgccaa tatatactaa atacgtttct acagtcaaat gctttaacgt
2761 ttcatgatta agtgactatt taccgtcaat cctttcccat tcctcccact aatccaactt
2821 tttaattact cttaaatcac cactaagcta gtaacgccta tcatgaatta gctctactaa
2881 atctagcaac ctttcaaatt tgcagtattg caggtgtctc tgtgtcttta aaatagttgc
2941 cttatgattt cttcggtttc aagatgatca aatagttata gatttcatgc tcacacatgc
3001 tcattagatg tgtacatact ttacttaccc aaatctattt tctcgcaaag attttgatgg
3061 taaagctgat ttggttctat tgaactaaat caaacgagtt tcagactgag tgattctaat
3121 ccggcccatt agcccctaaa cagacccact aattacgcag cttttaatag agtaattaca
3181 cctagtttac ccactaaacc actaagcact aattatctca caatctaatg agcttccctc
3241 gtaattactt gggctttcac tctaccattt atttgtaaca gtcaagtctc tactgtctct
3301 atataaactc tctaaagtta acacacaatt ctcatcacaa acaaatcaac caaagcaact
3361 tctactcttt cttctttcga ccttatcaat ctgttgagaa atctagatgg attacaagga
3421 ccacgacggg gattacaagg accacgacat tgattacaag gatgatgatg acaagatggc
3481 tccgaagaag aagaggaagg ttggcatcca cggggtgcca gctgctgaca agaagtactc
3541 gatcggcctc gatattggga ctaactctgt tggctgggcc gtgatcaccg acgagtacaa
3601 ggtgccctca aagaagttca aggtcctggg caacaccgat cggcattcca tcaagaagaa
3661 tctcattggc gctctcctgt tcgacagcgg cgagacggct gaggctacgc ggctcaagcg
3721 caccgcccgc aggcggtaca cgcgcaggaa gaatcgcatc tgctacctgc aggagatttt
3781 ctccaacgag atggcgaagg ttgacgattc tttcttccac aggctggagg agtcattcct
3841 cgtggaggag gataagaagc acgagcggca tccaatcttc ggcaacattg tcgacgaggt
3901 tgcctaccac gagaagtacc ctacgatcta ccatctgcgg aagaagctcg tggactccac
3961 agataaggcg gacctccgcc tgatctacct cgctctggcc cacatgatta agttcagggg
4021 ccatttcctg atcgaggggg atctcaaccc ggacaatagc gatgttgaca agctgttcat
4081 ccagctcgtg cagacgtaca accagctctt cgaggagaac cccattaatg cgtcaggcgt
4141 cgacgcgaag gctatcctgt ccgctaggct ctcgaagtct cggcgcctcg agaacctgat
4201 cgcccagctg ccgggcgaga agaagaacgg cctgttcggg aatctcattg cgctcagcct
4261 ggggctcacg cccaacttca agtcgaattt cgatctcgct gaggacgcca agctgcagct
4321 ctccaaggac acatacgacg atgacctgga taacctcctg gcccagatcg gcgatcagta
4381 cgcggacctg ttcctcgctg ccaagaatct gtcggacgcc atcctcctgt ctgatattct
4441 cagggtgaac accgagatta cgaaggctcc gctctcagcc tccatgatca agcgctacga
4501 cgagcaccat caggatctga ccctcctgaa ggcgctggtc aggcagcagc tccccgagaa
4561 gtacaaggag atcttcttcg atcagtcgaa gaacggctac gctgggtaca ttgacggcgg
4621 ggcctctcag gaggagttct acaagttcat caagccgatt ctggagaaga tggacggcac
4681 ggaggagctg ctggtgaagc tcaatcgcga ggacctcctg aggaagcagc ggacattcga
4741 taacggcagc atcccacacc agattcatct cggggagctg cacgctatcc tgaggaggca
4801 ggaggacttc taccctttcc tcaaggataa ccgcgagaag atcgagaaga ttctgacttt
4861 caggatcccg tactacgtcg gcccactcgc taggggcaac tcccgcttcg cttggatgac
4921 ccgcaagtca gaggagacga tcacgccgtg gaacttcgag gaggtggtcg acaagggcgc
4981 tagcgctcag tcgttcatcg agaggatgac gaatttcgac aagaacctgc caaatgagaa
5041 ggtgctccct aagcactcgc tcctgtacga gtacttcaca gtctacaacg agctgactaa
5101 ggtgaagtat gtgaccgagg gcatgaggaa gccggctttc ctgtctgggg agcagaagaa
5161 ggccatcgtg gacctcctgt tcaagaccaa ccggaaggtc acggttaagc agctcaagga
5221 ggactacttc aagaagattg agtgcttcga ttcggtcgag atctctggcg ttgaggaccg
5281 cttcaacgcc tccctgggga cctaccacga tctcctgaag atcattaagg ataaggactt
5341 cctggacaac gaggagaatg aggatatcct cgaggacatt gtgctgacac tcactctgtt
5401 cgaggaccgg gagatgatcg aggagcgcct gaagacttac gcccatctct tcgatgacaa
5461 ggtcatgaag cagctcaaga ggaggaggta caccggctgg gggaggctga gcaggaagct
5521 catcaacggc attcgggaca agcagtccgg gaagacgatc ctcgacttcc tgaagagcga
5581 tggcttcgcg aaccgcaatt tcatgcagct gattcacgat gacagcctca cattcaagga
5641 ggatatccag aaggctcagg tgagcggcca gggggactcg ctgcacgagc atatcgcgaa
5701 cctcgctggc tcgccagcta tcaagaaggg gattctgcag accgtgaagg ttgtggacga
5761 gctggtgaag gtcatgggca ggcacaagcc tgagaacatc gtcattgaga tggcccggga
5821 gaatcagacc acgcagaagg gccagaagaa ctcacgcgag aggatgaaga ggatcgagga
5881 gggcattaag gagctggggt cccagatcct caaggagcac ccggtggaga acacgcagct
5941 gcagaatgag aagctctacc tgtactacct ccagaatggc cgcgatatgt atgtggacca
6001 ggagctggat attaacaggc tcagcgatta cgacgtcgat catatcgttc cacagtcatt
6061 cctgaaggat gactccattg acaacaaggt cctcaccagg tcggacaaga accggggcaa
6121 gtctgataat gttccttcag aggaggtcgt taagaagatg aagaactact ggcgccagct
6181 cctgaatgcc aagctgatca cgcagcggaa gttcgataac ctcacaaagg ctgagagggg
6241 cgggctctct gagctggaca aggcgggctt catcaagagg cagctggtcg agacacggca
6301 gatcactaag cacgttgcgc agattctcga ctcacggatg aacactaagt acgatgagaa
6361 tgacaagctg atccgcgagg tgaaggtcat caccctgaag tcaaagctcg tctccgactt
6421 caggaaggat ttccagttct acaaggttcg ggagatcaac aattaccacc atgcccatga
6481 cgcgtacctg aacgcggtgg tcggcacagc tctgatcaag aagtacccaa agctcgagag
6541 cgagttcgtg tacggggact acaaggttta cgatgtgagg aagatgatcg ccaagtcgga
6601 gcaggagatt ggcaaggcta ccgccaagta cttcttctac tctaacatta tgaatttctt
6661 caagacagag atcactctgg ccaatggcga gatccggaag cgccccctca tcgagacgaa
6721 cggcgagacg ggggagatcg tgtgggacaa gggcagggat ttcgcgaccg tcaggaaggt
6781 tctctccatg ccacaagtga atatcgtcaa gaagacagag gtccagactg gcgggttctc
6841 taaggagtca attctgccta agcggaacag cgacaagctc atcgcccgca agaaggactg
6901 ggatccgaag aagtacggcg ggttcgacag ccccactgtg gcctactcgg tcctggttgt
6961 ggcgaaggtt gagaagggca agtccaagaa gctcaagagc gtgaaggagc tgctggggat
7021 cacgattatg gagcgctcca gcttcgagaa gaacccgatc gatttcctgg aggcgaaggg
7081 ctacaaggag gtgaagaagg acctgatcat taagctcccc aagtactcac tcttcgagct
7141 ggagaacggc aggaagcgga tgctggcttc cgctggcgag ctgcagaagg ggaacgagct
7201 ggctctgccg tccaagtatg tgaacttcct ctacctggcc tcccactacg agaagctcaa
7261 gggcagcccc gaggacaacg agcagaagca gctgttcgtc gagcagcaca agcattacct
7321 cgacgagatc attgagcaga tttccgagtt ctccaagcgc gtgatcctgg ccgacgcgaa
7381 tctggataag gtcctctccg cgtacaacaa gcaccgcgac aagccaatca gggagcaggc
7441 tgagaatatc attcatctct tcaccctgac gaacctcggc gcccctgctg ctttcaagta
7501 cttcgacaca actatcgatc gcaagaggta cacaagcact aaggaggtcc tggacgcgac
7561 cctcatccac cagtcgatta ccggcctcta cgagacgcgc atcgacctgt ctcagctcgg
7621 gggcgacaag cggccagcgg cgacgaagaa ggcggggcag gcgaagaaga agaagtgagc
7681 tcagagcttt cgttcgtatc atcggtttcg acaacgttcg tcaagttcaa tgcatcagtt
7741 tcattgcgca cacaccagaa tcctactgag tttgagtatt atggcattgg gaaaactgtt
7801 tttcttgtac catttgttgt gcttgtaatt tactgtgttt tttattcggt tttcgctatc
7861 gaactgtgaa atggaaatgg atggagaaga gttaatgaat gatatggtcc ttttgttcat
7921 tctcaaatta atattatttg ttttttctct tatttgttgt gtgttgaatt tgaaattata
7981 agagatatgc aaacattttg ttttgagtaa aaatgtgtca aatcgtggcc tctaatgacc
8041 gaagttaata tgaggagtaa aacacttgta gttgtaccat tatgcttatt cactaggcaa
8101 caaatatatt ttcagaccta gaaaagctgc aaatgttact gaatacaagt atgtcctctt
8161 gtgttttaga catttatgaa ctttccttta tgtaattttc cagaatcctt gtcagattct
8221 aatcattgct ttataattat agttatactc atggatttgt agttgagtat gaaaatattt
8281 tttaatgcat tttatgactt gccaattgat tgacaacgaa ttcgtaatca tgtcatagct
8341 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat
8401 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc
8461 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
8521 cgcggggaga ggcggtttgc gtattggcta gagcagcttg ccaacatggt ggagcacgac
8581 actctcgtct actccaagaa tatcaaagat acagtctcag aagaccaaag ggctattgag
8641 acttttcaac aaagggtaat atcgggaaac ctcctcggat tccattgccc agctatctgt
8701 cacttcatca aaaggacagt agaaaaggaa ggtggcacct acaaatgcca tcattgcgat
8761 aaaggaaagg ctatcgttca agatgcctct gccgacagtg gtcccaaaga tggaccccca
8821 cccacgagga gcatcgtgga aaaagaagac gttccaacca cgtcttcaaa gcaagtggat
8881 tgatgtgaac atggtggagc acgacactct cgtctactcc aagaatatca aagatacagt
8941 ctcagaagac caaagggcta ttgagacttt tcaacaaagg gtaatatcgg gaaacctcct
9001 cggattccat tgcccagcta tctgtcactt catcaaaagg acagtagaaa aggaaggtgg
9061 cacctacaaa tgccatcatt gcgataaagg aaaggctatc gttcaagatg cctctgccga
9121 cagtggtccc aaagatggac ccccacccac gaggagcatc gtggaaaaag aagacgttcc
9181 aaccacgtct tcaaagcaag tggattgatg tgatatctcc actgacgtaa gggatgacgc
9241 acaatcccac tatccttcgc aagacccttc ctctatataa ggaagttcat ttcatttgga
9301 gaggacacgc tgaaatcacc agtctctctc tacaaatcta tctctctcga gctttcgcag
9361 atccgggggg caatgagata tgaaaaagcc tgaactcacc gcgacgtctg tcgagaagtt
9421 tctgatcgaa aagttcgaca gcgtctccga cctgatgcag ctctcggagg gcgaagaatc
9481 tcgtgctttc agcttcgatg taggagggcg tggatatgtc ctgcgggtaa atagctgcgc
9541 cgatggtttc tacaaagatc gttatgttta tcggcacttt gcatcggccg cgctcccgat
9601 tccggaagtg cttgacattg gggagtttag cgagagcctg acctattgca tctcccgccg
9661 ttcacagggt gtcacgttgc aagacctgcc tgaaaccgaa ctgcccgctg ttctacaacc
9721 ggtcgcggag gctatggatg cgatcgctgc ggccgatctt agccagacga gcgggttcgg
9781 cccattcgga ccgcaaggaa tcggtcaata cactacatgg cgtgatttca tatgcgcgat
9841 tgctgatccc catgtgtatc actggcaaac tgtgatggac gacaccgtca gtgcgtccgt
9901 cgcgcaggct ctcgatgagc tgatgctttg ggccgaggac tgccccgaag tccggcacct
9961 cgtgcacgcg gatttcggct ccaacaatgt cctgacggac aatggccgca taacagcggt
10021 cattgactgg agcgaggcga tgttcgggga ttcccaatac gaggtcgcca acatcttctt
10081 ctggaggccg tggttggctt gtatggagca gcagacgcgc tacttcgagc ggaggcatcc
10141 ggagcttgca ggatcgccac gactccgggc gtatatgctc cgcattggtc ttgaccaact
10201 ctatcagagc ttggttgacg gcaatttcga tgatgcagct tgggcgcagg gtcgatgcga
10261 cgcaatcgtc cgatccggag ccgggactgt cgggcgtaca caaatcgccc gcagaagcgc
10321 ggccgtctgg accgatggct gtgtagaagt actcgccgat agtggaaacc gacgccccag
10381 cactcgtccg agggcaaaga aatagagtag atgccgaccg ggatctgtcg atcgacaagc
10441 tcgagtttct ccataataat gtgtgagtag ttcccagata agggaattag ggttcctata
10501 gggtttcgct catgtgttga gcatataaga aacccttagt atgtatttgt atttgtaaaa
10561 tacttctatc aataaaattt ctaattccta aaaccaaaat ccagtactaa aatccagatc
10621 ccccgaatta attcggcgtt aattcagtac attaaaaacg tccgcaatgt gttattaagt
10681 tgtctaagcg tcaatttgtt tacaccacaa tatatcctgc caccagccag ccaacagctc
10741 cccgaccggc agctcggcac aaaatcacca ctcgatacag gcagcccatc agtccgggac
10801 ggcgtcagcg ggagagccgt tgtaaggcgg cagactttgc tcatgttacc gatgctattc
10861 ggaagaacgg caactaagct gccgggtttg aaacacggat gatctcgcgg agggtagcat
10921 gttgattgta acgatgacag agcgttgctg cctgtgatca ccgcggtttc aaaatcggct
10981 ccgtcgatac tatgttatac gccaactttg aaaacaactt tgaaaaagct gttttctggt
11041 atttaaggtt ttagaatgca aggaacagtg aattggagtt cgtcttgtta taattagctt
11101 cttggggtat ctttaaatac tgtagaaaag aggaaggaaa taataaatgg ctaaaatgag
11161 aatatcaccg gaattgaaaa aactgatcga aaaataccgc tgcgtaaaag atacggaagg
11221 aatgtctcct gctaaggtat ataagctggt gggagaaaat gaaaacctat atttaaaaat
11281 gacggacagc cggtataaag ggaccaccta tgatgtggaa cgggaaaagg acatgatgct
11341 atggctggaa ggaaagctgc ctgttccaaa ggtcctgcac tttgaacggc atgatggctg
11401 gagcaatctg ctcatgagtg aggccgatgg cgtcctttgc tcggaagagt atgaagatga
11461 acaaagccct gaaaagatta tcgagctgta tgcggagtgc atcaggctct ttcactccat
11521 cgacatatcg gattgtccct atacgaatag cttagacagc cgcttagccg aattggatta
11581 cttactgaat aacgatctgg ccgatgtgga ttgcgaaaac tgggaagaag acactccatt
11641 taaagatccg cgcgagctgt atgatttttt aaagacggaa aagcccgaag aggaacttgt
11701 cttttcccac ggcgacctgg gagacagcaa catctttgtg aaagatggca aagtaagtgg
11761 ctttattgat cttgggagaa gcggcagggc ggacaagtgg tatgacattg ccttctgcgt
11821 ccggtcgatc agggaggata tcggggaaga acagtatgtc gagctatttt ttgacttact
11881 ggggatcaag cctgattggg agaaaataaa atattatatt ttactggatg aattgtttta
11941 gtacctagaa tgcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
12001 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
12061 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
12121 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct
12181 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
12241 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
12301 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
12361 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
12421 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
12481 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
12541 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
12601 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
12661 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac
12721 cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt
12781 gagcgaggaa gcggaagagc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat
12841 ttcacaccgc atatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca
12901 gtatacactc cgctatcgct acgtgactgg gtcatggctg cgccccgaca cccgccaaca
12961 cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg
13021 accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg
13081 cagggtgcct tgatgtgggc gccggcggtc gagtggcgac ggcgcggctt gtccgcgccc
13141 tggtagattg cctggccgta ggccagccat ttttgagcgg ccagcggccg cgataggccg
13201 acgcgaagcg gcggggcgta gggagcgcag cgaccgaagg gtaggcgctt tttgcagctc
13261 ttcggctgtg cgctggccag acagttatgc acaggccagg cgggttttaa gagttttaat
13321 aagttttaaa gagttttagg cggaaaaatc gccttttttc tcttttatat cagtcactta
13381 catgtgtgac cggttcccaa tgtacggctt tgggttccca atgtacgggt tccggttccc
13441 aatgtacggc tttgggttcc caatgtacgt gctatccaca ggaaacagac cttttcgacc
13501 tttttcccct gctagggcaa tttgccctag catctgctcc gtacattagg aaccggcgga
13561 tgcttcgccc tcgatcaggt tgcggtagcg catgactagg atcgggccag cctgccccgc
13621 ctcctccttc aaatcgtact ccggcaggtc atttgacccg atcagcttgc gcacggtgaa
13681 acagaacttc ttgaactctc cggcgctgcc actgcgttcg tagatcgtct tgaacaacca
13741 tctggcttct gccttgcctg cggcgcggcg tgccaggcgg tagagaaaac ggccgatgcc
13801 gggatcgatc aaaaagtaat cggggtgaac cgtcagcacg tccgggttct tgccttctgt
13861 gatctcgcgg tacatccaat cagctagctc gatctcgatg tactccggcc gcccggtttc
13921 gctctttacg atcttgtagc ggctaatcaa ggcttcaccc tcggataccg tcaccaggcg
13981 gccgttcttg gccttcttcg tacgctgcat ggcaacgtgc gtggtgttta accgaatgca
14041 ggtttctacc aggtcgtctt tctgctttcc gccatcggct cgccggcaga acttgagtac
14101 gtccgcaacg tgtggacgga acacgcggcc gggcttgtct cccttccctt cccggtatcg
14161 gttcatggat tcggttagat gggaaaccgc catcagtacc aggtcgtaat cccacacact
14221 ggccatgccg gccggccctg cggaaacctc tacgtgcccg tctggaagct cgtagcggat
14281 cacctcgcca gctcgtcggt cacgcttcga cagacggaaa acggccacgt ccatgatgct
14341 gcgactatcg cgggtgccca cgtcatagag catcggaacg aaaaaatctg gttgctcgtc
14401 gcccttgggc ggcttcctaa tcgacggcgc accggctgcc ggcggttgcc gggattcttt
14461 gcggattcga tcagcggccg cttgccacga ttcaccgggg cgtgcttctg cctcgatgcg
14521 ttgccgctgg gcggcctgcg cggccttcaa cttctccacc aggtcatcac ccagcgccgc
14581 gccgatttgt accgggccgg atggtttgcg accgctcacg ccgattcctc gggcttgggg
14641 gttccagtgc cattgcaggg ccggcaggca acccagccgc ttacgcctgg ccaaccgccc
14701 gttcctccac acatggggca ttccacggcg tcggtgcctg gttgttcttg attttccatg
14761 ccgcctcctt tagccgctaa aattcatcta ctcatttatt catttgctca tttactctgg
14821 tagctgcgcg atgtattcag atagcagctc ggtaatggtc ttgccttggc gtaccgcgta
14881 catcttcagc ttggtgtgat cctccgccgg caactgaaag ttgacccgct tcatggctgg
14941 cgtgtctgcc aggctggcca acgttgcagc cttgctgctg cgtgcgctcg gacggccggc
15001 acttagcgtg tttgtgcttt tgctcatttt ctctttacct cattaactca aatgagtttt
15061 gatttaattt cagcggccag cgcctggacc tcgcgggcag cgtcgccctc gggttctgat
15121 tcaagaacgg ttgtgccggc ggcggcagtg cctgggtagc tcacgcgctg cgtgatacgg
15181 gactcaagaa tgggcagctc gtacccggcc agcgcctcgg caacctcacc gccgatgcgc
15241 gtgcctttga tcgcccgcga cacgacaaag gccgcttgta gccttccatc cgtgacctca
15301 atgcgctgct taaccagctc caccaggtcg gcggtggccc atatgtcgta agggcttggc
15361 tgcaccggaa tcagcacgaa gtcggctgcc ttgatcgcgg acacagccaa gtccgccgcc
15421 tggggcgctc cgtcgatcac tacgaagtcg cgccggccga tggccttcac gtcgcggtca
15481 atcgtcgggc ggtcgatgcc gacaacggtt agcggttgat cttcccgcac ggccgcccaa
15541 tcgcgggcac tgccctgggg atcggaatcg actaacagaa catcggcccc ggcgagttgc
15601 agggcgcggg ctagatgggt tgcgatggtc gtcttgcctg acccgccttt ctggttaagt
15661 acagcgataa ccttcatgcg ttccccttgc gtatttgttt atttactcat cgcatcatat
15721 acgcagcgac cgcatgacgc aagctgtttt actcaaatac acatcacctt tttagacggc
15781 ggcgctcggt ttcttcagcg gccaagctgg ccggccaggc cgccagcttg gcatcagaca
15841 aaccggccag gatttcatgc agccgcacgg ttgagacgtg cgcgggcggc tcgaacacgt
15901 acccggccgc gatcatctcc gcctcgatct cttcggtaat gaaaaacggt tcgtcctggc
15961 cgtcctggtg cggtttcatg cttgttcctc ttggcgttca ttctcggcgg ccgccagggc
16021 gtcggcctcg gtcaatgcgt cctcacggaa ggcaccgcgc cgcctggcct cggtgggcgt
16081 cacttcctcg ctgcgctcaa gtgcgcggta cagggtcgag cgatgcacgc caagcagtgc
16141 agccgcctct ttcacggtgc ggccttcctg gtcgatcagc tcgcgggcgt gcgcgatctg
16201 tgccggggtg agggtagggc gggggccaaa cttcacgcct cgggccttgg cggcctcgcg
16261 cccgctccgg gtgcggtcga tgattaggga acgctcgaac tcggcaatgc cggcgaacac
16321 ggtcaacacc atgcggccgg ccggcgtggt ggtgtcggcc cacggctctg ccaggctacg
16381 caggcccgcg ccggcctcct ggatgcgctc ggcaatgtcc agtaggtcgc gggtgctgcg
16441 ggccaggcgg tctagcctgg tcactgtcac aacgtcgcca gggcgtaggt ggtcaagcat
16501 cctggccagc tccgggcggt cgcgcctggt gccggtgatc ttctcggaaa acagcttggt
16561 gcagccggcc gcgtgcagtt cggcccgttg gttggtcaag tcctggtcgt cggtgctgac
16621 gcgggcatag cccagcaggc cagcggcggc gctcttgttc atggcgtaat gtctccggtt
16681 ctagtcgcaa gtattctact ttatgcgact aaaacacgcg acaagaaaac gccaggaaaa
16741 gggcagggcg gcagcctgtc gcgtaactta ggacttgtgc gacatgtcgt tttcagaaga
16801 cggctgcact gaacgtcaga agccgactgc actatagcag cggaggggtt ggatcaaagt
16861 actttgatcc cgaggggaac cctgtggttg gcatgcacat acaaatggac gaacggataa
16921 accttttcac gcccttttaa atatccgatt attctaataa acgctctttt ctcttaggtt
16981 tacccgccaa tatatcctgt caaacactga tagtttaaac tgaaggcggg aaacgacaat
17041 ctgatccaag ctcaagctgc tctagcattc gccattcagg ctgcgcaact gttgggaagg
17101 gcgatcggtg cg
//
