| 质粒名称: | pYLCRISPR/Cas9Pubi-B |
|---|---|
| 出品公司: | 爱迪基因 |
| 目录编号: | 66188 |
| 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: | Basta |
| 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: | plant-codon optimized with higher GC contents (62.5%) in the 5’ region (400 bp) and 54.2% overall GC content |
| GenBank编号 | -- |
| 启动子 | maize ubiquitin promoter (Pubi) |
| 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/Cas9Pubi-B was a gift from Yao-Guang Liu (Addgene plasmid # 66188 ; http://n2t.net/addgene:66188 ; RRID:Addgene_66188) |
| 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-B 17089 bp ds-DNA circular SYN 30-JAN-2019
DEFINITION expression of Cas9 in plants, maize ubiquitin promoter (Pubi), basta
selection.
ACCESSION .
VERSION .
KEYWORDS pYLCRISPR/Cas9Pubi-B
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 17089)
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 17089)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Jan 30, 2019 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..17089
/organism="synthetic DNA construct"
/mol_type="other DNA"
misc_feature 1..25
/label=LB T-DNA repeat
/note="left border repeat from nopaline C58 T-DNA"
polyA_signal 103..277
/label=CaMV poly(A) signal
/note="cauliflower mosaic virus polyadenylation signal"
CDS complement(284..835)
/codon_start=1
/gene="Streptomyces hygroscopicus bar"
/product="phosphinothricin acetyltransferase"
/label=BlpR
/note="confers resistance to bialophos or phosphinothricin"
/translation="MSPERRPADIRRATEADMPAVCTIVNHYIETSTVNFRTEPQEPQE
WTDDLVRLRERYPWLVAEVDGEVAGIAYAGPWKARNAYDWTAESTVYVSPRHQRTGLGS
TLYTHLLKSLEAQGFKSVVAVIGLPNDPSVRMHEALGYAPRGMLRAAGFKHGNWHDVGF
WQLDFSLPVPPRPVLPVTEI"
promoter complement(880..1556)
/label=CaMV 35S promoter (enhanced)
/note="cauliflower mosaic virus 35S promoter with a
duplicated enhancer region"
primer_bind complement(923..943)
/label=35S promoter
/note="CaMV 35S promoter, forward primer"
protein_bind 1747..1768
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 1783..1813
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 1821..1837
/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 1826..1848
/label=M13/pUC Reverse
/note="In lacZ gene"
primer_bind 1845..1861
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind 1845..1861
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
CDS 4003..4023
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 4048..8148
/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 5234..5242
/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 8149..8196
/codon_start=1
/product="bipartite nuclear localization signal from
nucleoplasmin"
/label=nucleoplasmin NLS
/translation="KRPAATKKAGQAKKKK"
terminator 8209..8461
/label=NOS terminator
/note="nopaline synthase terminator and poly(A) signal"
protein_bind 8579..8600
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 8615..8645
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 8653..8669
/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 8711..8729
/label=SP6 promoter
/note="promoter for bacteriophage SP6 RNA polymerase"
primer_bind 8711..8728
/label=SP6
/note="SP6 promoter, forward primer"
CDS 8803..9105
/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
SRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
primer_bind 8984..9003
/label=ccdB-fwd
/note="ccdB gene, forward primer"
protein_bind complement(9315..9348)
/label=loxP
/bound_moiety="Cre recombinase"
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (GCATACAT)."
primer_bind complement(9392..9409)
/label=M13 Forward
/note="In lacZ gene. Also called M13-F20 or M13 (-21)
Forward"
primer_bind complement(9392..9408)
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(9401..9423)
/label=M13/pUC Forward
/note="In lacZ gene"
misc_feature 9611..9635
/label=RB T-DNA repeat
/note="right border repeat from nopaline C58 T-DNA"
CDS 10936..11565
/codon_start=1
/product="stability protein from the Pseudomonas plasmid
pVS1 (Heeb et al., 2000)"
/label=pVS1 StaA
/translation="MKVIAVLNQKGGSGKTTIATHLARALQLAGADVLLVDSDPQGSAR
DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP
VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI
LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI"
CDS 11994..13067
/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 13133..13327
/label=pVS1 oriV
/note="origin of replication for the Pseudomonas plasmid
pVS1 (Heeb et al., 2000)"
primer_bind complement(13563..13585)
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
misc_feature 13671..13811
/label=bom
/note="basis of mobility region from pBR322"
primer_bind 13726..13745
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind complement(13826..13843)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(13997..14585)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(14077..14096)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS complement(14672..15466)
/codon_start=1
/gene="aphA-3"
/product="aminoglycoside phosphotransferase"
/label=KanR
/note="confers resistance to kanamycin"
/translation="MAKMRISPELKKLIEKYRCVKDTEGMSPAKVYKLVGENENLYLKM
TDSRYKGTTYDVEREKDMMLWLEGKLPVPKVLHFERHDGWSNLLMSEADGVLCSEEYED
EQSPEKIIELYAECIRLFHSIDISDCPYTNSLDSRLAELDYLLNNDLADVDCENWEEDT
PFKDPRELYDFLKTEKPEEELVFSHGDLGDSNIFVKDGKVSGFIDLGRSGRADKWYDIA
FCVRSIREDIGEEQYVELFFDLLGIKPDWEKIKYYILLDELF"
ORIGIN
1 tggcaggata tattgtggtg taaacaaatt gacgcttaga caacttaata acacattgcg
61 gacgttttta atgtactgaa ttaacgccga attaattcgg gggatctgga ttttagtact
121 ggattttggt tttaggaatt agaaatttta ttgatagaag tattttacaa atacaaatac
181 atactaaggg tttcttatat gctcaacaca tgagcgaaac cctataggaa ccctaattcc
241 cttatctggg aactactcac acattattat ggagaaactc gagtcaaatc tcggtgacgg
301 gcaggaccgg acggggcggt accggcaggc tgaagtccag ctgccagaaa cccacgtcat
361 gccagttccc gtgcttgaag ccggccgccc gcagcatgcc gcggggggca tatccgagcg
421 cctcgtgcat gcgcacgctc gggtcgttgg gcagcccgat gacagcgacc acgctcttga
481 agccctgtgc ctccagggac ttcagcaggt gggtgtagag cgtggagccc agtcccgtcc
541 gctggtggcg gggggagacg tacacggtcg actcggccgt ccagtcgtag gcgttgcgtg
601 ccttccaggg gcccgcgtag gcgatgccgg cgacctcgcc gtccacctcg gcgacgagcc
661 agggatagcg ctcccgcaga cggacgaggt cgtccgtcca ctcctgcggt tcctgcggct
721 cggtacggaa gttgaccgtg cttgtctcga tgtagtggtt gacgatggtg cagaccgccg
781 gcatgtccgc ctcggtggca cggcggatgt cggccgggcg tcgttctggg ctcatggtag
841 actcgagaga gatagatttg tagagagaga ctggtgattt cagcgtgtcc tctccaaatg
901 aaatgaactt ccttatatag aggaagggtc ttgcgaagga tagtgggatt gtgcgtcatc
961 ccttacgtca gtggagatat cacatcaatc cacttgcttt gaagacgtgg ttggaacgtc
1021 ttctttttcc acgatgctct tcgtgggtgg gggtccatct ttgggaccac tgtcggcaga
1081 ggcatcttga acgatagcct ttcctttatc gcaatgatgg catttgtagg tgccaccttc
1141 cttttctact gtccttttga tgaagtgaca gatagctggg caatggaatc cgaggaggtt
1201 tcccgatatt accctttgtt gaaaagtctc aatagccctt tggtcttctg agactgtatc
1261 tttgatattc ttggagtaga cgagagtgtc gtgctccacc atgttcacat caatccactt
1321 gctttgaaga cgtggttgga acgtcttctt tttccacgat gctcctcgtg ggtgggggtc
1381 catctttggg accactgtcg gcagaggcat cttgaacgat agcctttcct ttatcgcaat
1441 gatggcattt gtaggtgcca ccttcctttt ctactgtcct tttgatgaag tgacagatag
1501 ctgggcaatg gaatccgagg aggtttcccg atattaccct ttgttgaaaa gtctcaatag
1561 ccctttggtc ttctgagact gtatctttga tattcttgga gtagacgaga gtgtcgtgct
1621 ccaccatgtt ggcaagctgc tctagccaat acgcaaaccg cctctccccg cgcgttggcc
1681 gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa
1741 cgcaattaat gtgagttagc tcactcatta ggcaccccag gctttacact ttatgcttcc
1801 ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa acagctatga
1861 catgattacg aattcgagct cggtaccgta actataacgg tcctaaggta gcgaaggatc
1921 cgctcgctac cttaagagag gatatccctc catcctataa tgtaggctat aggaactagg
1981 gcaaggccgg ccatgcggcc gcaagctggg tgcagcgtga cccggtcgtg cccctctcta
2041 gagataatga gcattgcatg tctaagttat aaaaaattac cacatatttt ttttgtcaca
2101 cttgtttgaa gtgcagttta tctatcttta tacatatatt taaactttac tctacgaata
2161 atataatcta tagtactaca ataatatcag tgttttagag aatcatataa atgaacagtt
2221 agacatggtc taaaggacaa ttgagtattt tgacaacagg actctacagt tttatctttt
2281 tagtgtgcat gtgttctcct ttttttttgc aaatagcttc acctatataa tacttcatcc
2341 attttattag tacatccatt tagggtttag ggttaatggt ttttatagac taattttttt
2401 agtacatcta ttttattcta ttttagcctc taaattaaga aaactaaaac tctattttag
2461 tttttttatt taataattta gatataaaat agaataaaat aaagtgacta aaaattaaac
2521 aaataccctt taagaaatta aaaaaactaa ggaaacattt ttcttgtttc gagtagataa
2581 tgccagcctg ttaaacgccg tcgacgagtc taacggacac caaccagcga accagcagcg
2641 tcgcgtcggg ccaagcgaag cagacggcac ggcatctctg tcgctgcctc tggacccctc
2701 tcgagagttc cgctccaccg ttggacttgc tccgctgtcg gcatccagaa attgcgtggc
2761 ggagcggcag acgtgagccg gcacggcagg cggcctcctc ctcctctcac ggcaccggca
2821 gctacggggg attcctttcc caccgctcct tcgctttccc ttcctcgccc gccgtaataa
2881 atagacaccc cctccacacc ctctttcccc aacctcgtgt tgttcggagc gcacacacac
2941 acaaccagat ctcccccaaa tccacccgtc ggcacctccg cttcaaggta cgccgctcgt
3001 cctccccccc cccctnctct accttctcta gatcggcgtt ccggtccatg gttagggccc
3061 ggtagttcta cttctgttca tgtttgtgtt agatccgtgt ttgtgttaga tccgtgctgc
3121 tagcgttcgt acacggatgc gacctgtacg tcagacacgt tctgattgct aacttgccag
3181 tgtttctctt tggggaatcc tgggatggct ctagccgttc cgcagacggg atcgatttca
3241 tgattttttt tgtttcgttg catagggttt ggtttgccct tttcctttat ttcaatatat
3301 gccgtgcact tgtttgtcgg gtcatctttt catgcttttt tttgtcttgg ttgtgatgat
3361 gtggtctggt tgggcggtcg ttctagatcg gagtagaatt ctgtttcaaa ctacctggtg
3421 gatttattaa ttttggatct gtatgtgtgt gccatacata ttcatagtta cgaattgaag
3481 atgatggatg gaaatatcga tctaggatag gtatacatgt tgatgcgggt tttactgatg
3541 catatacaga gatgcttttt gttcgcttgg ttgtgatgat gtggtgtggt tgggcggtcg
3601 ttcattcgtt ctagatcgga gtagaatact gtttcaaact acctggtgta tttattaatt
3661 ttggaactgt atgtgtgtgt catacatctt catagttacg agtttaagat ggatggaaat
3721 atcgatctag gataggtata catgttgatg tgggttttac tgatgcatat acatgatggc
3781 atatgcagca tctattcata tgctctaacc ttgagtacct atctattata ataaacaagt
3841 atgttttata attattttga tcttgatata cttggatgat ggcatatgca gcagctatat
3901 gtggattttt ttagccctgc cttcatacgc tatttatttg cttggtactg tttcttttgt
3961 cgatgctcac cctgttgttt ggtgttactt ctgcagatgg ctcctaagaa gaagcggaag
4021 gttggtattc acggggtgcc tgcggctgac aagaagtact ccatcggcct cgacatcggc
4081 accaacagcg tcggctgggc ggtgatcacc gacgagtaca aggtcccgtc caagaagttc
4141 aaggtcctgg gcaacaccga ccgccactcc atcaagaaga acctcatcgg cgccctcctc
4201 ttcgactccg gcgagacggc ggaggcgacc cgcctcaagc gcaccgcccg ccgccgctac
4261 acccgccgca agaaccgcat ctgctacctc caggagatct tctccaacga gatggcgaag
4321 gtcgacgact ccttcttcca ccgcctcgag gagtccttcc tcgtggagga ggacaagaag
4381 cacgagcgcc accccatctt cggcaacatc gtcgacgagg tcgcctacca cgagaagtac
4441 cccactatct accaccttcg taagaagctt gttgactcta ctgataaggc tgatcttcgt
4501 ctcatctacc ttgctctcgc tcacatgatc aagttccgtg gtcacttcct tatcgagggt
4561 gaccttaacc ctgataactc cgacgtggac aagctcttca tccagctcgt ccagacctac
4621 aaccagctct tcgaggagaa ccctatcaac gcttccggtg tcgacgctaa ggcgatcctt
4681 tccgctaggc tctccaagtc caggcgtctc gagaacctca tcgcccagct ccctggtgag
4741 aagaagaacg gtcttttcgg taacctcatc gctctctccc tcggtctgac ccctaacttc
4801 aagtccaact tcgacctcgc tgaggacgct aagcttcagc tctccaagga tacctacgac
4861 gatgatctcg acaacctcct cgctcagatt ggagatcagt acgctgatct cttccttgct
4921 gctaagaacc tctccgatgc tatcctcctt tcggatatcc ttagggttaa cactgagatc
4981 actaaggctc ctctttctgc ttccatgatc aagcgctacg acgagcacca ccaggacctc
5041 accctcctca aggctcttgt tcgtcagcag ctccccgaga agtacaagga gatcttcttc
5101 gaccagtcca agaacggcta cgccggttac attgacggtg gagctagcca ggaggagttc
5161 tacaagttca tcaagccaat ccttgagaag atggatggta ctgaggagct tctcgttaag
5221 cttaaccgtg aggacctcct taggaagcag aggactttcg ataacggctc tatccctcac
5281 cagatccacc ttggtgagct tcacgccatc cttcgtaggc aggaggactt ctaccctttc
5341 ctcaaggaca accgtgagaa gatcgagaag atccttactt tccgtattcc ttactacgtt
5401 ggtcctcttg ctcgtggtaa ctcccgtttc gcttggatga ctaggaagtc cgaggagact
5461 atcacccctt ggaacttcga ggaggttgtt gacaagggtg cttccgccca gtccttcatc
5521 gagcgcatga ccaacttcga caagaacctc cccaacgaga aggtcctccc caagcactcc
5581 ctcctctacg agtacttcac ggtctacaac gagctcacca aggtcaagta cgtcaccgag
5641 ggtatgcgca agcctgcctt cctctccggc gagcagaaga aggctatcgt tgacctcctc
5701 ttcaagacca accgcaaggt caccgtcaag cagctcaagg aggactactt caagaagatc
5761 gagtgcttcg actccgtcga gatcagcggc gttgaggacc gtttcaacgc ttctctcggt
5821 acctaccacg atctcctcaa gatcatcaag gacaaggact tcctcgacaa cgaggagaac
5881 gaggacatcc tcgaggacat cgtcctcact cttactctct tcgaggatag ggagatgatc
5941 gaggagaggc tcaagactta cgctcatctc ttcgatgaca aggttatgaa gcagctcaag
6001 cgtcgccgtt acaccggttg gggtaggctc tcccgcaagc tcatcaacgg tatcagggat
6061 aagcagagcg gcaagactat cctcgacttc ctcaagtctg atggtttcgc taacaggaac
6121 ttcatgcagc tcatccacga tgactctctt accttcaagg aggatattca gaaggctcag
6181 gtgtccggtc agggcgactc tctccacgag cacattgcta accttgctgg ttcccctgct
6241 atcaagaagg gcatccttca gactgttaag gttgtcgatg agcttgtcaa ggttatgggt
6301 cgtcacaagc ctgagaacat cgtcatcgag atggctcgtg agaaccagac tacccagaag
6361 ggtcagaaga actcgaggga gcgcatgaag aggattgagg agggtatcaa ggagcttggt
6421 tctcagatcc ttaaggagca ccctgtcgag aacacccagc tccagaacga gaagctctac
6481 ctctactacc tccagaacgg tagggatatg tacgttgacc aggagctcga catcaacagg
6541 ctttctgact acgacgtcga ccacattgtt cctcagtctt tccttaagga tgactccatc
6601 gacaacaagg tcctcacgag gtccgacaag aacaggggta agtcggacaa cgtcccttcc
6661 gaggaggttg tcaagaagat gaagaactac tggaggcagc ttctcaacgc taagctcatt
6721 acccagagga agttcgacaa cctcacgaag gctgagaggg gtggcctttc cgagcttgac
6781 aaggctggtt tcatcaagag gcagcttgtt gagacgaggc agattaccaa gcacgttgct
6841 cagatcctcg attctaggat gaacaccaag tacgacgaga acgacaagct catccgcgag
6901 gtcaaggtga tcaccctcaa gtccaagctc gtctccgact tccgcaagga cttccagttc
6961 tacaaggtcc gcgagatcaa caactaccac cacgctcacg atgcttacct taacgctgtc
7021 gttggtaccg ctcttatcaa gaagtaccct aagcttgagt ccgagttcgt ctacggtgac
7081 tacaaggtct acgacgttcg taagatgatc gccaagtccg agcaggagat cggcaaggcc
7141 accgccaagt acttcttcta ctccaacatc atgaacttct tcaagaccga gatcaccctc
7201 gccaacggcg agatccgcaa gcgccctctt atcgagacga acggtgagac tggtgagatc
7261 gtttgggaca agggtcgcga cttcgctact gttcgcaagg tcctttctat gcctcaggtt
7321 aacatcgtca agaagaccga ggtccagacc ggtggcttct ccaaggagtc tatccttcca
7381 aagagaaact cggacaagct catcgctagg aagaaggatt gggaccctaa gaagtacggt
7441 ggtttcgact cccctactgt cgcctactcc gtcctcgtgg tcgccaaggt ggagaagggt
7501 aagtcgaaga agctcaagtc cgtcaaggag ctcctcggca tcaccatcat ggagcgctcc
7561 tccttcgaga agaacccgat cgacttcctc gaggccaagg gctacaagga ggtcaagaag
7621 gacctcatca tcaagctccc caagtactct cttttcgagc tcgagaacgg tcgtaagagg
7681 atgctggctt ccgctggtga gctccagaag ggtaacgagc ttgctcttcc ttccaagtac
7741 gtgaacttcc tctacctcgc ctcccactac gagaagctca agggttcccc tgaggataac
7801 gagcagaagc agctcttcgt ggagcagcac aagcactacc tcgacgagat catcgagcag
7861 atctccgagt tctccaagcg cgtcatcctc gctgacgcta acctcgacaa ggtcctctcc
7921 gcctacaaca agcaccgcga caagcccatc cgcgagcagg ccgagaacat catccacctc
7981 ttcacgctca cgaacctcgg cgcccctgct gctttcaagt acttcgacac caccatcgac
8041 aggaagcgtt acacgtccac caaggaggtt ctcgacgcta ctctcatcca ccagtccatc
8101 accggtcttt acgagactcg tatcgacctt tcccagcttg gtggtgataa gcgtcctgct
8161 gccaccaaaa aggccggaca ggctaagaaa aagaagtagg atcctcccga tcgttcaaac
8221 atttggcaat aaagtttctt aagattgaat cctgttgccg gtcttgcgat gattatcata
8281 taatttctgt tgaattacgt taagcatgta ataattaaca tgtaatgcat gacgttattt
8341 atgaggtggg tttttatgat tagagtcccg caattataca tttaatacgc gatagaaaac
8401 aaaatatagc gcgcaaacta ggataaatta tcgcgcgcgg tgtcatctat gttactagat
8461 cgggagcacc ggtaaggcgc gccgtagtgc tcgagagacc tctgaagtgg ccgattcatt
8521 aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta
8581 atgtgagtta gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta
8641 tgttgtgtgg aattgtgagc ggataacaat ttcacacaag aaacagctat gaccatgatt
8701 acgccaagct atttaggtga cactatagaa tactcaagct atgcatcaag ctcaatgggt
8761 ctagtctgta gatacccatc acactggcga ccgctcgaac atcagtttaa ggtttacacc
8821 tataaaagag agagccgtta tcgtctgttt gtggatgtac agagtgatat tattgacacg
8881 ccggggcgac ggatggtgat ccccctggcc agtgcacgtc tgctgtcaga taaagtctcc
8941 cgtgaacttt acccggtggt gcatatcggg gatgaaagct ggcgcatgat gaccaccgat
9001 atggccagtg tgcctgtctc cgttatcggg gaagaagtgg ctgatctcag ccaccgcgaa
9061 aatgacatca aaaacgccat taacctgatg ttctggggaa tataaatgtc aggcctgaat
9121 ggcgaatgga cgcgccctgt agcggcgcat taagcgcggc gggtgagcgt gggtctcgcg
9181 gtatcattgg cgcgcctctc gagctagcgg ccgcatgcat cgatctccta catcgtataa
9241 attagcctat acgaagttat tgcatctatg tcgggtgcgg agaaagaggt aatgaaatgg
9301 cagtattaga tctgataact tcgtataatg tatgctatac gaagttatga ctgcaggtcg
9361 acacccataa tagctgtttg ccaagcttgg cactggccgt cgttttacaa cgtcgtgact
9421 gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct
9481 ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg
9541 gcgaatgcta gagcagcttg agcttggatc agattgtcgt ttcccgcctt cagtttaaac
9601 tatcagtgtt tgacaggata tattggcggg taaacctaag agaaaagagc gtttattaga
9661 ataatcggat atttaaaagg gcgtgaaaag gtttatccgt tcgtccattt gtatgtgcat
9721 gccaaccaca gggttcccct cgggatcaaa gtactttgat ccaacccctc cgctgctata
9781 gtgcagtcgg cttctgacgt tcagtgcagc cgtcttctga aaacgacatg tcgcacaagt
9841 cctaagttac gcgacaggct gccgccctgc ccttttcctg gcgttttctt gtcgcgtgtt
9901 ttagtcgcat aaagtagaat acttgcgact agaaccggag acattacgcc atgaacaaga
9961 gcgccgccgc tggcctgctg ggctatgccc gcgtcagcac cgacgaccag gacttgacca
10021 accaacgggc cgaactgcac gcggccggct gcaccaagct gttttccgag aagatcaccg
10081 gcaccaggcg cgaccgcccg gagctggcca ggatgcttga ccacctacgc cctggcgacg
10141 ttgtgacagt gaccaggcta gaccgcctgg cccgcagcac ccgcgaccta ctggacattg
10201 ccgagcgcat ccaggaggcc ggcgcgggcc tgcgtagcct ggcagagccg tgggccgaca
10261 ccaccacgcc ggccggccgc atggtgttga ccgtgttcgc cggcattgcc gagttcgagc
10321 gttccctaat catcgaccgc acccggagcg ggcgcgaggc cgccaaggcc cgaggcgtga
10381 agtttggccc ccgccctacc ctcaccccgg cacagatcgc gcacgcccgc gagctgatcg
10441 accaggaagg ccgcaccgtg aaagaggcgg ctgcactgct tggcgtgcat cgctcgaccc
10501 tgtaccgcgc acttgagcgc agcgaggaag tgacgcccac cgaggccagg cggcgcggtg
10561 ccttccgtga ggacgcattg accgaggccg acgccctggc ggccgccgag aatgaacgcc
10621 aagaggaaca agcatgaaac cgcaccagga cggccaggac gaaccgtttt tcattaccga
10681 agagatcgag gcggagatga tcgcggccgg gtacgtgttc gagccgcccg cgcacgtctc
10741 aaccgtgcgg ctgcatgaaa tcctggccgg tttgtctgat gccaagctgg cggcctggcc
10801 ggccagcttg gccgctgaag aaaccgagcg ccgccgtcta aaaaggtgat gtgtatttga
10861 gtaaaacagc ttgcgtcatg cggtcgctgc gtatatgatg cgatgagtaa ataaacaaat
10921 acgcaagggg aacgcatgaa ggttatcgct gtacttaacc agaaaggcgg gtcaggcaag
10981 acgaccatcg caacccatct agcccgcgcc ctgcaactcg ccggggccga tgttctgtta
11041 gtcgattccg atccccaggg cagtgcccgc gattgggcgg ccgtgcggga agatcaaccg
11101 ctaaccgttg tcggcatcga ccgcccgacg attgaccgcg acgtgaaggc catcggccgg
11161 cgcgacttcg tagtgatcga cggagcgccc caggcggcgg acttggctgt gtccgcgatc
11221 aaggcagccg acttcgtgct gattccggtg cagccaagcc cttacgacat atgggccacc
11281 gccgacctgg tggagctggt taagcagcgc attgaggtca cggatggaag gctacaagcg
11341 gcctttgtcg tgtcgcgggc gatcaaaggc acgcgcatcg gcggtgaggt tgccgaggcg
11401 ctggccgggt acgagctgcc cattcttgag tcccgtatca cgcagcgcgt gagctaccca
11461 ggcactgccg ccgccggcac aaccgttctt gaatcagaac ccgagggcga cgctgcccgc
11521 gaggtccagg cgctggccgc tgaaattaaa tcaaaactca tttgagttaa tgaggtaaag
11581 agaaaatgag caaaagcaca aacacgctaa gtgccggccg tccgagcgca cgcagcagca
11641 aggctgcaac gttggccagc ctggcagaca cgccagccat gaagcgggtc aactttcagt
11701 tgccggcgga ggatcacacc aagctgaaga tgtacgcggt acgccaaggc aagaccatta
11761 ccgagctgct atctgaatac atcgcgcagc taccagagta aatgagcaaa tgaataaatg
11821 agtagatgaa ttttagcggc taaaggaggc ggcatggaaa atcaagaaca accaggcacc
11881 gacgccgtgg aatgccccat gtgtggagga acgggcggtt ggccaggcgt aagcggctgg
11941 gttgcctgcc ggccctgcaa tggcactgga acccccaagc ccgaggaatc ggcgtgagcg
12001 gtcgcaaacc atccggcccg gtacaaatcg gcgcggcgct gggtgatgac ctggtggaga
12061 agttgaaggc cgcgcaggcc gcccagcggc aacgcatcga ggcagaagca cgccccggtg
12121 aatcgtggca agcggccgct gatcgaatcc gcaaagaatc ccggcaaccg ccggcagccg
12181 gtgcgccgtc gattaggaag ccgcccaagg gcgacgagca accagatttt ttcgttccga
12241 tgctctatga cgtgggcacc cgcgatagtc gcagcatcat ggacgtggcc gttttccgtc
12301 tgtcgaagcg tgaccgacga gctggcgagg tgatccgcta cgagcttcca gacgggcacg
12361 tagaggtttc cgcagggccg gccggcatgg ccagtgtgtg ggattacgac ctggtactga
12421 tggcggtttc ccatctaacc gaatccatga accgataccg ggaagggaag ggagacaagc
12481 ccggccgcgt gttccgtcca cacgttgcgg acgtactcaa gttctgccgg cgagccgatg
12541 gcggaaagca gaaagacgac ctggtagaaa cctgcattcg gttaaacacc acgcacgttg
12601 ccatgcagcg tacgaagaag gccaagaacg gccgcctggt gacggtatcc gagggtgaag
12661 ccttgattag ccgctacaag atcgtaaaga gcgaaaccgg gcggccggag tacatcgaga
12721 tcgagctagc tgattggatg taccgcgaga tcacagaagg caagaacccg gacgtgctga
12781 cggttcaccc cgattacttt ttgatcgatc ccggcatcgg ccgttttctc taccgcctgg
12841 cacgccgcgc cgcaggcaag gcagaagcca gatggttgtt caagacgatc tacgaacgca
12901 gtggcagcgc cggagagttc aagaagttct gtttcaccgt gcgcaagctg atcgggtcaa
12961 atgacctgcc ggagtacgat ttgaaggagg aggcggggca ggctggcccg atcctagtca
13021 tgcgctaccg caacctgatc gagggcgaag catccgccgg ttcctaatgt acggagcaga
13081 tgctagggca aattgcccta gcaggggaaa aaggtcgaaa agttctcttt cctgtggata
13141 gcacgtacat tgggaaccca aagccgtaca ttgggaaccg gaacccgtac attgggaacc
13201 caaagccgta cattgggaac cggtcacaca tgtaagtgac tgatataaaa gagaaaaaag
13261 gcgatttttc cgcctaaaac tctttaaaac ttattaaaac tcttaaaacc cgcctggcct
13321 gtgcataact gtctggccag cgcacagccg aagagctgca aaaagcgcct acccttcggt
13381 cgctgcgctc cctacgcccc gccgcttcgc gtcggcctat cgcggccgct ggccgctcaa
13441 aaatggctgg cctacggcca ggcaatctac cagggcgcgg acaagccgcg ccgtcgccac
13501 tcgaccgccg gcgcccacat caaggcaccc tgcctcgcgc gtttcggtga tgacggtgaa
13561 aacctctgac acatgcagct cccggagacg gtcacagctt gtctgtaagc ggatgccggg
13621 agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg cgcagccatg
13681 acccagtcac gtagcgatag cggagtgtat actggcttaa ctatgcggca tcagagcaga
13741 ttgtactgag agtgcaccat atgcggtgtg aaataccgca cagatgcgta aggagaaaat
13801 accgcatcag gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
13861 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
13921 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
13981 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
14041 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
14101 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
14161 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
14221 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
14281 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
14341 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
14401 tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc
14461 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
14521 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
14581 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
14641 gttaagggat tttggtcatg cattctaggt actaaaacaa ttcatccagt aaaatataat
14701 attttatttt ctcccaatca ggcttgatcc ccagtaagtc aaaaaatagc tcgacatact
14761 gttcttcccc gatatcctcc ctgatcgacc ggacgcagaa ggcaatgtca taccacttgt
14821 ccgccctgcc gcttctccca agatcaataa agccacttac tttgccatct ttcacaaaga
14881 tgttgctgtc tcccaggtcg ccgtgggaaa agacaagttc ctcttcgggc ttttccgtct
14941 ttaaaaaatc atacagctcg cgcggatctt taaatggagt gtcttcttcc cagttttcgc
15001 aatccacatc ggccagatcg ttattcagta agtaatccaa ttcggctaag cggctgtcta
15061 agctattcgt atagggacaa tccgatatgt cgatggagtg aaagagcctg atgcactccg
15121 catacagctc gataatcttt tcagggcttt gttcatcttc atactcttcc gagcaaagga
15181 cgccatcggc ctcactcatg agcagattgc tccagccatc atgccgttca aagtgcagga
15241 cctttggaac aggcagcttt ccttccagcc atagcatcat gtccttttcc cgttccacat
15301 cataggtggt ccctttatac cggctgtccg tcatttttaa atataggttt tcattttctc
15361 ccaccagctt atatacctta gcaggagaca ttccttccgt atcttttacg cagcggtatt
15421 tttcgatcag ttttttcaat tccggtgata ttctcatttt agccatttat tatttccttc
15481 ctcttttcta cagtatttaa agatacccca agaagctaag gaaggtgcga acaagtccct
15541 gatatgagat catgtttgtc atctggagcc atagaacagg gttcatcatg agtcatcaac
15601 ttaccttcgc cgacagtgaa ttcagcagta agcgccgtca gaccagaaaa gagattttct
15661 tgtcccgcat ggagcagatt ctgccatggc aaaacatggt ggaagtcatc gagccgtttt
15721 accccaaggc tggtaatggc cggcgacctt atccgctgga aaccatgcta cgcattcact
15781 gcatgcagca ttggtacaac ctgagcgatg gcgcgatgga agatgctctg tacgaaatcg
15841 cctccatgcg tctgtttgcc cggttatccc tggatagcgc cttgccggac cgcaccacca
15901 tcatgaattt ccgccacctg ctggagcagc atcaactggc ccgccaattg ttcaagacca
15961 tcaatcgctg gctggccgaa gcaggcgtca tgatgactca aggcaccttg gtcgatgcca
16021 ccatcattga ggcacccagc tcgaccaaga acaaagagca gcaacgcgat ccggagatgc
16081 atcagaccaa gaaaggcaat cagtggcact ttggcatgaa ggcccacatt ggtgtcgatg
16141 ccaagagtgg cctgacccac agcctggtca ccaccgcggc caacgagcat gacctcaatc
16201 agctgggtaa tctgctgcat ggagaggagc aatttgtctc agccgatgcc ggctaccaag
16261 gggcgccaca gcgcgaggag ctggccgagg tggatgtgga ctggctgatc gccgagcgcc
16321 ccggcaaggt aagaaccttg aaacagcatc cacgcaagaa caaaacggcc atcaacatcg
16381 aatacatgaa agccagcatc cgggccaggg tggagcaccc atttcgcatc atcaagcgac
16441 agttcggctt cgtgaaagcc agatacaagg ggttgctgaa aaacgataac caactggcga
16501 tgttattcac gctggccaac ctgtttcggg cggaccaaat gatacgtcag tgggagagat
16561 ctcactaaaa actggggata acgccttaaa tggcgaagaa acggtctaaa taggctgatt
16621 caaggcattt acgggagaaa aaatcggctc aaacatgaag aaatgaaatg actgagtcag
16681 ccgagaagaa tttccccgct tattcgcacc ttccctaatt ataacaagac gaactccaat
16741 tcactgttcc ttgcattcta aaaccttaaa taccagaaaa cagctttttc aaagttgttt
16801 tcaaagttgg cgtataacat agtatcgacg gagccgattt tgaaaccgcg gtgatcacag
16861 gcagcaacgc tctgtcatcg ttacaatcaa catgctaccc tccgcgagat catccgtgtt
16921 tcaaacccgg cagcttagtt gccgttcttc cgaatagcat cggtaacatg agcaaagtct
16981 gccgccttac aacggctctc ccgctgacgc cgtcccggac tgatgggctg cctgtatcga
17041 gtggtgattt tgtgccgagc tgccggtcgg ggagctgttg gctggctgg
//
