| 质粒名称: | pHSN6A01 |
|---|---|
| 出品公司: | 爱迪基因 |
| 目录编号: | 50586 |
| 存储实验室: | Qi-Jun Chen |
|
载体骨架基本信息 |
|
| 载体名称: | pGreen-like binary vector |
| 载体出品公司: | Mullineaux Lab, see http://www.pgreen.ac.uk/ |
| 载体抗性: | 卡那霉素和大观霉素 |
| 载体类型: | CRISPR ; 植物表达 |
| 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: | dCas9-VP64,Cas9 |
| 物种来源: | Synthetic |
| 基因突变: | dCas9 = defective in DNA cleavage; 4×minimal VP16 activation domain; maize codon–optimized dCas9-VP64 vectors were designed for targeted activation of the genes of interest |
| 启动子 | 2×35Sp |
| 标签/融合蛋白: |
3xFLAG（插入件上的N端子）
NLS（插入件上的N端子）
NLS（插入件上的C端子）
HA（插入件上的C端子）
|
| 5' 测序引物 | hSpCas9-R1, CGCTCGTGCTTCTTATCCTC |
|
文献引用方法 |
|
| 材料和方法部分: | pHSN6A01 was a gift from Qi-Jun Chen (Addgene plasmid # 50586 ; http://n2t.net/addgene:50586 ; RRID:Addgene_50586) |
| 参考文献部分: | A CRISPR/Cas9 toolkit for multiplex genome editing in plants. Xing HL, Dong L, Wang ZP, Zhang HY, Han CY, Liu B, Wang XC, Chen QJ. BMC Plant Biol. 2014 Nov 29;14(1):327. 10.1186/s12870-014-0327-y PubMed 25432517 |
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 pHSN6A01 12697 bp ds-DNA circular SYN 02-OCT-2019
DEFINITION CRISPR/Cas based plant genome editing and gene regulation; expresses
dCas9-VP64, gRNA scaffold for insertion of target sequence (AtU6-26
promoter), Hyg resistance.
ACCESSION .
VERSION .
KEYWORDS pHSN6A01
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 12697)
AUTHORS Xing HL, Dong L, Wang ZP, Zhang HY, Han CY, Liu B, Wang XC, Chen QJ
TITLE A CRISPR/Cas9 toolkit for multiplex genome editing in plants.
JOURNAL BMC Plant Biol. 2014 Nov 29;14(1):327.
PUBMED 25432517
REFERENCE 2 (bases 1 to 12697)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Oct 2, 2019 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..12697
/organism="synthetic DNA construct"
/mol_type="other DNA"
misc_feature 17..41
/label=RB T-DNA repeat
/note="right border repeat from nopaline C58 T-DNA"
CDS 826..1617
/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 1769..1844
/label=gRNA scaffold
/note="guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system"
promoter 2458..2802
/label=CaMV 35S promoter
/note="strong constitutive promoter from cauliflower mosaic
virus"
CDS 2985..3050
/codon_start=1
/product="three tandem FLAG(R) epitope tags, followed by an
enterokinase cleavage site"
/label=3xFLAG
/translation="DYKDHDGDYKDHDIDYKDDDDK"
CDS 3057..3077
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 3102..7202
/codon_start=1
/product="catalytically dead mutant of the Cas9
endonuclease from the Streptococcus pyogenes Type II
CRISPR/Cas system"
/label=dCas9
/note="RNA-guided DNA-binding protein that lacks
endonuclease activity due to the D10A mutation in the RuvC
catalytic domain and the H840A mutation in the HNH
catalytic domain"
/translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
ATLIHQSITGLYETRIDLSQLGGD"
RBS 7115..7123
/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 7203..7250
/codon_start=1
/product="bipartite nuclear localization signal from
nucleoplasmin"
/label=nucleoplasmin NLS
/translation="KRPAATKKAGQAKKKK"
CDS 7269..7289
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 7305..7325
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 7332..7481
/codon_start=1
/product="tetrameric repeat of the minimal activation
domain of herpes simplex virus VP16 (Beerli et al., 1998)"
/label=VP64
/translation="DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDF
DLDML"
CDS 7488..7508
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 7515..7541
/codon_start=1
/product="HA (human influenza hemagglutinin) epitope tag"
/label=HA
/translation="YPYDVPDYA"
terminator 7594..7846
/label=NOS terminator
/note="nopaline synthase terminator and poly(A) signal"
promoter 7963..8308
/label=CaMV 35S promoter
/note="strong constitutive promoter from cauliflower mosaic
virus"
CDS 8381..9406
/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 9447..9621
/label=CaMV poly(A) signal
/note="cauliflower mosaic virus polyadenylation signal"
misc_feature 9771..9795
/label=LB T-DNA repeat
/note="left border repeat from nopaline C58 T-DNA"
primer_bind complement(10043..10066)
/label=pENTR-F
/note="pENTR vectors, forward primer"
terminator 10074..10101
/label=rrnB T2 terminator
/note="transcription terminator T2 from the E. coli rrnB
gene"
terminator 10193..10279
/gene="Escherichia coli rrnB"
/label=rrnB T1 terminator
/note="transcription terminator T1 from the E. coli rrnB
gene"
rep_origin 10448..10883
/label=pSa ori
/note="origin of replication from bacterial plasmid pSa"
rep_origin complement(10998..11586)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(11078..11097)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS complement(11676..12485)
/codon_start=1
/gene="aph(3')-Ia"
/product="aminoglycoside phosphotransferase"
/label=KanR
/note="confers resistance to kanamycin in bacteria or G418
(Geneticin(R)) in eukaryotes"
/translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
FQVLEEYPDSGENIVDALAVSLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
primer_bind 12399..12418
/label=Kan-R
/note="Kanamycin resistance gene, reverse primer"
primer_bind 12477..12496
/label=pENTR-R
/note="pENTR vectors, reverse primer"
ORIGIN
1 cgctcttttc tcttaggttt acccgccaat atatcctgtc aaacactgat agtttaaact
61 gaaggcggga aacgacaatc tgatccaagc tcaagctaag ctcacgtgac ggaattaagc
121 ttcgacttgc cttccgcaca atacatcatt tcttcttagc tttttttctt cttcttcgtt
181 catacagttt ttttttgttt atcagcttac attttcttga accgtagctt tcgttttctt
241 ctttttaact ttccattcgg agtttttgta tcttgtttca tagtttgtcc caggattaga
301 atgattaggc atcgaacctt caagaatttg attgaataaa acatcttcat tcttaagata
361 tgaagataat cttcaaaagg cccctgggaa tctgaaagaa gagaagcagg cccatttata
421 tgggaaagaa caatagtatt tcttatatag gcccatttaa gttgaaaaca atcttcaaaa
481 gtcccacatc gcttagataa gaaaacgaag ctgagtttat atacagctag agtcgaagta
541 gtgattggga gaccaaccca gtggacataa gcctgttcgg ttcgtaagct gtaatgcaag
601 tagcgtatgc gctcacgcaa ctggtccaga accttgaccg aacgcagcgg tggtaacggc
661 gcagtggcgg ttttcatggc ttgttatgac tgtttttttg gggtacagtc tatgcctcgg
721 gcatccaagc agcaagcgcg ttacgccgtg ggtcgatgtt tgatgttatg gagcagcaac
781 gatgttacgc agcagggcag tcgccctaaa acaaagttaa acatcatggg ggaagcggtg
841 atcgccgaag tatcgactca actatcagag gtagttggcg tcatcgagcg ccatctcgaa
901 ccgacgttgc tggccgtaca tttgtacggc tccgcagtgg atggcggcct gaagccacac
961 agtgatattg atttgctggt tacggtgacc gtaaggcttg atgaaacaac gcggcgagct
1021 ttgatcaacg accttttgga aacttcggct tcccctggag agagcgagat tctccgcgct
1081 gtagaagtca ccattgttgt gcacgacgac atcattccgt ggcgttatcc agctaagcgc
1141 gaactgcaat ttggagaatg gcagcgcaat gacattcttg caggtatctt cgagccagcc
1201 acgatcgaca ttgatctggc tatcttgctg acaaaagcaa gagaacatag cgttgccttg
1261 gtaggtccag cggcggagga actctttgat ccggttcctg aacaggatct atttgaggcg
1321 ctaaatgaaa ccttaacgct atggaactcg ccgcccgact gggctggcga tgagcgaaat
1381 gtagtgctta cgttgtcccg catttggtac agcgcagtaa ccggcaaaat cgcgccgaag
1441 gatgtcgctg ccgactgggc aatggagcgc ctgccggccc agtatcagcc cgtcatactt
1501 gaagctagac aggcttatct tggacaagaa gaagatcgct tggcctcgcg cgcagatcag
1561 ttggaagaat ttgtccacta cgtgaaaggc gagatcacca aggtagtcgg caaataatgt
1621 ctagctagaa attcgttcaa gccgacgccg cttcgcggcg cggcttaact caagcgttag
1681 atgcactaag cacataattg ctcacagcca aactatcagg tcaagtctgc ttttattatt
1741 tttaagcgtg cataataagc cggtctcggt tttagagcta gaaatagcaa gttaaaataa
1801 ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt ttgcaaaatt
1861 ttccagatcg atttcttctt cctctgttct tcggcgttca atttctgggg ttttctcttc
1921 gttttctgta actgaaacct aaaatttgac ctaaaaaaaa tctcaaataa tatgattcag
1981 tggttttgta cttttcagtt agttgagttt tgcagttccg atgagataaa ccaataagct
2041 tgcatgcctg caggtcaaca tggtggagca cgacacactt gtctactcca aaaatatcaa
2101 agatacagtc tcagaagacc aaagggcaat tgagactttt caacaaaggg taatatccgg
2161 aaacctcctc ggattccatt gcccagctat ctgtcacttt attgtgaaga tagtggaaaa
2221 ggaaggtggc tcctacaaat gccatcattg cgataaagga aaggccatcg ttgaagatgc
2281 ctctgccgac agtggtccca aagatggacc cccacccacg aggagcatcg tggaaaaaga
2341 agacgttcca accacgtctt caaagcaagt ggattgatgt gataacatgg tggagcacga
2401 cacacttgtc tactccaaaa atatcaaaga tacagtctca gaagaccaaa gggcaattga
2461 gacttttcaa caaagggtaa tatccggaaa cctcctcgga ttccattgcc cagctatctg
2521 tcactttatt gtgaagatag tggaaaagga aggtggctcc tacaaatgcc atcattgcga
2581 taaaggaaag gccatcgttg aagatgcctc tgccgacagt ggtcccaaag atggaccccc
2641 acccacgagg agcatcgtgg aaaaagaaga cgttccaacc acgtcttcaa agcaagtgga
2701 ttgatgtgat atctccactg acgtaaggga tgacgcacaa tcccactatc cttcgcaaga
2761 cccttcctct atataaggaa gttcatttca tttggagagg acctcgacct caacacaaca
2821 tatacaaaac aaacgaatct caagcaatca agcattctac ttctattgca gcaatttaaa
2881 tcatttcttt taaagcaaaa gcaattttct gaaaattttc accatttacg aacgatactc
2941 gagggggatc cccaatactt gtatggccgc ggccgctcta gatggattac aaggaccacg
3001 acggggatta caaggaccac gacattgatt acaaggatga tgatgacaag atggctccga
3061 agaagaagag gaaggttggc atccacgggg tgccagctgc tgacaagaag tactcgatcg
3121 gcctcgccat tgggactaac tctgttggct gggccgtgat caccgacgag tacaaggtgc
3181 cctcaaagaa gttcaaggtc ctgggcaaca ccgatcggca ttccatcaag aagaatctca
3241 ttggcgctct cctgttcgac agcggcgaga cggctgaggc tacgcggctc aagcgcaccg
3301 cccgcaggcg gtacacgcgc aggaagaatc gcatctgcta cctgcaggag attttctcca
3361 acgagatggc gaaggttgac gattctttct tccacaggct ggaggagtca ttcctcgtgg
3421 aggaggataa gaagcacgag cggcatccaa tcttcggcaa cattgtcgac gaggttgcct
3481 accacgagaa gtaccctacg atctaccatc tgcggaagaa gctcgtggac tccacagata
3541 aggcggacct ccgcctgatc tacctcgctc tggcccacat gattaagttc aggggccatt
3601 tcctgatcga gggggatctc aacccggaca atagcgatgt tgacaagctg ttcatccagc
3661 tcgtgcagac gtacaaccag ctcttcgagg agaaccccat taatgcgtca ggcgtcgacg
3721 cgaaggctat cctgtccgct aggctctcga agtctcggcg cctcgagaac ctgatcgccc
3781 agctgccggg cgagaagaag aacggcctgt tcgggaatct cattgcgctc agcctggggc
3841 tcacgcccaa cttcaagtcg aatttcgatc tcgctgagga cgccaagctg cagctctcca
3901 aggacacata cgacgatgac ctggataacc tcctggccca gatcggcgat cagtacgcgg
3961 acctgttcct cgctgccaag aatctgtcgg acgccatcct cctgtctgat attctcaggg
4021 tgaacaccga gattacgaag gctccgctct cagcctccat gatcaagcgc tacgacgagc
4081 accatcagga tctgaccctc ctgaaggcgc tggtcaggca gcagctcccc gagaagtaca
4141 aggagatctt cttcgatcag tcgaagaacg gctacgctgg gtacattgac ggcggggcct
4201 ctcaggagga gttctacaag ttcatcaagc cgattctgga gaagatggac ggcacggagg
4261 agctgctggt gaagctcaat cgcgaggacc tcctgaggaa gcagcggaca ttcgataacg
4321 gcagcatccc acaccagatt catctcgggg agctgcacgc tatcctgagg aggcaggagg
4381 acttctaccc tttcctcaag gataaccgcg agaagatcga gaagattctg actttcagga
4441 tcccgtacta cgtcggccca ctcgctaggg gcaactcccg cttcgcttgg atgacccgca
4501 agtcagagga gacgatcacg ccgtggaact tcgaggaggt ggtcgacaag ggcgctagcg
4561 ctcagtcgtt catcgagagg atgacgaatt tcgacaagaa cctgccaaat gagaaggtgc
4621 tccctaagca ctcgctcctg tacgagtact tcacagtcta caacgagctg actaaggtga
4681 agtatgtgac cgagggcatg aggaagccgg ctttcctgtc tggggagcag aagaaggcca
4741 tcgtggacct cctgttcaag accaaccgga aggtcacggt taagcagctc aaggaggact
4801 acttcaagaa gattgagtgc ttcgattcgg tcgagatctc tggcgttgag gaccgcttca
4861 acgcctccct ggggacctac cacgatctcc tgaagatcat taaggataag gacttcctgg
4921 acaacgagga gaatgaggat atcctcgagg acattgtgct gacactcact ctgttcgagg
4981 accgggagat gatcgaggag cgcctgaaga cttacgccca tctcttcgat gacaaggtca
5041 tgaagcagct caagaggagg aggtacaccg gctgggggag gctgagcagg aagctcatca
5101 acggcattcg ggacaagcag tccgggaaga cgatcctcga cttcctgaag agcgatggct
5161 tcgcgaaccg caatttcatg cagctgattc acgatgacag cctcacattc aaggaggata
5221 tccagaaggc tcaggtgagc ggccaggggg actcgctgca cgagcatatc gcgaacctcg
5281 ctggctcgcc agctatcaag aaggggattc tgcagaccgt gaaggttgtg gacgagctgg
5341 tgaaggtcat gggcaggcac aagcctgaga acatcgtcat tgagatggcc cgggagaatc
5401 agaccacgca gaagggccag aagaactcac gcgagaggat gaagaggatc gaggagggca
5461 ttaaggagct ggggtcccag atcctcaagg agcacccggt ggagaacacg cagctgcaga
5521 atgagaagct ctacctgtac tacctccaga atggccgcga tatgtatgtg gaccaggagc
5581 tggatattaa caggctcagc gattacgacg tcgatgccat cgttccacag tcattcctga
5641 aggatgactc cattgacaac aaggtcctca ccaggtcgga caagaaccgg ggcaagtctg
5701 ataatgttcc ttcagaggag gtcgttaaga agatgaagaa ctactggcgc cagctcctga
5761 atgccaagct gatcacgcag cggaagttcg ataacctcac aaaggctgag aggggcgggc
5821 tctctgagct ggacaaggcg ggcttcatca agaggcagct ggtcgagaca cggcagatca
5881 ctaagcacgt tgcgcagatt ctcgactcac ggatgaacac taagtacgat gagaatgaca
5941 agctgatccg cgaggtgaag gtcatcaccc tgaagtcaaa gctcgtctcc gacttcagga
6001 aggatttcca gttctacaag gttcgggaga tcaacaatta ccaccatgcc catgacgcgt
6061 acctgaacgc ggtggtcggc acagctctga tcaagaagta cccaaagctc gagagcgagt
6121 tcgtgtacgg ggactacaag gtttacgatg tgaggaagat gatcgccaag tcggagcagg
6181 agattggcaa ggctaccgcc aagtacttct tctactctaa cattatgaat ttcttcaaga
6241 cagagatcac tctggccaat ggcgagatcc ggaagcgccc cctcatcgag acgaacggcg
6301 agacggggga gatcgtgtgg gacaagggca gggatttcgc gaccgtcagg aaggttctct
6361 ccatgccaca agtgaatatc gtcaagaaga cagaggtcca gactggcggg ttctctaagg
6421 agtcaattct gcctaagcgg aacagcgaca agctcatcgc ccgcaagaag gactgggatc
6481 cgaagaagta cggcgggttc gacagcccca ctgtggccta ctcggtcctg gttgtggcga
6541 aggttgagaa gggcaagtcc aagaagctca agagcgtgaa ggagctgctg gggatcacga
6601 ttatggagcg ctccagcttc gagaagaacc cgatcgattt cctggaggcg aagggctaca
6661 aggaggtgaa gaaggacctg atcattaagc tccccaagta ctcactcttc gagctggaga
6721 acggcaggaa gcggatgctg gcttccgctg gcgagctgca gaaggggaac gagctggctc
6781 tgccgtccaa gtatgtgaac ttcctctacc tggcctccca ctacgagaag ctcaagggca
6841 gccccgagga caacgagcag aagcagctgt tcgtcgagca gcacaagcat tacctcgacg
6901 agatcattga gcagatttcc gagttctcca agcgcgtgat cctggccgac gcgaatctgg
6961 ataaggtcct ctccgcgtac aacaagcacc gcgacaagcc aatcagggag caggctgaga
7021 atatcattca tctcttcacc ctgacgaacc tcggcgcccc tgctgctttc aagtacttcg
7081 acacaactat cgatcgcaag aggtacacaa gcactaagga ggtcctggac gcgaccctca
7141 tccaccagtc gattaccggc ctctacgaga cgcgcatcga cctgtctcag ctcgggggcg
7201 acaagcggcc agcggcgacg aagaaggcgg ggcaggcgaa gaagaagaag tccgggtcat
7261 caggcagccc taagaagaag aggaaggtcg gcagcagcgg cagccccaag aagaagagga
7321 aggtcgggtc ggacgccctc gacgatttcg acctcgatat gctgggctcc gacgccctgg
7381 acgacttcga cctcgatatg ctcgggagcg acgcgctcga cgactttgac ctcgatatgc
7441 tcgggtcgga cgctctggac gattttgacc tcgatatgct ggggtcgcct aagaagaagc
7501 ggaaggttgg ctcataccca tacgatgtgc ccgactacgc ctcctgagct caggcctcca
7561 tacaagtatt ggggatccac tacgaatttc cccgatcgtt caaacatttg gcaataaagt
7621 ttcttaagat tgaatcctgt tgccggtctt gcgatgatta tcatataatt tctgttgaat
7681 tacgttaagc atgtaataat taacatgtaa tgcatgacgt tatttatgag atgggttttt
7741 atgattagag tcccgcaatt atacatttaa tacgcgatag aaaacaaaat atagcgcgca
7801 aactaggata aattatcgcg cgcggtgtca tctatgttac tagatcggga attcgaggcg
7861 gtttgcgtat tggctagagc agcttgccaa catggtggag cacgacactc tcgtctactc
7921 caagaatatc aaagatacag tctcagaaga ccaaagggct attgagactt ttcaacaaag
7981 ggtaatatcg ggaaacctcc tcggattcca ttgcccagct atctgtcact tcatcaaaag
8041 gacagtagaa aaggaaggtg gcacctacaa atgccatcat tgcgataaag gaaaggctat
8101 cgttcaagat gcctctgccg acagtggtcc caaagatgga cccccaccca cgaggagcat
8161 cgtggaaaaa gaagacgttc caaccacgtc ttcaaagcaa gtggattgat gtgatatctc
8221 cactgacgta agggatgacg cacaatccca ctatccttcg caagaccctt cctctatata
8281 aggaagttca tttcatttgg agaggacacg ctgaaatcac cagtctctct ctacaaatct
8341 atctctctcg agctttcgca gatccggggg gcaatgagat atgaaaaagc ctgaactcac
8401 cgcgacgtct gtcgagaagt ttctgatcga aaagttcgac agcgtctccg acctgatgca
8461 gctctcggag ggcgaagaat ctcgtgcttt cagcttcgat gtaggagggc gtggatatgt
8521 cctgcgggta aatagctgcg ccgatggttt ctacaaagat cgttatgttt atcggcactt
8581 tgcatcggcc gcgctcccga ttccggaagt gcttgacatt ggggagttta gcgagagcct
8641 gacctattgc atctcccgcc gttcacaggg tgtcacgttg caagacctgc ctgaaaccga
8701 actgcccgct gttctacaac cggtcgcgga ggctatggat gcgatcgctg cggccgatct
8761 tagccagacg agcgggttcg gcccattcgg accgcaagga atcggtcaat acactacatg
8821 gcgtgatttc atatgcgcga ttgctgatcc ccatgtgtat cactggcaaa ctgtgatgga
8881 cgacaccgtc agtgcgtccg tcgcgcaggc tctcgatgag ctgatgcttt gggccgagga
8941 ctgccccgaa gtccggcacc tcgtgcacgc ggatttcggc tccaacaatg tcctgacgga
9001 caatggccgc ataacagcgg tcattgactg gagcgaggcg atgttcgggg attcccaata
9061 cgaggtcgcc aacatcttct tctggaggcc gtggttggct tgtatggagc agcagacgcg
9121 ctacttcgag cggaggcatc cggagcttgc aggatcgcca cgactccggg cgtatatgct
9181 ccgcattggt cttgaccaac tctatcagag cttggttgac ggcaatttcg atgatgcagc
9241 ttgggcgcag ggtcgatgcg acgcaatcgt ccgatccgga gccgggactg tcgggcgtac
9301 acaaatcgcc cgcagaagcg cggccgtctg gaccgatggc tgtgtagaag tactcgccga
9361 tagtggaaac cgacgcccca gcactcgtcc gagggcaaag aaatagagta gatgccgacc
9421 gggatctgtc gatcgacaag ctcgagtttc tccataataa tgtgtgagta gttcccagat
9481 aagggaatta gggttcctat agggtttcgc tcatgtgttg agcatataag aaacccttag
9541 tatgtatttg tatttgtaaa atacttctat caataaaatt tctaattcct aaaaccaaaa
9601 tccagtacta aaatccagat cccccgaatt aattcggcgt taattcagta cattaaaaac
9661 gtccgcaatg tgttattaag ttgtcactag tcaggttaac tcaattcggc gttaattcag
9721 tacattaaaa acgtccgcaa tgtgttatta agttgtctaa gcgtcaattt gtttacacca
9781 caatatatcc tgccaccagc cagccaacag ctccccgacc ggcagctcgg cacaaaatca
9841 ccactcgata caggcagccc atcagtccgg gacggcgtca gcgggagagc cgttgtaagg
9901 cggcagactt tgctcatgtt accgatgcta ttcggaagaa cggcaactaa gctgccgggt
9961 ttgaaacacg gatgatctcg cggagggtag catgttgatt gtaacgatga cagagcgttg
10021 ctgcctgtga tcacttaagt aactaactaa caggaagagt ttgtagaaac gcaaaaaggc
10081 catccgtcag gatggccttc tgcttagttt gatgcctggc agtttatggc gggcgtcctg
10141 cccgccaccc tccgggccgt tgcttcacaa cgttcaaatc cgctcccggc ggatttgtcc
10201 tactcaggag agcgttcacc gacaaacaac agataaaacg aaaggcccag tcttccgact
10261 gagcctttcg ttttatttga tgcctggcag ttccctactc tcgcttagta gttagacgtc
10321 cccgagatcc atgctagacc atgaatccag aagcccgaga ggttgccgcc tttcgggctt
10381 tttctttttc aaaaaaaaaa atttataaaa cgatctgttg cggccggccg ccgggttgtg
10441 ggcaaaggcg ctcgacggtg ggcaaccgct tgcggttgtc cacgggcgga gccggtgcgc
10501 gtagcgcatt gtccacaagc caagggcgac caataattga tatatatatt cataattgaa
10561 aagctaattg aacatactac ttgctgtaac tacttgccgg agcgaggggt gtttgcaagc
10621 tgttgatctg aaagggctat tagcgttctc acgtgccttt ttgattagcg atttcacgtg
10681 accttattag cgatttcacg tactccgatt agcgatttca cgtaccctga ttagcgattt
10741 cacgtggata gtttttggag cgggccggaa agccccgtga atcaaggctt tgcggggcat
10801 tagcggtttc acgtggataa ctaccctcta tccacaggct tccggggata aaaaagcccg
10861 ctcgacggcg ggctgttgga tggggatcta gcggtaatac ggttatccac agaatcaggg
10921 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
10981 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
11041 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
11101 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
11161 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
11221 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
11281 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
11341 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
11401 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct
11461 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
11521 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
11581 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cggggcccaa
11641 tctgaataat gttacaacca attaaccaat tctgattaga aaaactcatc gagcatcaaa
11701 tgaaactgca atttattcat atcaggatta tcaataccat atttttgaaa aagccgtttc
11761 tgtaatgaag gagaaaactc accgaggcag ttccatagga tggcaagatc ctggtatcgg
11821 tctgcgattc cgactcgtcc aacatcaata caacctatta atttcccctc gtcaaaaata
11881 aggttatcaa gtgagaaatc accatgagtg acgactgaat ccggtgagaa tggcaaaagt
11941 ttatgcattt ctttccagac ttgttcaaca ggccagccat tacgctcgtc atcaaaatca
12001 ctcgcatcaa ccaaaccgtt attcattcgt gattgcgcct gagcgagacg aaatacgcga
12061 tcgctgttaa aaggacaatt acaaacagga atcgaatgca accggcgcag ggacactgcc
12121 agcgcatcaa caatattttc acctgaatca ggatattctt ctaatacctg gaatgctgtt
12181 tttccgggga tcgcagtggt gagtaaccat gcatcatcag gagtacggat aaaatgcttg
12241 atggtcggaa gaggcataaa ttccgtcagc cagtttagtc tgaccatctc atctgtaaca
12301 tcattggcaa cgctaccttt gccatgtttc agaaacaact ctggcgcatc gggcttccca
12361 tacaagcgat agattgtcgc acctgattgc ccgacattat cgcgagccca tttataccca
12421 tataaatcag catccatgtt ggaatttaat cgcggcctcg acgtttcccg ttgaatatgg
12481 ctcataacac cccttgtatt actgtttatg taagcagaca gttttattgt tcatgatgat
12541 atatttttat cttgtgcaat gtaacatcag agattttgag acacgggcca gagctgcagt
12601 ttgatcccga ggggaaccct gtggttgaca tgcacataca aatggacgaa cggataaacc
12661 ttttcacgcc cttttaaata tccgttattc taataaa
//
