| 质粒名称: | BICstim-Gag-dCAS9-VPR |
|---|---|
| 出品公司： | addgene |
| 目录编号: | 120922 |
| 存储实验室: | Philippe Mangeot, Théophile Ohlmann, Emiliano Ricci |
|
载体骨架基本信息 |
|
| 载体名称: | pcDNA3-like |
| 空载体大小: | 12435 bp |
| 完整质粒大小: | 12435 bp |
| 载体类型: | 哺乳细胞表达 |
| 载体抗性: | 氨苄霉素 |
| 生长温度: | 37 ℃ |
| 宿主菌: | DH5a |
| 拷贝数: | 高拷贝 |
|
插入基因信息 |
|
| 插入基因名称: | GAG (FMLV)-dCAS9-VPR |
| 物种来源: | H. sapiens (human) |
| 启动子: | CMV |
| 标签/融合蛋白: | none (N terminal on backbone) |
| 克隆方法: | 限制性内切酶 |
| 5’克隆位点: | Xho I not unique(未破坏) |
| 3’克隆位点: | KpnI(未破坏) |
| 5’测序引物 | ACTACATCCTGGTCATCATCC |
| 3’测序引物 | CCACCTTCTGATAGGCAGCC |
|
文献引用方法 |
|
| 材料和方法部分: |
BICstim-Gag-dCAS9-VPR was a gift from Philippe Mangeot & Théophile Ohlmann & Emiliano Ricci (Addgene plasmid # 120922 ; http://n2t.net/addgene:120922 ; RRID:Addgene_120922)
|
| 参考文献部分: | Genome editing in primary cells and in vivo using viral-derived Nanoblades loaded with Cas9-sgRNA ribonucleoproteins. Mangeot PE, Risson V, Fusil F, Marnef A, Laurent E, Blin J, Mournetas V, Massourides E, Sohier TJM, Corbin A, Aube F, Teixeira M, Pinset C, Schaeffer L, Legube G, Cosset FL, Verhoeyen E, Ohlmann T, Ricci EP. Nat Commun. 2019 Jan 3;10(1):45. doi: 10.1038/s41467-018-07845-z. 10.1038/s41467-018-07845-z PubMed 30604748 |
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 BICstim-Gag-dCAS9-VPR 12435 bp ds-DNA circular SYN 12-MAY-2021
DEFINITION encodes a GAG-dCAS9-VPR fusion for targeted transcriptional
activation..
ACCESSION .
VERSION .
KEYWORDS BICstim-Gag-dCAS9-VPR
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 12435)
AUTHORS Mangeot PE, Risson V, Fusil F, Marnef A, Laurent E, Blin J,
Mournetas V, Massourides E, Sohier TJM, Corbin A, Aube F, Teixeira
M, Pinset C, Schaeffer L, Legube G, Cosset FL, Verhoeyen E, Ohlmann
T, Ricci EP
TITLE Genome editing in primary cells and in vivo using viral-derived
Nanoblades loaded with Cas9-sgRNA ribonucleoproteins.
JOURNAL Nat Commun. 2019 Jan 3;10(1):45. doi: 10.1038/s41467-018-07845-z.
PUBMED 30604748
REFERENCE 2 (bases 1 to 12435)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58
http://www.biofeng.com
FEATURES Location/Qualifiers
source 1..12435
/organism="synthetic DNA construct"
/mol_type="other DNA"
enhancer 51..430
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 431..634
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
primer_bind 585..605
/label=CMV-F
/note="Human CMV immediate early promoter, forward primer"
intron 746..1318
/label=-beta-globin intron
/note="intron from rabbit beta-globin gene"
promoter 3057..3095
/label=minimal CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
regulatory 3204..3213
/regulatory_class="other"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
CDS 3210..7313
/codon_start=1
/product="catalytically dead mutant of the Cas9
endonuclease from the Streptococcus pyogenes Type II
CRISPR/Cas system"
/label=Cas9m4
/note="RNA-guided DNA-binding protein that lacks
endonuclease activity due to the mutations D10A, D839A,
H840A, and N863A (Mali et al., 2013)"
/translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
LYYLQNGRDMYVDQELDINRLSDYDVAAIVPQSFLKDDSIDNKVLTRSDKARGKSDNVP
SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKNVKELLGI
TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
DATLIHQSITGLYETRIDLSQLGGD"
CDS 7326..7346
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
protein_bind 7383..7407
/gene="mutant version of attB"
/label=attB1
/bound_moiety="BP Clonase(TM)"
/note="recombination site for the Gateway(R) BP reaction"
CDS 7437..7586
/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 7611..7631
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 8061..8417
/codon_start=1
/product="transcriptional activation domain of human RelA,
also known as p65 (O'Shea and Perkins, 2008)"
/label=RelA (p65) AD
/translation="PTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNS
EFQQLLNQGIPVAPHTTEPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGD
EDFSSIADMDFSALL"
CDS 8436..9005
/codon_start=1
/product="transcriptional activation domain from the human
herpesvirus 4 (Epstein-Barr virus) replication and
transcription activator Rta/BRLF1 (Hardwick et al., 1992;
Chavez et al., 2015)"
/label=Rta AD
/translation="RDSREGMFLPKPEAGSAISDVFEGREVCQPKRIRPFHPPGSPWAN
RPLPASLAPTPTGPVHEPVGSLTPAPVPQPLDPAPAVTPEASHLLEDPDEETSQAVKAL
REMADTVIPQKEEAAICGQMDLSHPPPRGHLDELTTTLESMTEDLNLDSPLTPELNEIL
DTFLNDECLLHAMHISTGLSIFDTSLF"
primer_bind complement(9094..9113)
/label=Bglob-pA-R
/note="Rabbit beta-globin polyA region, reverse primer"
polyA_signal 9159..9214
/label=-beta-globin poly(A) signal
/note="rabbit beta-globin polyadenylation signal (Gil and
Proudfoot, 1987)"
primer_bind complement(9213..9232)
/label=rbglobpA-R
/note="Rabbit beta-globin polyA, reverse primer. Also
called rb-glob-pA-term-R"
primer_bind complement(9572..9588)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(9572..9588)
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind complement(9585..9607)
/label=M13/pUC Reverse
/note="In lacZ gene"
protein_bind 9596..9612
/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(9620..9650)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 9665..9686
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(9803..9820)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(9974..10562)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(10054..10073)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS complement(10733..11593)
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/label=AmpR
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
primer_bind 11356..11375
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
promoter complement(11594..11698)
/gene="bla"
/label=AmpR promoter
rep_origin complement(11724..12179)
/direction=LEFT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(11861..11882)
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind 12073..12092
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 12306..12328
/label=M13/pUC Forward
/note="In lacZ gene"
primer_bind 12320..12337
/label=M13 Forward
/note="In lacZ gene. Also called M13-F20 or M13 (-21)
Forward"
primer_bind 12321..12337
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
primer_bind 12347..12366
/label=T7
/note="T7 promoter, forward primer"
promoter 12347..12365
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
1 tcataatatg tacatttata ttggctcatg tccaacatta ccgccatgtt gacattgatt
61 attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga
121 gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg
181 cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg
241 acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca
301 tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc
361 ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc
421 tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc
481 acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa
541 tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag
601 gcgtgtacgg tgggaggtct atataagcag agctcgttta gtgaaccgtc agatcgcctg
661 gagacgccat ccacgctgtt ttgacctcca tagaagacac cgggaccgat ccagcctccg
721 gtcgaccgat cctgagaact tcagggtgag tttggggacc cttgattgtt ctttcttttt
781 cgctattgta aaattcatgt tatatggagg gggcaaagtt ttcagggtgt tgtttagaat
841 gggaagatgt cccttgtatc accatggacc ctcatgataa ttttgtttct ttcactttct
901 actctgttga caaccattgt ctcctcttat tttcttttca ttttctgtaa ctttttcgtt
961 aaactttagc ttgcatttgt aacgaatttt taaattcact tttgtttatt tgtcagattg
1021 taagtacttt ctctaatcac ttttttttca aggcaatcag ggtatattat attgtacttc
1081 agcacagttt tagagaacaa ttgttataat taaatgataa ggtagaatat ttctgcatat
1141 aaattctggc tggcgtggaa atattcttat tggtagaaac aactacaccc tggtcatcat
1201 cctgcctttc tctttatggt tacaatgata tacactgttt gagatgagga taaaatactc
1261 tgagtccaaa ccgggcccct ctgctaacca tgttcatgcc ttcttctctt tcctacagct
1321 cctgggcaac gtgctggttg ttgtgctgtc tcatcatttt ggcaaagaat tcctcgaggg
1381 ccggatccga attcctgtat ttgtctgaaa acatgggcca ggctgttacc acccccttaa
1441 gtttgacttt agaccactgg aaggatgtcg aacggacagc ccacaacctg tcggtagagg
1501 ttagaaaaag gcgctgggtt acattctgct ctgcagaatg gccaaccttc aacgtcggat
1561 ggccacgaga cggcactttt aacccagaca ttattacaca ggttaagatc aaggtcttct
1621 cacctggccc acatggacat ccggatcagg tcccctacat cgtgacctgg gaagctatag
1681 cagtagaccc ccctccctgg gtcagaccct tcgtgcaccc taaacctccc ctctctcttc
1741 ccccttcagc cccctctctc ccacctgaac ccccactctc gaccccgccc cagtcctccc
1801 tctatccggc tctcacttct cctttaaaca ccaaacctag gcctcaagtc cttcctgata
1861 gcggaggacc actcattgat ctactcacgg aggaccctcc gccttaccgg gacccagggc
1921 caccctctcc tgacgggaac ggcgatagcg gagaagtggc ccctacagaa ggagcccctg
1981 acccttcccc aatggtatcc cgcctgcggg gaagaaaaga accccccgtg gcggattcta
2041 ctacctctca ggcgttcccc cttcgcctgg gagggaatgg acagtatcaa tactggccat
2101 tttcctcctc tgacctctat aactggaaaa ataacaaccc ctctttctcc gaggacccag
2161 ctaaattgac agctttgatc gagtccgttc tccttactca tcagcccact tgggatgact
2221 gccaacagct attagggacc ctgctgacgg gagaagaaaa acagcgagtg ctcctagagg
2281 cccgaaaggc ggttcgaggg gaggacggac gcccaactca gctgcccaat gacattaatg
2341 atgcttttcc cttggaacgt cccgactggg actacaacac ccaacgaggt aggaaccacc
2401 tagtccacta tcgccagttg ctcctagcgg gtctccaaaa cgcgggcaga agccccacca
2461 atttggccaa ggtaaaaggg ataacccagg gacctaatga gtctccctca gcctttttag
2521 agagactcaa ggaggcctat cgcagataca ctccttatga ccctgaggac ccagggcaag
2581 aaaccaatgt ggccatgtca ttcatctggc agtccgcccc ggatatcggg cgaaagttag
2641 agcggttaga agatttgaag agtaagacct taggagactt agtgagggaa gctgaaaaga
2701 tctttaataa acgagaaacc ccggaagaaa gagaggaacg tattaggaga gaaacagagg
2761 aaaaggaaga acgccgtagg gcagaggatg tgcagagaga gaaggagagg gaccgcagaa
2821 gacatagaga aatgagtaag ttgctggcta ctgtcgttag cgggcagaga caggatagac
2881 agggaggaga gcgaaggagg ccccaactcg accacgacca gtgtgcctac tgcaaagaaa
2941 agggacattg ggctagagat tgccccaaga agccaagagg accccgggga ccacgacccc
3001 aggcctccct cctgacgcgt agttccctgt atccagccct cacacctacc ggtggcggta
3061 ggcgtgtacg gtgggaggtc tatataagca gagctcgttt agtgaaccgt cagatcgcct
3121 ggagacgcca tccacgctgt tttgacctcc atagaagaca ccgggaccga tccagcctcc
3181 ggactctaga ggatcgaacc cttgccacca tggacaagaa gtactccatt gggctcgcta
3241 tcggcacaaa cagcgtcggc tgggccgtca ttacggacga gtacaaggtg ccgagcaaaa
3301 aattcaaagt tctgggcaat accgatcgcc acagcataaa gaagaacctc attggcgccc
3361 tcctgttcga ctccggggag acggccgaag ccacgcggct caaaagaaca gcacggcgca
3421 gatatacccg cagaaagaat cggatctgct acctgcagga gatctttagt aatgagatgg
3481 ctaaggtgga tgactctttc ttccataggc tggaggagtc ctttttggtg gaggaggata
3541 aaaagcacga gcgccaccca atctttggca atatcgtgga cgaggtggcg taccatgaaa
3601 agtacccaac catatatcat ctgaggaaga agcttgtaga cagtactgat aaggctgact
3661 tgcggttgat ctatctcgcg ctggcgcata tgatcaaatt tcggggacac ttcctcatcg
3721 agggggacct gaacccagac aacagcgatg tcgacaaact ctttatccaa ctggttcaga
3781 cttacaatca gcttttcgaa gagaacccga tcaacgcatc cggagttgac gccaaagcaa
3841 tcctgagcgc taggctgtcc aaatcccggc ggctcgaaaa cctcatcgca cagctccctg
3901 gggagaagaa gaacggcctg tttggtaatc ttatcgccct gtcactcggg ctgaccccca
3961 actttaaatc taacttcgac ctggccgaag atgccaagct tcaactgagc aaagacacct
4021 acgatgatga tctcgacaat ctgctggccc agatcggcga ccagtacgca gacctttttt
4081 tggcggcaaa gaacctgtca gacgccattc tgctgagtga tattctgcga gtgaacacgg
4141 agatcaccaa agctccgctg agcgctagta tgatcaagcg ctatgatgag caccaccaag
4201 acttgacttt gctgaaggcc cttgtcagac agcaactgcc tgagaagtac aaggaaattt
4261 tcttcgatca gtctaaaaat ggctacgccg gatacattga cggcggagca agccaggagg
4321 aattttacaa atttattaag cccatcttgg aaaaaatgga cggcaccgag gagctgctgg
4381 taaagcttaa cagagaagat ctgttgcgca aacagcgcac tttcgacaat ggaagcatcc
4441 cccaccagat tcacctgggc gaactgcacg ctatcctcag gcggcaagag gatttctacc
4501 cctttttgaa agataacagg gaaaagattg agaaaatcct cacatttcgg ataccctact
4561 atgtaggccc cctcgcccgg ggaaattcca gattcgcgtg gatgactcgc aaatcagaag
4621 agaccatcac tccctggaac ttcgaggaag tcgtggataa gggggcctct gcccagtcct
4681 tcatcgaaag gatgactaac tttgataaaa atctgcctaa cgaaaaggtg cttcctaaac
4741 actctctgct gtacgagtac ttcacagttt ataacgagct caccaaggtc aaatacgtca
4801 cagaagggat gagaaagcca gcattcctgt ctggagagca gaagaaagct atcgtggacc
4861 tcctcttcaa gacgaaccgg aaagttaccg tgaaacagct caaagaagac tatttcaaaa
4921 agattgaatg tttcgactct gttgaaatca gcggagtgga ggatcgcttc aacgcatccc
4981 tgggaacgta tcacgatctc ctgaaaatca ttaaagacaa ggacttcctg gacaatgagg
5041 agaacgagga cattcttgag gacattgtcc tcacccttac gttgtttgaa gatagggaga
5101 tgattgaaga acgcttgaaa acttacgctc atctcttcga cgacaaagtc atgaaacagc
5161 tcaagaggcg ccgatataca ggatgggggc ggctgtcaag aaaactgatc aatgggatcc
5221 gagacaagca gagtggaaag acaatcctgg attttcttaa gtccgatgga tttgccaacc
5281 ggaacttcat gcagttgatc catgatgact ctctcacctt taaggaggac atccagaaag
5341 cacaagtttc tggccagggg gacagtcttc acgagcacat cgctaatctt gcaggtagcc
5401 cagctatcaa aaagggaata ctgcagaccg ttaaggtcgt ggatgaactc gtcaaagtaa
5461 tgggaaggca taagcccgag aatatcgtta tcgagatggc ccgagagaac caaactaccc
5521 agaagggaca gaagaacagt agggaaagga tgaagaggat tgaagagggt ataaaagaac
5581 tggggtccca aatccttaag gaacacccag ttgaaaacac ccagcttcag aatgagaagc
5641 tctacctgta ctacctgcag aacggcaggg acatgtacgt ggatcaggaa ctggacatca
5701 atcggctctc cgactacgac gtggctgcta tcgtgcccca gtcttttctc aaagatgatt
5761 ctattgataa taaagtgttg acaagatccg ataaagctag agggaagagt gataacgtcc
5821 cctcagaaga agttgtcaag aaaatgaaaa attattggcg gcagctgctg aacgccaaac
5881 tgatcacaca acggaagttc gataatctga ctaaggctga acgaggtggc ctgtctgagt
5941 tggataaagc cggcttcatc aaaaggcagc ttgttgagac acgccagatc accaagcacg
6001 tggcccaaat tctcgattca cgcatgaaca ccaagtacga tgaaaatgac aaactgattc
6061 gagaggtgaa agttattact ctgaagtcta agctggtctc agatttcaga aaggactttc
6121 agttttataa ggtgagagag atcaacaatt accaccatgc gcatgatgcc tacctgaatg
6181 cagtggtagg cactgcactt atcaaaaaat atcccaagct tgaatctgaa tttgtttacg
6241 gagactataa agtgtacgat gttaggaaaa tgatcgcaaa gtctgagcag gaaataggca
6301 aggccaccgc taagtacttc ttttacagca atattatgaa ttttttcaag accgagatta
6361 cactggccaa tggagagatt cggaagcgac cacttatcga aacaaacgga gaaacaggag
6421 aaatcgtgtg ggacaagggt agggatttcg cgacagtccg gaaggtcctg tccatgccgc
6481 aggtgaacat cgttaaaaag accgaagtac agaccggagg cttctccaag gaaagtatcc
6541 tcccgaaaag gaacagcgac aagctgatcg cacgcaaaaa agattgggac cccaagaaat
6601 acggcggatt cgattctcct acagtcgctt acagtgtact ggttgtggcc aaagtggaga
6661 aagggaagtc taaaaaactc aaaaacgtca aggaactgct gggcatcaca atcatggagc
6721 gatcaagctt cgaaaaaaac cccatcgact ttctcgaggc gaaaggatat aaagaggtca
6781 aaaaagacct catcattaag cttcccaagt actctctctt tgagcttgaa aacggccgga
6841 aacgaatgct cgctagtgcg ggcgagctgc agaaaggtaa cgagctggca ctgccctcta
6901 aatacgttaa tttcttgtat ctggccagcc actatgaaaa gctcaaaggg tctcccgaag
6961 ataatgagca gaagcagctg ttcgtggaac aacacaaaca ctaccttgat gagatcatcg
7021 agcaaataag cgaattctcc aaaagagtga tcctcgccga cgctaacctc gataaggtgc
7081 tttctgctta caataagcac agggataagc ccatcaggga gcaggcagaa aacattatcc
7141 acttgtttac tctgaccaac ttgggcgcgc ctgcagcctt caagtacttc gacaccacca
7201 tagacagaaa gcggtacacc tctacaaagg aggtcctgga cgccacactg attcatcagt
7261 caattacggg gctctatgaa acaagaatcg acctctctca gctcggtgga gacagcaggg
7321 ctgaccccaa gaagaagagg aaggtgtcgc cagggatccg tcgacttgac gcgttgatat
7381 caacaagttt gtacaaaaaa gcaggctaca aagaggccag cggttccgga cgggctgacg
7441 cattggacga ttttgatctg gatatgctgg gaagtgacgc cctcgatgat tttgaccttg
7501 acatgcttgg ttcggatgcc cttgatgact ttgacctcga catgctcggc agtgacgccc
7561 ttgatgattt cgacctggac atgctgatta actctagaag ttccggatct ccgaaaaaga
7621 aacgcaaagt tggtagccag tacctgcccg acaccgacga ccggcaccgg atcgaggaaa
7681 agcggaagcg gacctacgag acattcaaga gcatcatgaa gaagtccccc ttcagcggcc
7741 ccaccgaccc tagacctcca cctagaagaa tcgccgtgcc cagcagatcc agcgccagcg
7801 tgccaaaacc tgccccccag ccttacccct tcaccagcag cctgagcacc atcaactacg
7861 acgagttccc taccatggtg ttccccagcg gccagatctc tcaggcctct gctctggctc
7921 cagcccctcc tcaggtgctg cctcaggctc ctgctcctgc accagctcca gccatggtgt
7981 ctgcactggc tcaggcacca gcacccgtgc ctgtgctggc tcctggacct ccacaggctg
8041 tggctccacc agcccctaaa cctacacagg ccggcgaggg cacactgtct gaagctctgc
8101 tgcagctgca gttcgacgac gaggatctgg gagccctgct gggaaacagc accgatcctg
8161 ccgtgttcac cgacctggcc agcgtggaca acagcgagtt ccagcagctg ctgaaccagg
8221 gcatccctgt ggcccctcac accaccgagc ccatgctgat ggaatacccc gaggccatca
8281 cccggctcgt gacaggcgct cagaggcctc ctgatccagc tcctgcccct ctgggagcac
8341 caggcctgcc taatggactg ctgtctggcg acgaggactt cagctctatc gccgatatgg
8401 atttctcagc cttgctgggc tctggcagcg gcagccggga ttccagggaa gggatgtttt
8461 tgccgaagcc tgaggccggc tccgctatta gtgacgtgtt tgagggccgc gaggtgtgcc
8521 agccaaaacg aatccggcca tttcatcctc caggaagtcc atgggccaac cgcccactcc
8581 ccgccagcct cgcaccaaca ccaactggtc cagtacatga gccagtcggg tcactgaccc
8641 cggcaccagt ccctcagcca ctggatccag cgcccgcagt gactcccgag gccagtcacc
8701 tgttggagga tcccgatgaa gagacgagcc aggctgtcaa agcccttcgg gagatggccg
8761 atactgtgat tccccagaag gaagaggctg caatctgtgg ccaaatggac ctttcccatc
8821 cgcccccaag gggccatctg gatgagctga caaccacact tgagtccatg accgaggatc
8881 tgaacctgga ctcacccctg accccggaat tgaacgagat tctggatacc ttcctgaacg
8941 acgagtgcct cttgcatgcc atgcatatca gcacaggact gtccatcttc gacacatctc
9001 tgttttgagg tacctgcatc ctcaggtgca ggctgcctat cagaaggtgg tggctggtgt
9061 ggccaatgcc ctggctcaca aataccactg agatcttttt ccctctgcca aaaattatgg
9121 ggacatcatg aagccccttg agcatctgac ttctggctaa taaaggaaat ttattttcat
9181 tgcaatagtg tgttggaatt ttttgtgtct ctcactcgga aggacatatg ggagggcaaa
9241 tcatttaaaa catcagaatg agtatttggt ttagagtttg gcaacatatg cccatatgct
9301 ggctgccatg aacaaaggtt ggctataaag aggtcatcag tatatgaaac agccccctgc
9361 tgtccattcc ttattccata gaaaagcctt gacttgaggt tagatttttt ttatattttg
9421 ttttgtgtta tttttttctt taacatccct aaaattttcc ttacatgttt tactagccag
9481 atttttcctc ctctcctgac tactcccagt catagctgtc cctcttctct tatggagatc
9541 cctcgacgga tcggccgcaa ttcgtaatca tgtcatagct gtttcctgtg tgaaattgtt
9601 atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg
9661 cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg
9721 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
9781 gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
9841 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
9901 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
9961 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
10021 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
10081 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
10141 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
10201 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
10261 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
10321 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
10381 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
10441 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
10501 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
10561 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
10621 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
10681 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
10741 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
10801 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
10861 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
10921 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
10981 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
11041 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
11101 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
11161 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
11221 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
11281 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
11341 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
11401 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
11461 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
11521 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
11581 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
11641 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
11701 catttccccg aaaagtgcca cctaaattgt aagcgttaat attttgttaa aattcgcgtt
11761 aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta
11821 taaatcaaaa gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc
11881 actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg
11941 cccactacgt gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact
12001 aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt
12061 ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc
12121 ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc
12181 ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg cctcttcgct
12241 attacgccag ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg taacgccagg
12301 gttttcccag tcacgacgtt gtaaaacgac ggccagtgag cgcgcgtaat acgactcact
12361 atagggcgaa ttggagctcc accgcggtgg cggccgctct agagagcttg gcccattgca
12421 tacgttgtat ccata
//
