| 质粒名称: | PB-TRE-dCas9-VPR |
|---|---|
| 出品公司: | 爱迪基因 |
| 目录编号: | 63800 |
| 存储实验室: | George Church |
|
载体骨架基本信息 |
|
| 载体名称: | PB-TRE |
| 载体出品公司: | -- |
| 载体抗性: | 氨苄青霉素 |
| 空载体大小: | -- |
| 完整质粒大小: | -- |
| 载体修饰: | -- |
| 载体类型: | 哺乳动物表达，CRISPR，合成生物学；Piggybac |
| 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-VPR,VP64-p65-Rta |
| 物种来源: | H. sapiens (human), M. musculus (mouse) |
| 插入大小: | 5733 bp |
| 基因突变: | -- |
| GenBank编号 | -- |
| 启动子 | TRE |
| 标签/融合蛋白: | VPR（VP64-p65-Rta）（插接件上的C端子） |
| 克隆方法: | Gibson克隆 |
| 5' 克隆位点: | -- |
| 3' 克隆位点: | -- |
| 5' 测序引物 | GATTCCCCAGAAGGAAGAGG |
| 3' 测序引物 | -- |
|
文献引用方法 |
|
| 材料和方法部分: | PB-TRE-dCas9-VPR was a gift from George Church (Addgene plasmid # 63800 ; http://n2t.net/addgene:63800 ; RRID:Addgene_63800) |
| 参考文献部分: | Highly efficient Cas9-mediated transcriptional programming. Chavez A, Scheiman J, Vora S, Pruitt BW, Tuttle M, P R Iyer E, Lin S, Kiani S, Guzman CD, Wiegand DJ, Ter-Ovanesyan D, Braff JL, Davidsohn N, Housden BE, Perrimon N, Weiss R, Aach J, Collins JJ, Church GM. Nat Methods. 2015 Mar 2. doi: 10.1038/nmeth.3312. 10.1038/nmeth.3312 PubMed 25730490 |
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 PB-TRE-dCas9-VPR 13808 bp ds-DNA circular SYN 06-AUG-2018
DEFINITION SP-dCas9-VPR with doxycycline-inducible expression.
ACCESSION .
VERSION .
KEYWORDS PB-TRE-dCas9-VPR
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 13808)
AUTHORS Chavez A, Scheiman J, Vora S, Pruitt BW, Tuttle M, P R Iyer E, Lin
S, Kiani S, Guzman CD, Wiegand DJ, Ter-Ovanesyan D, Braff JL,
Davidsohn N, Housden BE, Perrimon N, Weiss R, Aach J, Collins JJ,
Church GM
TITLE Highly efficient Cas9-mediated transcriptional programming.
JOURNAL Nat Methods. 2015 Mar 2. doi: 10.1038/nmeth.3312.
PUBMED 25730490
REFERENCE 2 (bases 1 to 13808)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Aug 6, 2018 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..13808
/organism="synthetic DNA construct"
/mol_type="other DNA"
rep_origin complement(14..469)
/direction=LEFT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(151..172)
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind 363..382
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 596..618
/label=M13/pUC Forward
/note="In lacZ gene"
primer_bind 610..627
/label=M13 Forward
/note="In lacZ gene. Also called M13-F20 or M13 (-21)
Forward"
primer_bind 611..627
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
repeat_region 757..819
/label=piggyBac right (3') inverted repeat
/note="piggyBac transposon-specific inverted terminal
repeat sequence (ITR)"
promoter 1268..1646
/label=TRE3G promoter
/note="3rd-generation Tet-responsive promoter that can be
activated by binding of Tet-On(R) 3G"
protein_bind 1276..1294
/gene="tetO"
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O2 for the tetR and tetA genes"
protein_bind 1312..1330
/gene="tetO"
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O2 for the tetR and tetA genes"
protein_bind 1348..1366
/gene="tetO"
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O2 for the tetR and tetA genes"
protein_bind 1384..1402
/gene="tetO"
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O2 for the tetR and tetA genes"
protein_bind 1420..1438
/gene="tetO"
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O2 for the tetR and tetA genes"
protein_bind 1456..1474
/gene="tetO"
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O2 for the tetR and tetA genes"
protein_bind 1492..1510
/gene="tetO"
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O2 for the tetR and tetA genes"
primer_bind 1555..1579
/label=LNCX
/note="Human CMV promoter, forward primer"
regulatory 1673..1682
/regulatory_class="other"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
CDS 1679..5782
/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
ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
DATLIHQSITGLYETRIDLSQLGGD"
CDS 5795..5815
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 5840..5989
/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 6014..6034
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 6464..6820
/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 6839..7408
/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(7463..7482)
/label=TK-pA-R
/note="Thymidine kinase polyA, reverse primer"
polyA_signal 7507..7555
/label=HSV TK poly(A) signal
/note="herpes simplex virus thymidine kinase
polyadenylation signal (Cole and Stacy, 1985)"
promoter 7786..7997
/label=EF-1-alpha core promoter
/note="core promoter for human elongation factor
EF-1-alpha"
LTR 8010..8278
/label=5' LTR (truncated)
/note="truncated 5' long terminal repeat (LTR) from human
T-cell leukemia virus (HTLV) type 1"
CDS 8312..9055
/codon_start=1
/product="modified rtTA protein that binds tightly to
promoters containing the tet operator in the presence of
doxycycline"
/label=Tet-On(R) 3G
/translation="MSRLDKSKVINSALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV
KNKRALLDALPIEMLDRHHTHSCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTR
PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
DSMPPLLKQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPA
DALDDFDLDMLPADALDDFDLDMLPG"
CDS 9056..9109
/codon_start=1
/product="2A peptide from Thosea asigna virus capsid
protein"
/label=T2A
/note="Eukaryotic ribosomes fail to insert a peptide bond
between the Gly and Pro residues, yielding separate
polypeptides."
/translation="EGRGSLLTCGDVEENPGP"
CDS 9131..10150
/codon_start=1
/gene="aph(4)-Ia"
/product="aminoglycoside phosphotransferase from E. coli"
/label=HygR
/note="confers resistance to hygromycin"
/translation="KPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGYV
LRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPET
ELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQTV
MDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFGD
SQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGNF
DDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE"
primer_bind complement(10207..10227)
/label=WPRE-R
/note="WPRE, reverse primer"
polyA_signal 10261..10382
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(10298..10317)
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
primer_bind 10352..10371
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
repeat_region 11145..11179
/label=piggyBac left (5') inverted repeat
/note="piggyBac transposon-specific inverted terminal
repeat sequence (ITR)"
primer_bind complement(11670..11686)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(11670..11686)
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind complement(11683..11705)
/label=M13/pUC Reverse
/note="In lacZ gene"
protein_bind 11694..11710
/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(11718..11748)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 11763..11784
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(11901..11918)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(12072..12660)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(12152..12171)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS complement(12831..13691)
/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 13454..13473
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
promoter complement(13692..13796)
/gene="bla"
/label=AmpR promoter
ORIGIN
1 aaaagtgcca cctaaattgt aagcgttaat attttgttaa aattcgcgtt aaatttttgt
61 taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa
121 gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag
181 aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt
241 gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac
301 cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag
361 gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg
421 cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc ccattcgcca
481 ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg cctcttcgct attacgccag
541 ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg taacgccagg gttttcccag
601 tcacgacgtt gtaaaacgac ggccagtgag cgcgcctcgt tcattcacgt ttttgaaccc
661 gtggaggacg ggcagactcg cggtgcaaat gtgttttaca gcgtgatgga gcagatgaag
721 atgctcgaca cgctgcagaa cacgcagcta gattaaccct agaaagataa tcatattgtg
781 acgtacgtta aagataatca tgtgtaaaat tgacgcatgt gttttatcgg tctgtatatc
841 gaggtttatt tattaatttg aatagatatt aagttttatt atatttacac ttacatacta
901 ataataaatt caacaaacaa tttatttatg tttatttatt tattaaaaaa aacaaaaact
961 caaaatttct tctataaagt aacaaaactt ttatgaggga cagccccccc ccaaagcccc
1021 cagggatgta attacgtccc tcccccgcta gggggcagca gcgagccgcc cggggctccg
1081 ctccggtccg gcgctccccc cgcatccccg agccggcagc gtgcggggac agcccgggca
1141 cggggaaggt ggcacgggat cgctttcctc tgaacgcttc tcgctgctct ttgagcctgc
1201 agacacctgg ggggatacgg ggaaaaggcc tccacggcca ctagtctttc gtcttcaaga
1261 attcctcgag tttactccct atcagtgata gagaacgtat gaagagttta ctccctatca
1321 gtgatagaga acgtatgcag actttactcc ctatcagtga tagagaacgt ataaggagtt
1381 tactccctat cagtgataga gaacgtatga ccagtttact ccctatcagt gatagagaac
1441 gtatctacag tttactccct atcagtgata gagaacgtat atccagttta ctccctatca
1501 gtgatagaga acgtataagc tttaggcgtg tacggtgggc gcctataaaa gcagagctcg
1561 tttagtgaac cgtcagatcg cctggagcaa ttccacaaca cttttgtctt ataccaactt
1621 tccgtaccac ttcctaccct cgtaaaggtc tagagctagc gaattcgaat ttgccaccat
1681 ggacaagaag tactccattg ggctcgctat cggcacaaac agcgtcggct gggccgtcat
1741 tacggacgag tacaaggtgc cgagcaaaaa attcaaagtt ctgggcaata ccgatcgcca
1801 cagcataaag aagaacctca ttggcgccct cctgttcgac tccggggaga cggccgaagc
1861 cacgcggctc aaaagaacag cacggcgcag atatacccgc agaaagaatc ggatctgcta
1921 cctgcaggag atctttagta atgagatggc taaggtggat gactctttct tccataggct
1981 ggaggagtcc tttttggtgg aggaggataa aaagcacgag cgccacccaa tctttggcaa
2041 tatcgtggac gaggtggcgt accatgaaaa gtacccaacc atatatcatc tgaggaagaa
2101 gcttgtagac agtactgata aggctgactt gcggttgatc tatctcgcgc tggcgcatat
2161 gatcaaattt cggggacact tcctcatcga gggggacctg aacccagaca acagcgatgt
2221 cgacaaactc tttatccaac tggttcagac ttacaatcag cttttcgaag agaacccgat
2281 caacgcatcc ggagttgacg ccaaagcaat cctgagcgct aggctgtcca aatcccggcg
2341 gctcgaaaac ctcatcgcac agctccctgg ggagaagaag aacggcctgt ttggtaatct
2401 tatcgccctg tcactcgggc tgacccccaa ctttaaatct aacttcgacc tggccgaaga
2461 tgccaagctt caactgagca aagacaccta cgatgatgat ctcgacaatc tgctggccca
2521 gatcggcgac cagtacgcag accttttttt ggcggcaaag aacctgtcag acgccattct
2581 gctgagtgat attctgcgag tgaacacgga gatcaccaaa gctccgctga gcgctagtat
2641 gatcaagcgc tatgatgagc accaccaaga cttgactttg ctgaaggccc ttgtcagaca
2701 gcaactgcct gagaagtaca aggaaatttt cttcgatcag tctaaaaatg gctacgccgg
2761 atacattgac ggcggagcaa gccaggagga attttacaaa tttattaagc ccatcttgga
2821 aaaaatggac ggcaccgagg agctgctggt aaagcttaac agagaagatc tgttgcgcaa
2881 acagcgcact ttcgacaatg gaagcatccc ccaccagatt cacctgggcg aactgcacgc
2941 tatcctcagg cggcaagagg atttctaccc ctttttgaaa gataacaggg aaaagattga
3001 gaaaatcctc acatttcgga taccctacta tgtaggcccc ctcgcccggg gaaattccag
3061 attcgcgtgg atgactcgca aatcagaaga gaccatcact ccctggaact tcgaggaagt
3121 cgtggataag ggggcctctg cccagtcctt catcgaaagg atgactaact ttgataaaaa
3181 tctgcctaac gaaaaggtgc ttcctaaaca ctctctgctg tacgagtact tcacagttta
3241 taacgagctc accaaggtca aatacgtcac agaagggatg agaaagccag cattcctgtc
3301 tggagagcag aagaaagcta tcgtggacct cctcttcaag acgaaccgga aagttaccgt
3361 gaaacagctc aaagaagact atttcaaaaa gattgaatgt ttcgactctg ttgaaatcag
3421 cggagtggag gatcgcttca acgcatccct gggaacgtat cacgatctcc tgaaaatcat
3481 taaagacaag gacttcctgg acaatgagga gaacgaggac attcttgagg acattgtcct
3541 cacccttacg ttgtttgaag atagggagat gattgaagaa cgcttgaaaa cttacgctca
3601 tctcttcgac gacaaagtca tgaaacagct caagaggcgc cgatatacag gatgggggcg
3661 gctgtcaaga aaactgatca atgggatccg agacaagcag agtggaaaga caatcctgga
3721 ttttcttaag tccgatggat ttgccaaccg gaacttcatg cagttgatcc atgatgactc
3781 tctcaccttt aaggaggaca tccagaaagc acaagtttct ggccaggggg acagtcttca
3841 cgagcacatc gctaatcttg caggtagccc agctatcaaa aagggaatac tgcagaccgt
3901 taaggtcgtg gatgaactcg tcaaagtaat gggaaggcat aagcccgaga atatcgttat
3961 cgagatggcc cgagagaacc aaactaccca gaagggacag aagaacagta gggaaaggat
4021 gaagaggatt gaagagggta taaaagaact ggggtcccaa atccttaagg aacacccagt
4081 tgaaaacacc cagcttcaga atgagaagct ctacctgtac tacctgcaga acggcaggga
4141 catgtacgtg gatcaggaac tggacatcaa tcggctctcc gactacgacg tggctgctat
4201 cgtgccccag tcttttctca aagatgattc tattgataat aaagtgttga caagatccga
4261 taaagctaga gggaagagtg ataacgtccc ctcagaagaa gttgtcaaga aaatgaaaaa
4321 ttattggcgg cagctgctga acgccaaact gatcacacaa cggaagttcg ataatctgac
4381 taaggctgaa cgaggtggcc tgtctgagtt ggataaagcc ggcttcatca aaaggcagct
4441 tgttgagaca cgccagatca ccaagcacgt ggcccaaatt ctcgattcac gcatgaacac
4501 caagtacgat gaaaatgaca aactgattcg agaggtgaaa gttattactc tgaagtctaa
4561 gctggtctca gatttcagaa aggactttca gttttataag gtgagagaga tcaacaatta
4621 ccaccatgcg catgatgcct acctgaatgc agtggtaggc actgcactta tcaaaaaata
4681 tcccaagctt gaatctgaat ttgtttacgg agactataaa gtgtacgatg ttaggaaaat
4741 gatcgcaaag tctgagcagg aaataggcaa ggccaccgct aagtacttct tttacagcaa
4801 tattatgaat tttttcaaga ccgagattac actggccaat ggagagattc ggaagcgacc
4861 acttatcgaa acaaacggag aaacaggaga aatcgtgtgg gacaagggta gggatttcgc
4921 gacagtccgg aaggtcctgt ccatgccgca ggtgaacatc gttaaaaaga ccgaagtaca
4981 gaccggaggc ttctccaagg aaagtatcct cccgaaaagg aacagcgaca agctgatcgc
5041 acgcaaaaaa gattgggacc ccaagaaata cggcggattc gattctccta cagtcgctta
5101 cagtgtactg gttgtggcca aagtggagaa agggaagtct aaaaaactca aaagcgtcaa
5161 ggaactgctg ggcatcacaa tcatggagcg atcaagcttc gaaaaaaacc ccatcgactt
5221 tctcgaggcg aaaggatata aagaggtcaa aaaagacctc atcattaagc ttcccaagta
5281 ctctctcttt gagcttgaaa acggccggaa acgaatgctc gctagtgcgg gcgagctgca
5341 gaaaggtaac gagctggcac tgccctctaa atacgttaat ttcttgtatc tggccagcca
5401 ctatgaaaag ctcaaagggt ctcccgaaga taatgagcag aagcagctgt tcgtggaaca
5461 acacaaacac taccttgatg agatcatcga gcaaataagc gaattctcca aaagagtgat
5521 cctcgccgac gctaacctcg ataaggtgct ttctgcttac aataagcaca gggataagcc
5581 catcagggag caggcagaaa acattatcca cttgtttact ctgaccaact tgggcgcgcc
5641 tgcagccttc aagtacttcg acaccaccat agacagaaag cggtacacct ctacaaagga
5701 ggtcctggac gccacactga ttcatcagtc aattacgggg ctctatgaaa caagaatcga
5761 cctctctcag ctcggtggag acagcagggc tgaccccaag aagaagagga aggtggaggc
5821 cagcggttcc ggacgggctg acgcattgga cgattttgat ctggatatgc tgggaagtga
5881 cgccctcgat gattttgacc ttgacatgct tggttcggat gcccttgatg actttgacct
5941 cgacatgctc ggcagtgacg cccttgatga tttcgacctg gacatgctga ttaactctag
6001 aagttccgga tctccgaaaa agaaacgcaa agttggtagc cagtacctgc ccgacaccga
6061 cgaccggcac cggatcgagg aaaagcggaa gcggacctac gagacattca agagcatcat
6121 gaagaagtcc cccttcagcg gccccaccga ccctagacct ccacctagaa gaatcgccgt
6181 gcccagcaga tccagcgcca gcgtgccaaa acctgccccc cagccttacc ccttcaccag
6241 cagcctgagc accatcaact acgacgagtt ccctaccatg gtgttcccca gcggccagat
6301 ctctcaggcc tctgctctgg ctccagcccc tcctcaggtg ctgcctcagg ctcctgctcc
6361 tgcaccagct ccagccatgg tgtctgcact ggctcaggca ccagcacccg tgcctgtgct
6421 ggctcctgga cctccacagg ctgtggctcc accagcccct aaacctacac aggccggcga
6481 gggcacactg tctgaagctc tgctgcagct gcagttcgac gacgaggatc tgggagccct
6541 gctgggaaac agcaccgatc ctgccgtgtt caccgacctg gccagcgtgg acaacagcga
6601 gttccagcag ctgctgaacc agggcatccc tgtggcccct cacaccaccg agcccatgct
6661 gatggaatac cccgaggcca tcacccggct cgtgacaggc gctcagaggc ctcctgatcc
6721 agctcctgcc cctctgggag caccaggcct gcctaatgga ctgctgtctg gcgacgagga
6781 cttcagctct atcgccgaca tggacttctc cgcactgctg ggtagcggat cgggatctcg
6841 ggattccagg gaagggatgt ttttgccgaa gcctgaggcc ggctccgcta ttagtgacgt
6901 gtttgagggc cgcgaggtgt gccagccaaa acgaatccgg ccatttcatc ctccaggaag
6961 tccatgggcc aaccgcccac tccccgccag cctcgcacca acaccaaccg gtccagtaca
7021 tgagccagtc gggtcactga ccccggcacc agtccctcag ccactggatc cagcgcccgc
7081 agtgactccc gaggccagtc acctgttgga ggatcccgat gaagagacga gccaggctgt
7141 caaagccctt cgggagatgg ccgatactgt gattccccag aaggaagagg ctgcaatctg
7201 tggccaaatg gacctttccc atccgccccc aaggggccat ctggatgagc tgacaaccac
7261 acttgagtcc atgaccgagg atctgaacct ggactcaccc ctgaccccgg aattgaacga
7321 gattctggat accttcctga acgacgagtg cctcttgcat gccatgcata tcagcacagg
7381 actgtccatc ttcgacacat ctctgttttg aaagggttcg atccctaccg gttagtaatg
7441 agtttaaacg ggggaggcta actgaaacac ggaaggagac aataccggaa ggaacccgcg
7501 ctatgacggc aataaaaaga cagaataaaa cgcacgggtg ttgggtcgtt tgttcataaa
7561 cgcggggttc ggtcccaggg ctggcactct gtcgataccc caccgagacc ccattggggc
7621 caatacgccc gcgtttcttc cttttcccca ccccaccccc caagttcggg tgaaggccca
7681 gggctcgcag ccaacgtcgg ggcggcaggc cctgccatag cagatctgcg aaatcggatc
7741 cagctaaatc taagtggatc tgcgatcgct ccggtgcccg tcagtgggca gagcgcacat
7801 cgcccacagt ccccgagaag ttggggggag gggtcggcaa ttgaacgggt gcctagagaa
7861 ggtggcgcgg ggtaaactgg gaaagtgatg tcgtgtactg gctccgcctt tttcccgagg
7921 gtgggggaga accgtatata agtgcagtag tcgccgtgaa cgttcttttt cgcaacgggt
7981 ttgccgccag aacacagctg aagcttcgag gggctcgcat ctctccttca cgcgcccgcc
8041 gccctacctg aggccgccat ccacgccggt tgagtcgcgt tctgccgcct cccgcctgtg
8101 gtgcctcctg aactgcgtcc gccgtctagg taagtttaaa gctcaggtcg agaccgggcc
8161 tttgtccggc gctcccttgg agcctaccta gactcagccg gctctccacg ctttgcctga
8221 ccctgcttgc tcaactctac gtctttgttt cgttttctgt tctgcgccgt tacagatcta
8281 agctgtgacc ggcgcctact ctagagccgc catgtctaga ctggacaaga gcaaagtcat
8341 aaactctgct ctggaattac tcaatggagt cggtatcgaa ggcctgacga caaggaaact
8401 cgctcaaaag ctgggagttg agcagcctac cctgtactgg cacgtgaaga acaagcgggc
8461 cctgctcgat gccctgccaa tcgagatgct ggacaggcat catacccact cctgccccct
8521 ggaaggcgag tcatggcaag actttctgcg gaacaacgcc aagtcatacc gctgtgctct
8581 cctctcacat cgcgacgggg ctaaagtgca tctcggcacc cgcccaacag agaaacagta
8641 cgaaaccctg gaaaatcagc tcgcgttcct gtgtcagcaa ggcttctccc tggagaacgc
8701 actgtacgct ctgtccgccg tgggccactt tacactgggc tgcgtattgg aggaacagga
8761 gcatcaagta gcaaaagagg aaagagagac acctaccacc gattctatgc ccccacttct
8821 gaaacaagca attgagctgt tcgaccggca gggagccgaa cctgccttcc ttttcggcct
8881 ggaactaatc atatgtggcc tggagaaaca gctaaagtgc gaaagcggcg ggccgaccga
8941 cgcccttgac gattttgact tagacatgct cccagccgat gcccttgacg actttgacct
9001 tgatatgctg cctgctgacg ctcttgacga ttttgacctt gacatgctcc ccggggaggg
9061 cagaggaagt cttctaacat gcggtgacgt ggaggagaat cccggccctg aattcatgga
9121 tagatccgga aagcctgaac tcaccgcgac gtctgtcgag aagtttctga tcgaaaagtt
9181 cgacagcgtc tccgacctga tgcagctctc ggagggcgaa gaatctcgtg ctttcagctt
9241 cgatgtagga gggcgtggat atgtcctgcg ggtaaatagc tgcgccgatg gtttctacaa
9301 agatcgttat gtttatcggc actttgcatc ggccgcgctc ccgattccgg aagtgcttga
9361 cattggggaa ttcagcgaga gcctgaccta ttgcatctcc cgccgtgcac agggtgtcac
9421 gttgcaagac ctgcctgaaa ccgaactgcc cgctgttctg cagccggtcg cggaggccat
9481 ggatgcgatc gctgcggccg atcttagcca gacgagcggg ttcggcccat tcggaccgca
9541 aggaatcggt caatacacta catggcgtga tttcatatgc gcgattgctg atccccatgt
9601 gtatcactgg caaactgtga tggacgacac cgtcagtgcg tccgtcgcgc aggctctcga
9661 tgagctgatg ctttgggccg aggactgccc cgaagtccgg cacctcgtgc acgcggattt
9721 cggctccaac aatgtcctga cggacaatgg ccgcataaca gcggtcattg actggagcga
9781 ggcgatgttc ggggattccc aatacgaggt cgccaacatc ttcttctgga ggccgtggtt
9841 ggcttgtatg gagcagcaga cgcgctactt cgagcggagg catccggagc ttgcaggatc
9901 gccgcggctc cgggcgtata tgctccgcat tggtcttgac caactctatc agagcttggt
9961 tgacggcaat ttcgatgatg cagcttgggc gcagggtcga tgcgacgcaa tcgtccgatc
10021 cggagccggg actgtcgggc gtacacaaat cgcccgcaga agcgcggccg tctggaccga
10081 tggctgtgta gaagtactcg ccgatagtgg aaaccgacgc cccagcactc gtccgagggc
10141 aaaggaataa gccaaggaac ctctggatta caaaatttgt gaaagattga ctggtattct
10201 taactatgtt gctcctttta cgctatgtgg atacgctgct ttaatgcctt tgtatcagtt
10261 aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca
10321 aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct
10381 tatcatgtct ggaattgact caaatgatgt caattagtct atcagaagct catctggtct
10441 cccttccggg ggacaagaca tccctgttta atatttaaac agcagtgttc ccaaactggg
10501 ttcttatatc ccttgctctg gtcaaccagg ttgcagggtt tcctgtcctc acaggaacga
10561 agtccctaaa gaaacagtgg cagccaggtt tagccccgga attgactgga ttcctttttt
10621 agggcccatt ggtatggctt tttccccgta tccccccagg tgtctgcagg ctcaaagagc
10681 agcgagaagc gttcagagga aagcgatccc gtgccacctt ccccgtgccc gggctgtccc
10741 cgcacgctgc cggctcgggg atgcgggggg agcgccggac cggagcggag ccccgggcgg
10801 ctcgctgctg ccccctagcg ggggagggac gtaattacat ccctgggggc tttggggggg
10861 ggctgtccct gatatctata acaagaaaat atatatataa taagttatca cgtaagtaga
10921 acatgaaata acaatataat tatcgtatga gttaaatctt aaaagtcacg taaaagataa
10981 tcatgcgtca ttttgactca cgcggtcgtt atagttcaaa atcagtgaca cttaccgcat
11041 tgacaagcac gcctcacggg agctccaagc ggcgactgag atgtcctaaa tgcacagcga
11101 cggattcgcg ctatttagaa agagagagca atatttcaag aatgcatgcg tcaattttac
11161 gcagactatc tttctagggt taatctagct gcatcaggat catatcgtcg ggtctttttt
11221 ccggctcagt catcgcccaa gctggcgcta tctgggcatc ggggaggaag aagcccgtgc
11281 cttttcccgc gaggttgaag cggcatggaa agagtttgcc gaggatgact gctgctgcat
11341 tgacgttgag cgaaaacgca cgtttaccat gatgattcgg gaaggtgtgg ccatgcacgc
11401 ctttaacggt gaactgttcg ttcaggccac ctgggatacc agttcgtcgc ggcttttccg
11461 gacacagttc cggatggtca gcccgaagcg catcagcaac ccgaacaata ccggcgacag
11521 ccggaactgc cgtgccggtg tgcagattaa tgacagcggt gcggcgctgg gatattacgt
11581 cagcgaggac gggtatcctg gctggatgcc gcagaaatgg acatggatac cccgtgagtt
11641 acccggcggg cgcgcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat
11701 ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc
11761 taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
11821 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
11881 attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
11941 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
12001 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
12061 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
12121 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
12181 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
12241 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
12301 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
12361 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
12421 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
12481 aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg
12541 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
12601 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
12661 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
12721 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
12781 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
12841 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
12901 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
12961 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
13021 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
13081 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
13141 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
13201 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
13261 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
13321 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
13381 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
13441 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
13501 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
13561 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
13621 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
13681 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
13741 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
13801 tttccccg
//
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