| 质粒名称: | Inducible Caspex expression |
|---|---|
| 出品公司: | addgene |
| 目录编号: | 97421 |
| 存储实验室: | Steven Carr, Samuel Myers |
|
载体骨架基本信息 |
|
| 载体名称: | PB-TetON |
| 载体出品公司: | George Church lab |
| 空载体大小: | 7501 bp |
| 完整质粒大小: | 13424 bp |
| 载体类型: | 哺乳细胞表达 |
| 筛选标记: | Puromycin |
| 载体抗性: | 氨苄霉素 |
| 生长温度: | 37 ℃ |
| 宿主菌: | DH5a |
| 拷贝数: | 未知 |
|
插入基因信息 |
|
| 插入基因名称1: | xCaspe/CATG/Genomic Locus Proteomics |
| 物种来源: | S. pyogenes, Soybean |
| 基因突变: | dCas9: D10A and H840A, APEX2: K14D, W41F, E112K, A134P |
| 启动子: | TRE3G |
| 标签/融合蛋白: |
3xFLAG (N terminal on insert)
V5 (C terminal on insert)
|
| 克隆方法: | Ligation Independent Cloning |
| 5’测序引物 | ggcctccacggccactagt |
| 3’测序引物 | EF1a-rev |
| 插入基因名称2: | T2A-Gfp |
| 物种来源: | Jellyfish |
|
文献引用方法 |
|
| 材料和方法部分: | Inducible Caspex expression was a gift from Steven Carr & Samuel Myers (Addgene plasmid # 97421 ; http://n2t.net/addgene:97421 ; RRID:Addgene_97421) |
| 参考文献部分: | Discovery of proteins associated with a predefined genomic locus via dCas9-APEX-mediated proximity labeling. Myers SA, Wright J, Peckner R, Kalish BT, Zhang F, Carr SA. Nat Methods. 2018 May 7. pii: 10.1038/s41592-018-0007-1. doi: 10.1038/s41592-018-0007-1. 10.1038/s41592-018-0007-1 PubMed 29735997 |
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 Inducible Caspex expression 13424 bp ds-DNA circular SYN 12-MAY-2021
DEFINITION For stable clone generation of cells expressing the dCas9-APEX2
fusion protein for Genomic locus proteomics.
ACCESSION .
VERSION .
KEYWORDS Inducible Caspex expression
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 13424)
AUTHORS Myers SA, Wright J, Peckner R, Kalish BT, Zhang F, Carr SA
TITLE Discovery of proteins associated with a predefined genomic locus via
dCas9-APEX-mediated proximity labeling.
JOURNAL Nat Methods. 2018 May 7. pii: 10.1038/s41592-018-0007-1. doi:
10.1038/s41592-018-0007-1.
PUBMED 29735997
REFERENCE 2 (bases 1 to 13424)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58
http://www.biofeng.com
FEATURES Location/Qualifiers
source 1..13424
/organism="synthetic DNA construct"
/mol_type="other DNA"
primer_bind 1..17
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
repeat_region 147..209
/label=piggyBac right (3') inverted repeat
/note="piggyBac transposon-specific inverted terminal
repeat sequence (ITR)"
promoter 658..1036
/label=TRE3G promoter
/note="3rd-generation Tet-responsive promoter that can be
activated by binding of Tet-On(R) 3G"
protein_bind 666..684
/gene="tetO"
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O2 for the tetR and tetA genes"
protein_bind 702..720
/gene="tetO"
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O2 for the tetR and tetA genes"
protein_bind 738..756
/gene="tetO"
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O2 for the tetR and tetA genes"
protein_bind 774..792
/gene="tetO"
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O2 for the tetR and tetA genes"
protein_bind 810..828
/gene="tetO"
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O2 for the tetR and tetA genes"
protein_bind 846..864
/gene="tetO"
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O2 for the tetR and tetA genes"
protein_bind 882..900
/gene="tetO"
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O2 for the tetR and tetA genes"
primer_bind 945..969
/label=LNCX
/note="Human CMV promoter, forward primer"
regulatory 1049..1058
/regulatory_class="other"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
CDS 1058..1123
/codon_start=1
/product="three tandem FLAG(R) epitope tags, followed by an
enterokinase cleavage site"
/label=3xFLAG
/translation="DYKDHDGDYKDHDIDYKDDDDK"
CDS 1130..1150
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 1160..5263
/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="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
DATLIHQSITGLYETRIDLSQLGGD"
CDS 5276..5317
/codon_start=1
/product="epitope tag from simian virus 5"
/label=V5 tag
/translation="GKPIPNPLLGLDST"
CDS 5318..6064
/codon_start=1
/product="soybean ascorbate peroxidase, engineered to serve
as a monomeric tag for electron microscopy and live-cell
proteomics (Lam et al., 2015)"
/label=APEX2
/note="more active than the original APEX due to the A134P
mutation"
/translation="GKSYPTVSADYQDAVEKAKKKLRGFIAEKRCAPLMLRLAFHSAGT
FDKGTKTGGPFGTIKHPAELAHSANNGLDIAVRLLEPLKAEFPILSYADFYQLAGVVAV
EVTGGPKVPFHPGREDKPEPPPEGRLPDPTKGSDHLRDVFGKAMGLTDQDIVALSGGHT
IGAAHKERSGFEGPWTSNPLIFDNSYFTELLSGEKEGLLQLPSDKALLSDPVFRPLVDK
YAADEDAFFADYAEAHQKLSELGFADA"
CDS 6107..6127
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 6128..6181
/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 6188..6901
/codon_start=1
/product="enhanced GFP"
/label=EGFP
/translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
FVTAAGITLGMDELYK"
CDS 6188..6901
/codon_start=1
/product="the original enhanced GFP (Yang et al., 1996)"
/label=EGFP
/note="mammalian codon-optimized"
/translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
FVTAAGITLGMDELYK"
primer_bind complement(6230..6251)
/label=EGFP-N
/note="EGFP, reverse primer"
primer_bind complement(6491..6510)
/label=EXFP-R
/note="For distinguishing EGFP variants, reverse primer"
primer_bind 6838..6859
/label=EGFP-C
/note="EGFP, forward primer"
primer_bind complement(6988..7007)
/label=TK-pA-R
/note="Thymidine kinase polyA, reverse primer"
polyA_signal 7032..7080
/label=HSV TK poly(A) signal
/note="herpes simplex virus thymidine kinase
polyadenylation signal (Cole and Stacy, 1985)"
promoter 7311..7522
/label=EF-1-alpha core promoter
/note="core promoter for human elongation factor
EF-1-alpha"
LTR 7535..7803
/label=5' LTR (truncated)
/note="truncated 5' long terminal repeat (LTR) from human
T-cell leukemia virus (HTLV) type 1"
CDS 7837..8580
/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 8581..8634
/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 8635..9234
/codon_start=1
/gene="pac from Streptomyces alboniger"
/product="puromycin N-acetyltransferase"
/label=PuroR
/note="confers resistance to puromycin"
/translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
primer_bind complement(8635..8654)
/label=Puro-R
/note="Puromycin resistance gene, reverse primer. Also
called puro-variant-R"
primer_bind 9131..9151
/label=Puro-F
/note="Puromycin resistance gene, forward primer"
polyA_signal 9267..9388
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(9304..9323)
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
primer_bind 9358..9377
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
repeat_region 10151..10185
/label=piggyBac left (5') inverted repeat
/note="piggyBac transposon-specific inverted terminal
repeat sequence (ITR)"
primer_bind complement(10676..10692)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(10676..10692)
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind complement(10689..10711)
/label=M13/pUC Reverse
/note="In lacZ gene"
protein_bind 10700..10716
/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(10724..10754)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 10769..10790
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(10907..10924)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(11078..11666)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(11158..11177)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS complement(11837..12697)
/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 12460..12479
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
promoter complement(12698..12802)
/gene="bla"
/label=AmpR promoter
rep_origin complement(12828..13283)
/direction=LEFT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(12965..12986)
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind 13177..13196
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 13410..8
/label=M13/pUC Forward
/note="In lacZ gene"
primer_bind 13424..17
/label=M13 Forward
/note="In lacZ gene. Also called M13-F20 or M13 (-21)
Forward"
ORIGIN
1 gtaaaacgac ggccagtgag cgcgcctcgt tcattcacgt ttttgaaccc gtggaggacg
61 ggcagactcg cggtgcaaat gtgttttaca gcgtgatgga gcagatgaag atgctcgaca
121 cgctgcagaa cacgcagcta gattaaccct agaaagataa tcatattgtg acgtacgtta
181 aagataatca tgtgtaaaat tgacgcatgt gttttatcgg tctgtatatc gaggtttatt
241 tattaatttg aatagatatt aagttttatt atatttacac ttacatacta ataataaatt
301 caacaaacaa tttatttatg tttatttatt tattaaaaaa aacaaaaact caaaatttct
361 tctataaagt aacaaaactt ttatgaggga cagccccccc ccaaagcccc cagggatgta
421 attacgtccc tcccccgcta gggggcagca gcgagccgcc cggggctccg ctccggtccg
481 gcgctccccc cgcatccccg agccggcagc gtgcggggac agcccgggca cggggaaggt
541 ggcacgggat cgctttcctc tgaacgcttc tcgctgctct ttgagcctgc agacacctgg
601 ggggatacgg ggaaaaggcc tccacggcca ctagtctttc gtcttcaaga attcctcgag
661 tttactccct atcagtgata gagaacgtat gaagagttta ctccctatca gtgatagaga
721 acgtatgcag actttactcc ctatcagtga tagagaacgt ataaggagtt tactccctat
781 cagtgataga gaacgtatga ccagtttact ccctatcagt gatagagaac gtatctacag
841 tttactccct atcagtgata gagaacgtat atccagttta ctccctatca gtgatagaga
901 acgtataagc tttaggcgtg tacggtgggc gcctataaaa gcagagctcg tttagtgaac
961 cgtcagatcg cctggagcaa ttccacaaca cttttgtctt ataccaactt tccgtaccac
1021 ttcctaccct cgtaaaggtc tagagctagc caccatggac tacaaagacc atgacggtga
1081 ttataaagat catgacatcg attacaagga tgacgatgac aagatggccc ccaagaagaa
1141 gaggaaggtg ggccgcggaa tggacaagaa gtactccatt gggctcgcca tcggcacaaa
1201 cagcgtcggc tgggccgtca ttacggacga gtacaaggtg ccgagcaaaa aattcaaagt
1261 tctgggcaat accgatcgcc acagcataaa gaagaacctc attggcgccc tcctgttcga
1321 ctccggggaa accgccgaag ccacgcggct caaaagaaca gcacggcgca gatatacccg
1381 cagaaagaat cggatctgct acctgcagga gatctttagt aatgagatgg ctaaggtgga
1441 tgactctttc ttccataggc tggaggagtc ctttttggtg gaggaggata aaaagcacga
1501 gcgccaccca atctttggca atatcgtgga cgaggtggcg taccatgaaa agtacccaac
1561 catatatcat ctgaggaaga agcttgtaga cagtactgat aaggctgact tgcggttgat
1621 ctatctcgcg ctggcgcata tgatcaaatt tcggggacac ttcctcatcg agggggacct
1681 gaacccagac aacagcgatg tcgacaaact ctttatccaa ctggttcaga cttacaatca
1741 gcttttcgaa gagaacccga tcaacgcatc cggagttgac gccaaagcaa tcctgagcgc
1801 taggctgtcc aaatcccggc ggctcgaaaa cctcatcgca cagctccctg gggagaagaa
1861 gaacggcctg tttggtaatc ttatcgccct gtcactcggg ctgaccccca actttaaatc
1921 taacttcgac ctggccgaag atgccaagct tcaactgagc aaagacacct acgatgatga
1981 tctcgacaat ctgctggccc agatcggcga ccagtacgca gacctttttt tggcggcaaa
2041 gaacctgtca gacgccattc tgctgagtga tattctgcga gtgaacacgg agatcaccaa
2101 agctccgctg agcgctagta tgatcaagcg ctatgatgag caccaccaag acttgacttt
2161 gctgaaggcc cttgtcagac agcaactgcc tgagaagtac aaggaaattt tcttcgatca
2221 gtctaaaaat ggctacgccg gatacattga cggcggagca agccaggagg aattttacaa
2281 atttattaag cccatcttgg aaaaaatgga cggcaccgag gagctgctgg taaagcttaa
2341 cagagaagat ctgttgcgca aacagcgcac tttcgacaat ggaagcatcc cccaccagat
2401 tcacctgggc gaactgcacg ctatcctcag gcggcaagag gatttctacc cctttttgaa
2461 agataacagg gaaaagattg agaaaatcct cacatttcgg ataccctact atgtaggccc
2521 cctcgcccgg ggaaattcca gattcgcgtg gatgactcgc aaatcagaag agaccatcac
2581 tccctggaac ttcgaggaag tcgtggataa gggggcctct gcccagtcct tcatcgaaag
2641 gatgactaac tttgataaaa atctgcctaa cgaaaaggtg cttcctaaac actctctgct
2701 gtacgagtac ttcacagttt ataacgagct caccaaggtc aaatacgtca cagaagggat
2761 gagaaagcca gcattcctgt ctggagagca gaagaaagct atcgtggacc tcctcttcaa
2821 gacgaaccgg aaagttaccg tgaaacagct caaagaagac tatttcaaaa agattgaatg
2881 tttcgactct gttgaaatca gcggagtgga ggatcgcttc aacgcatccc tgggaacgta
2941 tcacgatctc ctgaaaatca ttaaagacaa ggacttcctg gacaatgagg agaacgagga
3001 cattcttgag gacattgtcc tcacccttac gttgtttgaa gatagggaga tgattgaaga
3061 acgcttgaaa acttacgctc atctcttcga cgacaaagtc atgaaacagc tcaagaggcg
3121 ccgatataca ggatgggggc ggctgtcaag aaaactgatc aatgggatcc gagacaagca
3181 gagtggaaag acaatcctgg attttcttaa gtccgatgga tttgccaacc ggaacttcat
3241 gcagttgatc catgatgact ctctcacctt taaggaggac atccagaaag cacaagtttc
3301 tggccagggg gacagtcttc acgagcacat cgctaatctt gcaggtagcc cagctatcaa
3361 aaagggaata ctgcagaccg ttaaggtcgt ggatgaactc gtcaaagtaa tgggaaggca
3421 taagcccgag aatatcgtta tcgagatggc ccgagagaac caaactaccc agaagggaca
3481 gaagaacagt agggaaagga tgaagaggat tgaagagggt ataaaagaac tggggtccca
3541 aatccttaag gaacacccag ttgaaaacac ccagcttcag aatgagaagc tctacctgta
3601 ctacctgcag aacggcaggg acatgtacgt ggatcaggaa ctggacatca atcggctctc
3661 cgactacgac gtggatgcca tcgtgcccca gtcttttctc aaagatgatt ctattgataa
3721 taaagtgttg acaagatccg ataaaaatag agggaagagt gataacgtcc cctcagaaga
3781 agttgtcaag aaaatgaaaa attattggcg gcagctgctg aacgccaaac tgatcacaca
3841 acggaagttc gataatctga ctaaggctga acgaggtggc ctgtctgagt tggataaagc
3901 cggcttcatc aaaaggcagc ttgttgagac acgccagatc accaagcacg tggcccaaat
3961 tctcgattca cgcatgaaca ccaagtacga tgaaaatgac aaactgattc gagaggtgaa
4021 agttattact ctgaagtcta agctggtctc agatttcaga aaggactttc agttttataa
4081 ggtgagagag atcaacaatt accaccatgc gcatgatgcc tacctgaatg cagtggtagg
4141 cactgcactt atcaaaaaat atcccaagct tgaatctgaa tttgtttacg gagactataa
4201 agtgtacgat gttaggaaaa tgatcgcaaa gtctgagcag gaaataggca aggccaccgc
4261 taagtacttc ttttacagca atattatgaa ttttttcaag accgagatta cactggccaa
4321 tggagagatt cggaagcgac cacttatcga aacaaacgga gaaacaggag aaatcgtgtg
4381 ggacaagggt agggatttcg cgacagtccg gaaggtcctg tccatgccgc aggtgaacat
4441 cgttaaaaag accgaagtac agaccggagg cttctccaag gaaagtatcc tcccgaaaag
4501 gaacagcgac aagctgatcg cacgcaaaaa agattgggac cccaagaaat acggcggatt
4561 cgattctcct acagtcgctt acagtgtact ggttgtggcc aaagtggaga aagggaagtc
4621 taaaaaactc aaaagcgtca aggaactgct gggcatcaca atcatggagc gatcaagctt
4681 cgaaaaaaac cccatcgact ttctcgaggc gaaaggatat aaagaggtca aaaaagacct
4741 catcattaag cttcccaagt actctctctt tgagcttgaa aacggccgga aacgaatgct
4801 cgctagtgcg ggcgagctgc agaaaggtaa cgagctggca ctgccctcta aatacgttaa
4861 tttcttgtat ctggccagcc actatgaaaa gctcaaaggg tctcccgaag ataatgagca
4921 gaagcagctg ttcgtggaac aacacaaaca ctaccttgat gagatcatcg agcaaataag
4981 cgaattctcc aaaagagtga tcctcgccga cgctaacctc gataaggtgc tttctgctta
5041 caataagcac agggataagc ccatcaggga gcaggcagaa aacattatcc acttgtttac
5101 tctgaccaac ttgggcgcgc ctgcagcctt caagtacttc gacaccacca tagacagaaa
5161 gcggtacacc tctacaaagg aggtcctgga cgccacactg attcatcagt caattacggg
5221 gctctatgaa acaagaatcg acctctctca gctcggtgga gacgtgcggg gttctggcaa
5281 gcccatcccc aaccccctgc tgggcctgga cagcaccgga aagtcttacc caactgtgag
5341 tgctgattac caggacgccg ttgagaaggc gaagaagaag ctcagaggct tcatcgctga
5401 gaagagatgc gctcctctaa tgctccgttt ggcattccac tctgctggaa cctttgacaa
5461 gggcacgaag accggtggac ccttcggaac catcaagcac cctgccgaac tggctcacag
5521 cgctaacaac ggtcttgaca tcgctgttag gcttttggag ccactcaagg cggagttccc
5581 tattttgagc tacgccgatt tctaccagtt ggctggcgtt gttgccgttg aggtcacggg
5641 tggacctaag gttccattcc accctggaag agaggacaag cctgagccac caccagaggg
5701 tcgcttgcct gatcccacta agggttctga ccatttgaga gatgtgtttg gcaaagctat
5761 ggggcttact gaccaagata tcgttgctct atctgggggt cacactattg gagctgcaca
5821 caaggagcgt tctggatttg agggtccctg gacctctaat cctcttattt tcgacaactc
5881 atacttcacg gagttgttga gtggtgagaa ggaaggtctc cttcagctac cttctgacaa
5941 ggctcttttg tctgaccctg tattccgccc tctcgttgat aaatatgcag cggacgaaga
6001 tgccttcttt gctgattacg ctgaggctca ccaaaagctt tccgagcttg ggtttgctga
6061 tgccgaattc agcagggccg accccaagaa gaagaggagg gccgacccca agaagaagag
6121 gaaggtggag ggcagaggaa gtcttctaac atgcggtgac gtggaggaga atcccggccc
6181 tggtaccgtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct
6241 ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg gcgatgccac
6301 ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc
6361 caccctcgtg accaccctga cctacggcgt gcagtgcttc agccgctacc ccgaccacat
6421 gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat
6481 cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac
6541 cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg
6601 gcacaagctg gagtacaact acaacagcca caacgtctat atcatggccg acaagcagaa
6661 gaacggcatc aaggtgaact tcaagatccg ccacaacatc gaggacggca gcgtgcagct
6721 cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa
6781 ccactacctg agcacccagt ccgccctgag caaagacccc aacgagaagc gcgatcacat
6841 ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa
6901 gaccggttga ccggttagta atgagtttaa acgggggagg aagaccggtt gaccggttag
6961 taatgagttt aaacggggga ggctaactga aacacggaag gagacaatac cggaaggaac
7021 ccgcgctatg acggcaataa aaagacagaa taaaacgcac gggtgttggg tcgtttgttc
7081 ataaacgcgg ggttcggtcc cagggctggc actctgtcga taccccaccg agaccccatt
7141 ggggccaata cgcccgcgtt tcttcctttt ccccacccca ccccccaagt tcgggtgaag
7201 gcccagggct cgcagccaac gtcggggcgg caggccctgc catagcagat ctgcgaaatc
7261 ggatccagct gaatctaagt ggatctgcga tcgctccggt gcccgtcagt gggcagagcg
7321 cacatcgccc acagtccccg agaagttggg gggaggggtc ggcaattgaa cgggtgccta
7381 gagaaggtgg cgcggggtaa actgggaaag tgatgtcgtg tactggctcc gcctttttcc
7441 cgagggtggg ggagaaccgt atataagtgc agtagtcgcc gtgaacgttc tttttcgcaa
7501 cgggtttgcc gccagaacac agctgaagct tcgaggggct cgcatctctc cttcacgcgc
7561 ccgccgccct acctgaggcc gccatccacg ccggttgagt cgcgttctgc cgcctcccgc
7621 ctgtggtgcc tcctgaactg cgtccgccgt ctaggtaagt ttaaagctca ggtcgagacc
7681 gggcctttgt ccggcgctcc cttggagcct acctagactc agccggctct ccacgctttg
7741 cctgaccctg cttgctcaac tctacgtctt tgtttcgttt tctgttctgc gccgttacag
7801 atctaagctg tgaccggcgc ctactctaga gccgccatgt ctagactgga caagagcaaa
7861 gtcataaact ctgctctgga attactcaat ggagtcggta tcgaaggcct gacgacaagg
7921 aaactcgctc aaaagctggg agttgagcag cctaccctgt actggcacgt gaagaacaag
7981 cgggccctgc tcgatgccct gccaatcgag atgctggaca ggcatcatac ccactcctgc
8041 cccctggaag gcgagtcatg gcaagacttt ctgcggaaca acgccaagtc ataccgctgt
8101 gctctcctct cacatcgcga cggggctaaa gtgcatctcg gcacccgccc aacagagaaa
8161 cagtacgaaa ccctggaaaa tcagctcgcg ttcctgtgtc agcaaggctt ctccctggag
8221 aacgcactgt acgctctgtc cgccgtgggc cactttacac tgggctgcgt attggaggaa
8281 caggagcatc aagtagcaaa agaggaaaga gagacaccta ccaccgattc tatgccccca
8341 cttctgaaac aagcaattga gctgttcgac cggcagggag ccgaacctgc cttccttttc
8401 ggcctggaac taatcatatg tggcctggag aaacagctaa agtgcgaaag cggcgggccg
8461 accgacgccc ttgacgattt tgacttagac atgctcccag ccgatgccct tgacgacttt
8521 gaccttgata tgctgcctgc tgacgctctt gacgattttg accttgacat gctccccggg
8581 gagggcagag gaagtcttct aacatgcggt gacgtggagg agaatcccgg ccctatgacc
8641 gagtacaagc ccacggtgcg cctcgccacc cgcgacgacg tccccagggc cgtacgcacc
8701 ctcgccgccg cgttcgccga ctaccccgcc acgcgccaca ccgtcgatcc ggaccgccac
8761 atcgagcggg tcaccgagct gcaagaactc ttcctcacgc gcgtcgggct cgacatcggc
8821 aaggtgtggg tcgcggacga cggcgccgcg gtggcggtct ggaccacgcc ggagagcgtc
8881 gaagcggggg cggtgttcgc cgagatcggc ccgcgcatgg ccgagttgag cggttcccgg
8941 ctggccgcgc agcaacagat ggaaggcctc ctggcgccgc accggcccaa ggagcccgcg
9001 tggttcctgg ccaccgtcgg cgtctcgccc gaccaccagg gcaagggtct gggcagcgcc
9061 gtcgtgctcc ccggagtgga ggcggccgag cgcgccgggg tgcccgcctt cctggagacc
9121 tccgcgcccc gcaacctccc cttctacgag cggctcggct tcaccgtcac cgccgacgtc
9181 gaggtgcccg aaggaccgcg cacctggtgc atgacccgca agcccggtgc ctgaaatcaa
9241 cctctggatt acaaaatttg tgggtaaact tgtttattgc agcttataat ggttacaaat
9301 aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg
9361 gtttgtccaa actcatcaat gtatcttatc atgtctggaa ttgactcaaa tgatgtcaat
9421 tagtctatca gaagctcatc tggtctccct tccgggggac aagacatccc tgtttaatat
9481 ttaaacagca gtgttcccaa actgggttct tatatccctt gctctggtca accaggttgc
9541 agggtttcct gtcctcacag gaacgaagtc cctaaagaaa cagtggcagc caggtttagc
9601 cccggaattg actggattcc ttttttaggg cccattggta tggctttttc cccgtatccc
9661 cccaggtgtc tgcaggctca aagagcagcg agaagcgttc agaggaaagc gatcccgtgc
9721 caccttcccc gtgcccgggc tgtccccgca cgctgccggc tcggggatgc ggggggagcg
9781 ccggaccgga gcggagcccc gggcggctcg ctgctgcccc ctagcggggg agggacgtaa
9841 ttacatccct gggggctttg ggggggggct gtccctgata tctataacaa gaaaatatat
9901 atataataag ttatcacgta agtagaacat gaaataacaa tataattatc gtatgagtta
9961 aatcttaaaa gtcacgtaaa agataatcat gcgtcatttt gactcacgcg gtcgttatag
10021 ttcaaaatca gtgacactta ccgcattgac aagcacgcct cacgggagct ccaagcggcg
10081 actgagatgt cctaaatgca cagcgacgga ttcgcgctat ttagaaagag agagcaatat
10141 ttcaagaatg catgcgtcaa ttttacgcag actatctttc tagggttaat ctagctgcat
10201 caggatcata tcgtcgggtc ttttttccgg ctcagtcatc gcccaagctg gcgctatctg
10261 ggcatcgggg aggaagaagc ccgtgccttt tcccgcgagg ttgaagcggc atggaaagag
10321 tttgccgagg atgactgctg ctgcattgac gttgagcgaa aacgcacgtt taccatgatg
10381 attcgggaag gtgtggccat gcacgccttt aacggtgaac tgttcgttca ggccacctgg
10441 gataccagtt cgtcgcggct tttccggaca cagttccgga tggtcagccc gaagcgcatc
10501 agcaacccga acaataccgg cgacagccgg aactgccgtg ccggtgtgca gattaatgac
10561 agcggtgcgg cgctgggata ttacgtcagc gaggacgggt atcctggctg gatgccgcag
10621 aaatggacat ggataccccg tgagttaccc ggcgggcgcg cttggcgtaa tcatggtcat
10681 agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa
10741 gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc
10801 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
10861 aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact
10921 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
10981 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
11041 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
11101 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
11161 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
11221 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
11281 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
11341 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
11401 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
11461 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa
11521 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
11581 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
11641 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
11701 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
11761 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
11821 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
11881 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
11941 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
12001 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
12061 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
12121 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
12181 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
12241 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
12301 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
12361 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
12421 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
12481 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
12541 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
12601 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
12661 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
12721 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
12781 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctaaa ttgtaagcgt
12841 taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata
12901 ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtgt
12961 tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg
13021 aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcaccctaat caagtttttt
13081 ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc gatttagagc
13141 ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga aaggagcggg
13201 cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac ccgccgcgct
13261 taatgcgccg ctacagggcg cgtcccattc gccattcagg ctgcgcaact gttgggaagg
13321 gcgatcggtg cgggcctctt cgctattacg ccagctggcg aaagggggat gtgctgcaag
13381 gcgattaagt tgggtaacgc cagggttttc ccagtcacga cgtt
//
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