| 质粒名称: | pL-CRISPR.EFS.GFP |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 57818 |
| 存储实验室: | Benjamin Ebert |
|
载体骨架基本信息 |
|
| 载体名称: | pLKO.005 |
| 载体出品公司: | -- |
| 空载体大小: | -- |
| 完整质粒大小: | 11707 bp |
| 载体修饰: | -- |
| 载体类型: | 慢病毒,CRISPR |
| 筛选标记: | -- |
| 载体抗性: | 氨苄霉素 |
| 生长温度: | 37 ℃ |
| 宿主菌: | Stbl3 |
| 拷贝数: | 高拷贝 |
|
插入基因信息 |
|
| 插入基因名称: | SpCas9 |
| 物种来源: | S. pyogenes |
| 插入大小: | 4300 bp |
| 基因突变: | -- |
| GenBank编号 | -- |
| 启动子: | EFS |
| 标签/融合蛋白: | FLAG（插入件上的N端子） |
| 克隆方法: | 限制性内切酶 |
| 5’克隆位点: | BamHI(未破坏) |
| 3’克隆位点: | Nhei(未破坏) |
| 5’测序引物 | ATAAGTGCAGTAGTCGCCGTG |
| 3’测序引物 | AAAGCAGCGTATCCACATAGC |
|
文献引用方法 |
|
| 材料和方法部分: | pL-CRISPR.EFS.GFP was a gift from Benjamin Ebert (Addgene plasmid # 57818 ; http://n2t.net/addgene:57818 ; RRID:Addgene_57818) |
| 参考文献部分: | Generation of mouse models of myeloid malignancy with combinatorial genetic lesions using CRISPR-Cas9 genome editing. Heckl D, Kowalczyk MS, Yudovich D, Belizaire R, Puram RV, McConkey ME, Thielke A, Aster JC, Regev A, Ebert BL. Nat Biotechnol. 2014 Jun 22. doi: 10.1038/nbt.2951. 10.1038/nbt.2951 PubMed 24952903 |
买家导航
pL-CRISPR.EFS.GFP载体质粒基本信息
pL-CRISPR.EFS.GFP质粒图谱载体图谱和pL-CRISPR.EFS.GFP载体序列质粒序列多克隆位点信息
pL-CRISPR.EFS.GFP质粒载体简介
pL-CRISPR.EFS.GFP质粒序列载体序列
LOCUS pL-CRISPR.EFS.GFP 11707 bp ds-DNA circular SYN 12-MAY-2021
DEFINITION Lentiviral CRISPR-Cas9 delivery for SpCas9 and sgRNA. Coexpresses
eGFP via P2A cleavage site. EFS Promoter driven.
ACCESSION .
VERSION .
KEYWORDS pL-CRISPR.EFS.GFP
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 11707)
AUTHORS Heckl D, Kowalczyk MS, Yudovich D, Belizaire R, Puram RV, McConkey
ME, Thielke A, Aster JC, Regev A, Ebert BL
TITLE Generation of mouse models of myeloid malignancy with combinatorial
genetic lesions using CRISPR-Cas9 genome editing.
JOURNAL Nat Biotechnol. 2014 Jun 22. doi: 10.1038/nbt.2951.
PUBMED 24952903
REFERENCE 2 (bases 1 to 11707)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..11707
/organism="synthetic DNA construct"
/mol_type="other DNA"
promoter 5..231
/label=RSV promoter
/note="Rous sarcoma virus enhancer/promoter"
LTR 232..412
/label=5' LTR (truncated)
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
misc_feature 459..584
/label=HIV-1 Psi
/note="packaging signal of human immunodeficiency virus
type 1"
misc_feature 1077..1310
/label=RRE
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
CDS 1495..1539
/codon_start=1
/product="antigenic peptide corresponding to amino acids
655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik
et al., 2013)"
/label=gp41 peptide
/note="recognized by the 2H10 single-chain llama nanobody"
/translation="KNEQELLELDKWASL"
promoter 1717..1957
/label=U6 promoter
/note="RNA polymerase III promoter for human U6 snRNA"
primer_bind 1717..1737
/label=hU6-F
/note="Human U6 promoter, forward primer"
primer_bind 1888..1907
/label=LKO.1 5'
/note="Human U6 promoter, forward primer"
misc_RNA 1984..2059
/label=gRNA scaffold
/note="guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system"
misc_feature 2107..2224
/label=cPPT/CTS
/note="central polypurine tract and central termination
sequence of HIV-1"
promoter 2293..2504
/label=EF-1-alpha core promoter
/note="core promoter for human elongation factor
EF-1-alpha"
regulatory 2524..2533
/regulatory_class="other"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
CDS 2533..2556
/codon_start=1
/product="FLAG(R) epitope tag, followed by an enterokinase
cleavage site"
/label=FLAG
/translation="DYKDDDDK"
CDS 2563..2583
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 2608..6708
/codon_start=1
/product="Cas9 (Csn1) endonuclease from the Streptococcus
pyogenes Type II CRISPR/Cas system"
/label=Cas9
/note="generates RNA-guided double strand breaks in DNA"
/translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
ATLIHQSITGLYETRIDLSQLGGD"
CDS 6709..6756
/codon_start=1
/product="bipartite nuclear localization signal from
nucleoplasmin"
/label=nucleoplasmin NLS
/translation="KRPAATKKAGQAKKKK"
CDS 6772..6828
/codon_start=1
/product="2A peptide from porcine teschovirus-1
polyprotein"
/label=P2A
/note="Eukaryotic ribosomes fail to insert a peptide bond
between the Gly and Pro residues, yielding separate
polypeptides."
/translation="ATNFSLLKQAGDVEENPGP"
CDS 6829..7545
/codon_start=1
/product="enhanced GFP"
/label=EGFP
/translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
EFVTAAGITLGMDELYK"
primer_bind complement(6874..6895)
/label=EGFP-N
/note="EGFP, reverse primer"
primer_bind complement(7135..7154)
/label=EXFP-R
/note="For distinguishing EGFP variants, reverse primer"
primer_bind 7482..7503
/label=EGFP-C
/note="EGFP, forward primer"
misc_feature 7564..8152
/label=WPRE
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
primer_bind complement(7617..7637)
/label=WPRE-R
/note="WPRE, reverse primer"
CDS complement(8035..8046)
/codon_start=1
/product="Factor Xa recognition and cleavage site"
/label=Factor Xa site
/translation="IEGR"
LTR 8224..8457
/label=3' LTR (Delta-U3)
/note="self-inactivating 3' long terminal repeat (LTR) from
HIV-1"
polyA_signal 8535..8656
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(8572..8591)
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
primer_bind 8626..8645
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
rep_origin 8696..8831
/label=SV40 ori
/note="SV40 origin of replication"
primer_bind 8758..8777
/label=SV40pro-F
/note="SV40 promoter/origin, forward primer"
primer_bind complement(8851..8870)
/label=T7
/note="T7 promoter, forward primer"
promoter complement(8852..8870)
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
primer_bind complement(8880..8897)
/label=M13 Forward
/note="In lacZ gene. Also called M13-F20 or M13 (-21)
Forward"
primer_bind complement(8880..8896)
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(8889..8911)
/label=M13/pUC Forward
/note="In lacZ gene"
rep_origin 9038..9493
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(9125..9144)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 9335..9356
/label=F1ori-F
/note="F1 origin, forward primer"
promoter 9519..9623
/gene="bla"
/label=AmpR promoter
CDS 9624..10484
/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 complement(9842..9861)
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
rep_origin 10655..11243
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 11144..11163
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
primer_bind 11397..11414
/label=L4440
/note="L4440 vector, forward primer"
protein_bind 11531..11552
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 11567..11597
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 11605..11621
/label=lac operator
/bound_moiety="lac repressor encoded by lacI"
/note="The lac repressor binds to the lac operator to
inhibit transcription in E. coli. This inhibition can be
relieved by adding lactose or
isopropyl-beta-D-thiogalactopyranoside (IPTG)."
primer_bind 11610..11632
/label=M13/pUC Reverse
/note="In lacZ gene"
primer_bind 11629..11645
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind 11629..11645
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind 11664..11684
/label=T3
/note="T3 promoter, forward primer"
promoter 11666..11684
/label=T3 promoter
/note="promoter for bacteriophage T3 RNA polymerase"
ORIGIN
1 ttaatgtagt cttatgcaat actcttgtag tcttgcaaca tggtaacgat gagttagcaa
61 catgccttac aaggagagaa aaagcaccgt gcatgccgat tggtggaagt aaggtggtac
121 gatcgtgcct tattaggaag gcaacagacg ggtctgacat ggattggacg aaccactgaa
181 ttgccgcatt gcagagatat tgtatttaag tgcctagctc gatacataaa cgggtctctc
241 tggttagacc agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag
301 cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct
361 ggtaactaga gatccctcag acccttttag tcagtgtgga aaatctctag cagtggcgcc
421 cgaacaggga cttgaaagcg aaagggaaac cagaggagct ctctcgacgc aggactcggc
481 ttgctgaagc gcgcacggca agaggcgagg ggcggcgact ggtgagtacg ccaaaaattt
541 tgactagcgg aggctagaag gagagagatg ggtgcgagag cgtcagtatt aagcggggga
601 gaattagatc gcgatgggaa aaaattcggt taaggccagg gggaaagaaa aaatataaat
661 taaaacatat agtatgggca agcagggagc tagaacgatt cgcagttaat cctggcctgt
721 tagaaacatc agaaggctgt agacaaatac tgggacagct acaaccatcc cttcagacag
781 gatcagaaga acttagatca ttatataata cagtagcaac cctctattgt gtgcatcaaa
841 ggatagagat aaaagacacc aaggaagctt tagacaagat agaggaagag caaaacaaaa
901 gtaagaccac cgcacagcaa gcggccgctg atcttcagac ctggaggagg agatatgagg
961 gacaattgga gaagtgaatt atataaatat aaagtagtaa aaattgaacc attaggagta
1021 gcacccacca aggcaaagag aagagtggtg cagagagaaa aaagagcagt gggaatagga
1081 gctttgttcc ttgggttctt gggagcagca ggaagcacta tgggcgcagc gtcaatgacg
1141 ctgacggtac aggccagaca attattgtct ggtatagtgc agcagcagaa caatttgctg
1201 agggctattg aggcgcaaca gcatctgttg caactcacag tctggggcat caagcagctc
1261 caggcaagaa tcctggctgt ggaaagatac ctaaaggatc aacagctcct ggggatttgg
1321 ggttgctctg gaaaactcat ttgcaccact gctgtgcctt ggaatgctag ttggagtaat
1381 aaatctctgg aacagatttg gaatcacacg acctggatgg agtgggacag agaaattaac
1441 aattacacaa gcttaataca ctccttaatt gaagaatcgc aaaaccagca agaaaagaat
1501 gaacaagaat tattggaatt agataaatgg gcaagtttgt ggaattggtt taacataaca
1561 aattggctgt ggtatataaa attattcata atgatagtag gaggcttggt aggtttaaga
1621 atagtttttg ctgtactttc tatagtgaat agagttaggc agggatattc accattatcg
1681 tttcagaccc acctcccaac cccgagggga cccagagagg gcctatttcc catgattcct
1741 tcatatttgc atatacgata caaggctgtt agagagataa ttagaattaa tttgactgta
1801 aacacaaaga tattagtaca aaatacgtga cgtagaaagt aataatttct tgggtagttt
1861 gcagttttaa aattatgttt taaaatggac tatcatatgc ttaccgtaac ttgaaagtat
1921 ttcgatttct tggctttata tatcttgtgg aaaggacgaa acaccggaga cgtgaccgtc
1981 tctgttttag agctagaaat agcaagttaa aataaggcta gtccgttatc aacttgaaaa
2041 agtggcaccg agtcggtgct tttttgaatt ctagatcttg agacaaatgg cagtattcat
2101 ccacaatttt aaaagaaaag gggggattgg ggggtacagt gcaggggaaa gaatagtaga
2161 cataatagca acagacatac aaactaaaga attacaaaaa caaattacaa aaattcaaaa
2221 ttttcgggtt tattacaggg acagcagaga tccactttgg cgccggctcg agtggctccg
2281 gtgcccgtca gtgggcagag cgcacatcgc ccacagtccc cgagaagttg gggggagggg
2341 tcggcaattg aaccggtgcc tagagaaggt ggcgcggggt aaactgggaa agtgatgtcg
2401 tgtactggct ccgccttttt cccgagggtg ggggagaacc gtatataagt gcagtagtcg
2461 ccgtgaacgt tctttttcgc aacgggtttg ccgccagaac acaggtgtcg tgacgcggga
2521 tccgccacca tggattacaa agacgatgac gataagatgg ccccaaagaa gaagcggaag
2581 gtcggtatcc acggagtccc agcagccgac aagaagtaca gcatcggcct ggacatcggc
2641 accaactctg tgggctgggc cgtgatcacc gacgagtaca aggtgcccag caagaaattc
2701 aaggtgctgg gcaacaccga ccggcacagc atcaagaaga acctgatcgg agccctgctg
2761 ttcgacagcg gcgaaacagc cgaggccacc cggctgaaga gaaccgccag aagaagatac
2821 accagacgga agaaccggat ctgctatctg caagagatct tcagcaacga gatggccaag
2881 gtggacgaca gcttcttcca cagactggaa gagtccttcc tggtggaaga ggataagaag
2941 cacgagcggc accccatctt cggcaacatc gtggacgagg tggcctacca cgagaagtac
3001 cccaccatct accacctgag aaagaaactg gtggacagca ccgacaaggc cgacctgcgg
3061 ctgatctatc tggccctggc ccacatgatc aagttccggg gccacttcct gatcgagggc
3121 gacctgaacc ccgacaacag cgacgtggac aagctgttca tccagctggt gcagacctac
3181 aaccagctgt tcgaggaaaa ccccatcaac gccagcggcg tggacgccaa ggccatcctg
3241 tctgccagac tgagcaagag cagacggctg gaaaatctga tcgcccagct gcccggcgag
3301 aagaagaatg gcctgttcgg aaacctgatt gccctgagcc tgggcctgac ccccaacttc
3361 aagagcaact tcgacctggc cgaggatgcc aaactgcagc tgagcaagga cacctacgac
3421 gacgacctgg acaacctgct ggcccagatc ggcgaccagt acgccgacct gtttctggcc
3481 gccaagaacc tgtccgacgc catcctgctg agcgacatcc tgagagtgaa caccgagatc
3541 accaaggccc ccctgagcgc ctctatgatc aagagatacg acgagcacca ccaggacctg
3601 accctgctga aagctctcgt gcggcagcag ctgcctgaga agtacaaaga gattttcttc
3661 gaccagagca agaacggcta cgccggctac attgacggcg gagccagcca ggaagagttc
3721 tacaagttca tcaagcccat cctggaaaag atggacggca ccgaggaact gctcgtgaag
3781 ctgaacagag aggacctgct gcggaagcag cggaccttcg acaacggcag catcccccac
3841 cagatccacc tgggagagct gcacgccatt ctgcggcggc aggaagattt ttacccattc
3901 ctgaaggaca accgggaaaa gatcgagaag atcctgacct tccgcatccc ctactacgtg
3961 ggccctctgg ccaggggaaa cagcagattc gcctggatga ccagaaagag cgaggaaacc
4021 atcaccccct ggaacttcga ggaagtggtg gacaagggcg cttccgccca gagcttcatc
4081 gagcggatga ccaacttcga taagaacctg cccaacgaga aggtgctgcc caagcacagc
4141 ctgctgtacg agtacttcac cgtgtataac gagctgacca aagtgaaata cgtgaccgag
4201 ggaatgagaa agcccgcctt cctgagcggc gagcagaaaa aggccatcgt ggacctgctg
4261 ttcaagacca accggaaagt gaccgtgaag cagctgaaag aggactactt caagaaaatc
4321 gagtgcttcg actccgtgga aatctccggc gtggaagatc ggttcaacgc ctccctgggc
4381 acataccacg atctgctgaa aattatcaag gacaaggact tcctggacaa tgaggaaaac
4441 gaggacattc tggaagatat cgtgctgacc ctgacactgt ttgaggacag agagatgatc
4501 gaggaacggc tgaaaaccta tgcccacctg ttcgacgaca aagtgatgaa gcagctgaag
4561 cggcggagat acaccggctg gggcaggctg agccggaagc tgatcaacgg catccgggac
4621 aagcagtccg gcaagacaat cctggatttc ctgaagtccg acggcttcgc caacagaaac
4681 ttcatgcagc tgatccacga cgacagcctg acctttaaag aggacatcca gaaagcccag
4741 gtgtccggcc agggcgatag cctgcacgag cacattgcca atctggccgg cagccccgcc
4801 attaagaagg gcatcctgca gacagtgaag gtggtggacg agctcgtgaa agtgatgggc
4861 cggcacaagc ccgagaacat cgtgatcgaa atggccagag agaaccagac cacccagaag
4921 ggacagaaga acagccgcga gagaatgaag cggatcgaag agggcatcaa agagctgggc
4981 agccagatcc tgaaagaaca ccccgtggaa aacacccagc tgcagaacga gaagctgtac
5041 ctgtactacc tgcagaatgg gcgggatatg tacgtggacc aggaactgga catcaaccgg
5101 ctgtccgact acgatgtgga ccatatcgtg cctcagagct ttctgaagga cgactccatc
5161 gacaacaagg tgctgaccag aagcgacaag aaccggggca agagcgacaa cgtgccctcc
5221 gaagaggtcg tgaagaagat gaagaactac tggcggcagc tgctgaacgc caagctgatt
5281 acccagagaa agttcgacaa tctgaccaag gccgagagag gcggcctgag cgaactggat
5341 aaggccggct tcatcaagag acagctggtg gaaacccggc agatcacaaa gcacgtggca
5401 cagatcctgg actcccggat gaacactaag tacgacgaga atgacaagct gatccgggaa
5461 gtgaaagtga tcaccctgaa gtccaagctg gtgtccgatt tccggaagga tttccagttt
5521 tacaaagtgc gcgagatcaa caactaccac cacgcccacg acgcctacct gaacgccgtc
5581 gtgggaaccg ccctgatcaa aaagtaccct aagctggaaa gcgagttcgt gtacggcgac
5641 tacaaggtgt acgacgtgcg gaagatgatc gccaagagcg agcaggaaat cggcaaggct
5701 accgccaagt acttcttcta cagcaacatc atgaactttt tcaagaccga gattaccctg
5761 gccaacggcg agatccggaa gcggcctctg atcgagacaa acggcgaaac cggggagatc
5821 gtgtgggata agggccggga ttttgccacc gtgcggaaag tgctgagcat gccccaagtg
5881 aatatcgtga aaaagaccga ggtgcagaca ggcggcttca gcaaagagtc tatcctgccc
5941 aagaggaaca gcgataagct gatcgccaga aagaaggact gggaccctaa gaagtacggc
6001 ggcttcgaca gccccaccgt ggcctattct gtgctggtgg tggccaaagt ggaaaagggc
6061 aagtccaaga aactgaagag tgtgaaagag ctgctgggga tcaccatcat ggaaagaagc
6121 agcttcgaga agaatcccat cgactttctg gaagccaagg gctacaaaga agtgaaaaag
6181 gacctgatca tcaagctgcc taagtactcc ctgttcgagc tggaaaacgg ccggaagaga
6241 atgctggcct ctgccggcga actgcagaag ggaaacgaac tggccctgcc ctccaaatat
6301 gtgaacttcc tgtacctggc cagccactat gagaagctga agggctcccc cgaggataat
6361 gagcagaaac agctgtttgt ggaacagcac aagcactacc tggacgagat catcgagcag
6421 atcagcgagt tctccaagag agtgatcctg gccgacgcta atctggacaa agtgctgtcc
6481 gcctacaaca agcaccggga taagcccatc agagagcagg ccgagaatat catccacctg
6541 tttaccctga ccaatctggg agcccctgcc gccttcaagt actttgacac caccatcgac
6601 cggaagaggt acaccagcac caaagaggtg ctggacgcca ccctgatcca ccagagcatc
6661 accggcctgt acgagacacg gatcgacctg tctcagctgg gaggcgacaa gcgtcctgct
6721 gctactaaga aagctggtca agctaagaaa aagaaagcta gcggcagcgg cgccaccaac
6781 ttcagcctgc tgaagcaggc cggcgacgtg gaggagaacc ccggccccat ggtgtctaag
6841 ggcgaagagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac
6901 ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc
6961 ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc
7021 ctgacctacg gcgtgcagtg cttcagccgc taccccgacc acatgaagca gcacgacttc
7081 ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac
7141 ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc
7201 gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac
7261 aactacaaca gccacaacgt ctatatcatg gccgacaagc agaagaacgg catcaaggtg
7321 aacttcaaga tccgccacaa catcgaggac ggcagcgtgc agctcgccga ccactaccag
7381 cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagcacc
7441 cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc
7501 gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaagtaaac gcgttaagtc
7561 gacaatcaac ctctggatta caaaatttgt gaaagattga ctggtattct taactatgtt
7621 gctcctttta cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc tattgcttcc
7681 cgtatggctt tcattttctc ctccttgtat aaatcctggt tgctgtctct ttatgaggag
7741 ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc
7801 actggttggg gcattgccac cacctgtcag ctcctttccg ggactttcgc tttccccctc
7861 cctattgcca cggcggaact catcgccgcc tgccttgccc gctgctggac aggggctcgg
7921 ctgttgggca ctgacaattc cgtggtgttg tcggggaaat catcgtcctt tccttggctg
7981 ctcgcctgtg ttgccacctg gattctgcgc gggacgtcct tctgctacgt cccttcggcc
8041 ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt
8101 cttcgccttc gccctcagac gagtcggatc tccctttggg ccgcctcccc gcgtcgactt
8161 taagaccaat gacttacaag gcagctgtag atcttagcca ctttttaaaa gaaaaggggg
8221 gactggaagg gctaattcac tcccaacgaa gacaagatct gctttttgct tgtactgggt
8281 ctctctggtt agaccagatc tgagcctggg agctctctgg ctaactaggg aacccactgc
8341 ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg
8401 actctggtaa ctagagatcc ctcagaccct tttagtcagt gtggaaaatc tctagcagta
8461 cgtatagtag ttcatgtcat cttattattc agtatttata acttgcaaag aaatgaatat
8521 cagagagtga gaggaacttg tttattgcag cttataatgg ttacaaataa agcaatagca
8581 tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac
8641 tcatcaatgt atcttatcat gtctggctct agctatcccg cccctaactc cgcccatccc
8701 gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat
8761 ttatgcagag gccgaggccg cctcggcctc tgagctattc cagaagtagt gaggaggctt
8821 ttttggaggc ctagggacgt acccaattcg ccctatagtg agtcgtatta cgcgcgctca
8881 ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc
8941 cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc
9001 ccttcccaac agttgcgcag cctgaatggc gaatgggacg cgccctgtag cggcgcatta
9061 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg
9121 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa
9181 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc
9241 aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt
9301 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca
9361 acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc
9421 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta
9481 acgcttacaa tttaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
9541 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc
9601 aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct
9661 tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag
9721 atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta
9781 agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc
9841 tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca
9901 tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg
9961 atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg
10021 ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca
10081 tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa
10141 acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa
10201 ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata
10261 aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat
10321 ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc
10381 cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata
10441 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt
10501 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga
10561 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
10621 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
10681 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
10741 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
10801 ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
10861 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
10921 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
10981 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
11041 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
11101 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
11161 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
11221 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
11281 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc
11341 gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg
11401 agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt
11461 ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc
11521 gcaacgcaat taatgtgagt tagctcactc attaggcacc ccaggcttta cactttatgc
11581 ttccggctcg tatgttgtgt ggaattgtga gcggataaca atttcacaca ggaaacagct
11641 atgaccatga ttacgccaag cgcgcaatta accctcacta aagggaacaa aagctggagc
11701 tgcaagc
//
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