| 质粒名称: | pL-CRISPR.EFS.tRFP |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 57819 |
| 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: | 11701 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: | -- |
| 标签/融合蛋白: | FLAG（插入件上的N端子） |
| 克隆方法: | -- |
| 5’克隆位点: | -- |
| 3’克隆位点: | -- |
| 5’测序引物 | -- |
| 3’测序引物 | -- |
|
文献引用方法 |
|
| 材料和方法部分: | pL-CRISPR.EFS.tRFP was a gift from Benjamin Ebert (Addgene plasmid # 57819 ; http://n2t.net/addgene:57819 ; RRID:Addgene_57819) |
| 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
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 pL-CRISPR.EFS.tRFP 11701 bp ds-DNA circular SYN 12-MAY-2021
DEFINITION Lentiviral CRISPR-Cas9 delivery for SpCas9 and sgRNA. Coexpresses
tagRFP via P2A cleavage site. EFS Promoter driven.
ACCESSION .
VERSION .
KEYWORDS pL-CRISPR.EFS.tRFP
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 11701)
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 11701)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..11701
/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..7542
/codon_start=1
/product="monomeric derivative of red fluorescent protein
from Entacmaea quadricolor (Merzlyak et al., 2007)"
/label=TagRFP
/note="mammalian codon-optimized"
/translation="MVSKGEELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTM
RIKVVEGGPLPFAFDILATSFMYGSRTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGG
VLTATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEANTEMLYPADGGLEGRSD
MALKLVGGGHLICNFKTTYRSKKPAKNLKMPGVYYVDHRLERIKEADKETYVEQHEVAV
ARYCDLPSKLGHKLN"
misc_feature 7558..8146
/label=WPRE
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
primer_bind complement(7611..7631)
/label=WPRE-R
/note="WPRE, reverse primer"
CDS complement(8029..8040)
/codon_start=1
/product="Factor Xa recognition and cleavage site"
/label=Factor Xa site
/translation="IEGR"
LTR 8218..8451
/label=3' LTR (Delta-U3)
/note="self-inactivating 3' long terminal repeat (LTR) from
HIV-1"
polyA_signal 8529..8650
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(8566..8585)
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
primer_bind 8620..8639
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
rep_origin 8690..8825
/label=SV40 ori
/note="SV40 origin of replication"
primer_bind 8752..8771
/label=SV40pro-F
/note="SV40 promoter/origin, forward primer"
primer_bind complement(8845..8864)
/label=T7
/note="T7 promoter, forward primer"
promoter complement(8846..8864)
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
primer_bind complement(8874..8891)
/label=M13 Forward
/note="In lacZ gene. Also called M13-F20 or M13 (-21)
Forward"
primer_bind complement(8874..8890)
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(8883..8905)
/label=M13/pUC Forward
/note="In lacZ gene"
rep_origin 9032..9487
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(9119..9138)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 9329..9350
/label=F1ori-F
/note="F1 origin, forward primer"
promoter 9513..9617
/gene="bla"
/label=AmpR promoter
CDS 9618..10478
/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(9836..9855)
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
rep_origin 10649..11237
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 11138..11157
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
primer_bind 11391..11408
/label=L4440
/note="L4440 vector, forward primer"
protein_bind 11525..11546
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 11561..11591
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 11599..11615
/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 11604..11626
/label=M13/pUC Reverse
/note="In lacZ gene"
primer_bind 11623..11639
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind 11623..11639
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind 11658..11678
/label=T3
/note="T3 promoter, forward primer"
promoter 11660..11678
/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 tgattaagga gaacatgcac atgaagctgt atatggaggg caccgtgaac
6901 aaccaccact tcaagtgcac atccgagggc gaaggcaagc cctacgaggg cacccagacc
6961 atgagaatca aggtggtcga gggcggccct ctccccttcg ccttcgacat cctggctacc
7021 agcttcatgt acggcagcag aaccttcatc aaccacaccc agggcatccc cgacttcttt
7081 aagcagtcct tccctgaggg cttcacatgg gagagagtca ccacatacga agacgggggc
7141 gtgctgaccg ctacccagga caccagcctc caggacggct gcctcatcta caacgtcaag
7201 atcagagggg tgaacttccc atccaacggc cctgtgatgc agaagaaaac actcggctgg
7261 gaggccaaca ccgagatgct gtaccccgct gacggcggcc tggaaggcag aagcgacatg
7321 gccctgaagc tcgtgggcgg gggccacctg atctgcaact tcaagaccac atacagatcc
7381 aagaaacccg ctaagaacct caagatgccc ggcgtctact atgtggacca cagactggaa
7441 agaatcaagg aggccgacaa agagacctac gtcgagcagc acgaggtggc tgtggccaga
7501 tactgcgacc tccctagcaa actggggcac aaacttaatt gaacgcgtta agtcgacaat
7561 caacctctgg attacaaaat ttgtgaaaga ttgactggta ttcttaacta tgttgctcct
7621 tttacgctat gtggatacgc tgctttaatg cctttgtatc atgctattgc ttcccgtatg
7681 gctttcattt tctcctcctt gtataaatcc tggttgctgt ctctttatga ggagttgtgg
7741 cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac ccccactggt
7801 tggggcattg ccaccacctg tcagctcctt tccgggactt tcgctttccc cctccctatt
7861 gccacggcgg aactcatcgc cgcctgcctt gcccgctgct ggacaggggc tcggctgttg
7921 ggcactgaca attccgtggt gttgtcgggg aaatcatcgt cctttccttg gctgctcgcc
7981 tgtgttgcca cctggattct gcgcgggacg tccttctgct acgtcccttc ggccctcaat
8041 ccagcggacc ttccttcccg cggcctgctg ccggctctgc ggcctcttcc gcgtcttcgc
8101 cttcgccctc agacgagtcg gatctccctt tgggccgcct ccccgcgtcg actttaagac
8161 caatgactta caaggcagct gtagatctta gccacttttt aaaagaaaag gggggactgg
8221 aagggctaat tcactcccaa cgaagacaag atctgctttt tgcttgtact gggtctctct
8281 ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca ctgcttaagc
8341 ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg
8401 gtaactagag atccctcaga cccttttagt cagtgtggaa aatctctagc agtacgtata
8461 gtagttcatg tcatcttatt attcagtatt tataacttgc aaagaaatga atatcagaga
8521 gtgagaggaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa
8581 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca
8641 atgtatctta tcatgtctgg ctctagctat cccgccccta actccgccca tcccgcccct
8701 aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc
8761 agaggccgag gccgcctcgg cctctgagct attccagaag tagtgaggag gcttttttgg
8821 aggcctaggg acgtacccaa ttcgccctat agtgagtcgt attacgcgcg ctcactggcc
8881 gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa tcgccttgca
8941 gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc
9001 caacagttgc gcagcctgaa tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg
9061 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct
9121 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
9181 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
9241 cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct
9301 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc
9361 aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg
9421 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgctt
9481 acaatttagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
9541 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
9601 attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg
9661 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg
9721 aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc
9781 ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat
9841 gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact
9901 attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca
9961 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact
10021 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg
10081 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg
10141 agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg
10201 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg
10261 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag
10321 ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc
10381 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga
10441 tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat
10501 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc
10561 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
10621 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
10681 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
10741 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc
10801 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
10861 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
10921 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
10981 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
11041 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
11101 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
11161 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
11221 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
11281 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta
11341 ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag
11401 tgagcgagga agcggaagag cgcccaatac gcaaaccgcc tctccccgcg cgttggccga
11461 ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg
11521 caattaatgt gagttagctc actcattagg caccccaggc tttacacttt atgcttccgg
11581 ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca cacaggaaac agctatgacc
11641 atgattacgc caagcgcgca attaaccctc actaaaggga acaaaagctg gagctgcaag
11701 c
//
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