| 质粒名称: | pL-CRISPR.SFFV.GFP |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 57827 |
| 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: | 13615 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: | -- |
| 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.SFFV.GFP was a gift from Benjamin Ebert (Addgene plasmid # 57827 ; http://n2t.net/addgene:57827 ; RRID:Addgene_57827) |
| 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.SFFV.GFP 13735 bp ds-DNA circular SYN 12-MAY-2021
DEFINITION Lentiviral CRISPR-Cas9 delivery for SpCas9 and sgRNA. Coexpresses
eGFP via P2A cleavage site. SFFV Promoter driven.
ACCESSION .
VERSION .
KEYWORDS pL-CRISPR.SFFV.GFP
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 13735)
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 13735)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..13735
/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 3847..3922
/label=gRNA scaffold
/note="guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system"
misc_feature 3970..4087
/label=cPPT/CTS
/note="central polypurine tract and central termination
sequence of HIV-1"
promoter 4145..4546
/label=SFFV promoter
/note="spleen focus-forming virus long terminal repeat
(LTR) promoter"
regulatory 4552..4561
/regulatory_class="other"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
CDS 4561..4584
/codon_start=1
/product="FLAG(R) epitope tag, followed by an enterokinase
cleavage site"
/label=FLAG
/translation="DYKDDDDK"
CDS 4591..4611
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 4636..8736
/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 8737..8784
/codon_start=1
/product="bipartite nuclear localization signal from
nucleoplasmin"
/label=nucleoplasmin NLS
/translation="KRPAATKKAGQAKKKK"
CDS 8800..8856
/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 8857..9573
/codon_start=1
/product="enhanced GFP"
/label=EGFP
/translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
EFVTAAGITLGMDELYK"
primer_bind complement(8902..8923)
/label=EGFP-N
/note="EGFP, reverse primer"
primer_bind complement(9163..9182)
/label=EXFP-R
/note="For distinguishing EGFP variants, reverse primer"
primer_bind 9510..9531
/label=EGFP-C
/note="EGFP, forward primer"
misc_feature 9592..10180
/label=WPRE
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
primer_bind complement(9645..9665)
/label=WPRE-R
/note="WPRE, reverse primer"
CDS complement(10063..10074)
/codon_start=1
/product="Factor Xa recognition and cleavage site"
/label=Factor Xa site
/translation="IEGR"
LTR 10252..10485
/label=3' LTR (Delta-U3)
/note="self-inactivating 3' long terminal repeat (LTR) from
HIV-1"
polyA_signal 10563..10684
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(10600..10619)
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
primer_bind 10654..10673
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
rep_origin 10724..10859
/label=SV40 ori
/note="SV40 origin of replication"
primer_bind 10786..10805
/label=SV40pro-F
/note="SV40 promoter/origin, forward primer"
primer_bind complement(10879..10898)
/label=T7
/note="T7 promoter, forward primer"
promoter complement(10880..10898)
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
primer_bind complement(10908..10925)
/label=M13 Forward
/note="In lacZ gene. Also called M13-F20 or M13 (-21)
Forward"
primer_bind complement(10908..10924)
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(10917..10939)
/label=M13/pUC Forward
/note="In lacZ gene"
rep_origin 11066..11521
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(11153..11172)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 11363..11384
/label=F1ori-F
/note="F1 origin, forward primer"
promoter 11547..11651
/gene="bla"
/label=AmpR promoter
CDS 11652..12512
/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(11870..11889)
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
rep_origin 12683..13271
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 13172..13191
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
primer_bind 13425..13442
/label=L4440
/note="L4440 vector, forward primer"
protein_bind 13559..13580
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 13595..13625
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 13633..13649
/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 13638..13660
/label=M13/pUC Reverse
/note="In lacZ gene"
primer_bind 13657..13673
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind 13657..13673
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind 13692..13712
/label=T3
/note="T3 promoter, forward primer"
promoter 13694..13712
/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 cggttgtaaa
1981 tgagcacaca aaatacacat gctaaaatat tatattctat gacctttata aaatcaacca
2041 aaatcttctt tttaataact ttagtatcaa taattagaat ttttatgttc ctttttgcaa
2101 acttttaata aaaatgagca aaataaaaaa acgctagttt tagtaactcg cgttgttttc
2161 ttcaccttta ataatagcta ctccaccact tgttcctaag cggtcagctc ctgcttcaat
2221 cattttttga gcatcttcaa atgttctaac tccaccagct gctttaacta aagcattgtc
2281 tttaacaact gacttcatta gtttaacatc ttcaaatgtt gcacctgatt ttgaaaatcc
2341 tgttgatgtt ttaacaaatt ctaatccagc ttcaacagct atttcacaag ctttcatgat
2401 ttcttctttt gttaataaac aattttccat aatacattta acaacatgtg atccagctgc
2461 tttttttaca gctttcatgt cttctaaaac taattcataa tttttgtctt ttaatgcacc
2521 aatatttaat accatatcaa tttctgttgc accatcttta attgcttcag aaacttcgaa
2581 tgcttttgta gctgttgtgc atgcacctag aggaaaacct acaacatttg ttattcctac
2641 atttgtgcct tttaataatt ctttacaata gcttgttcaa tatgaattaa cacaaactgt
2701 tgcaaaatca aattcaattg cttcatcaca taattgttta atttcagctt tcgtagcatc
2761 ttgttttaat aatgtgtgat ctatatattt gtttagtttc attttttctc ctatatattc
2821 atttttaatt ttaattcttt aataatttcg tctactttaa ctttagcgtt ttgaacagat
2881 tcaccaacac ctataaaata aatttttagt ttaggttcag ttccacttgg gcgaacagca
2941 aatcatgact tatcttctaa ataaaatttt agtaagtctt gtcctggcat attatacatt
3001 ccatcgatgt agtcttcaac attaacaact ttaagtccag caatttgagt taagggtgtt
3061 gctctcaatg atttcattaa tggttcaatt tttaatttct tttcttctgg tttaaaattc
3121 aagtttaaag tgaaagtgta atatgcaccc atttctttaa ataaatcttc taaatagtct
3181 actaatgttt tattttgttt tttataaaat caagcagcct ctgctattaa tatagaagct
3241 tgtattccat ctttatctct agctgagtca tcaattacat atccataact ttcttcataa
3301 gcaaaaacaa aatttaatcc gttatcttct tctttagcaa tttctctacc cattcattta
3361 aatccagtta aagtttttac aatattaact ccatattttt catgagcgat tctatcaccc
3421 aaatcacttg ttacaaaact tgaatataga gccggatttt ttggaatgct atttaagcgt
3481 tttagatttg ataattttca atcaattaaa attggtcctg tttgatttcc atctaatctt
3541 acaaaatgac catcatgttt tattgccatt ccaaatctgt cagcatctgg gtcattcata
3601 ataataatat ctgcatcatg tttaatacca tattcaagcg gtatttttca tgcaggatca
3661 aattctggat ttggatttac aacattttta aatgtttcat cttcaaatgc atgctcttca
3721 acctcaataa cgttatatcc tgattcacgt aatatttttg gggtaaattt agttcctgtt
3781 ccattaactg cgctaaaaat aatttttaaa tcttttttag cttcttgctc ttttttgtac
3841 gtctctgttt tagagctaga aatagcaagt taaaataagg ctagtccgtt atcaacttga
3901 aaaagtggca ccgagtcggt gcttttttga attctagatc ttgagacaaa tggcagtatt
3961 catccacaat tttaaaagaa aaggggggat tggggggtac agtgcagggg aaagaatagt
4021 agacataata gcaacagaca tacaaactaa agaattacaa aaacaaatta caaaaattca
4081 aaattttcgg gtttattaca gggacagcag agatccactt tggcgccggc tcgagcgagc
4141 tgcagtaacg ccattttgca aggcatggaa aaataccaaa ccaagaatag agaagttcag
4201 atcaagggcg ggtacatgaa aatagctaac gttgggccaa acaggatatc tgcggtgagc
4261 agtttcggcc ccggcccggg gccaagaaca gatggtcacc gcagtttcgg ccccggcccg
4321 aggccaagaa cagatggtcc ccagatatgg cccaaccctc agcagtttct taagacccat
4381 cagatgtttc caggctcccc caaggacctg aaatgaccct gcgccttatt tgaattaacc
4441 aatcagcctg cttctcgctt ctgttcgcgc gcttctgctt cccgagctct ataaaagagc
4501 tcacaacccc tcactcggcg cgccagtcct ccgacagact gagtcggatc cgccaccatg
4561 gattacaaag acgatgacga taagatggcc ccaaagaaga agcggaaggt cggtatccac
4621 ggagtcccag cagccgacaa gaagtacagc atcggcctgg acatcggcac caactctgtg
4681 ggctgggccg tgatcaccga cgagtacaag gtgcccagca agaaattcaa ggtgctgggc
4741 aacaccgacc ggcacagcat caagaagaac ctgatcggag ccctgctgtt cgacagcggc
4801 gaaacagccg aggccacccg gctgaagaga accgccagaa gaagatacac cagacggaag
4861 aaccggatct gctatctgca agagatcttc agcaacgaga tggccaaggt ggacgacagc
4921 ttcttccaca gactggaaga gtccttcctg gtggaagagg ataagaagca cgagcggcac
4981 cccatcttcg gcaacatcgt ggacgaggtg gcctaccacg agaagtaccc caccatctac
5041 cacctgagaa agaaactggt ggacagcacc gacaaggccg acctgcggct gatctatctg
5101 gccctggccc acatgatcaa gttccggggc cacttcctga tcgagggcga cctgaacccc
5161 gacaacagcg acgtggacaa gctgttcatc cagctggtgc agacctacaa ccagctgttc
5221 gaggaaaacc ccatcaacgc cagcggcgtg gacgccaagg ccatcctgtc tgccagactg
5281 agcaagagca gacggctgga aaatctgatc gcccagctgc ccggcgagaa gaagaatggc
5341 ctgttcggaa acctgattgc cctgagcctg ggcctgaccc ccaacttcaa gagcaacttc
5401 gacctggccg aggatgccaa actgcagctg agcaaggaca cctacgacga cgacctggac
5461 aacctgctgg cccagatcgg cgaccagtac gccgacctgt ttctggccgc caagaacctg
5521 tccgacgcca tcctgctgag cgacatcctg agagtgaaca ccgagatcac caaggccccc
5581 ctgagcgcct ctatgatcaa gagatacgac gagcaccacc aggacctgac cctgctgaaa
5641 gctctcgtgc ggcagcagct gcctgagaag tacaaagaga ttttcttcga ccagagcaag
5701 aacggctacg ccggctacat tgacggcgga gccagccagg aagagttcta caagttcatc
5761 aagcccatcc tggaaaagat ggacggcacc gaggaactgc tcgtgaagct gaacagagag
5821 gacctgctgc ggaagcagcg gaccttcgac aacggcagca tcccccacca gatccacctg
5881 ggagagctgc acgccattct gcggcggcag gaagattttt acccattcct gaaggacaac
5941 cgggaaaaga tcgagaagat cctgaccttc cgcatcccct actacgtggg ccctctggcc
6001 aggggaaaca gcagattcgc ctggatgacc agaaagagcg aggaaaccat caccccctgg
6061 aacttcgagg aagtggtgga caagggcgct tccgcccaga gcttcatcga gcggatgacc
6121 aacttcgata agaacctgcc caacgagaag gtgctgccca agcacagcct gctgtacgag
6181 tacttcaccg tgtataacga gctgaccaaa gtgaaatacg tgaccgaggg aatgagaaag
6241 cccgccttcc tgagcggcga gcagaaaaag gccatcgtgg acctgctgtt caagaccaac
6301 cggaaagtga ccgtgaagca gctgaaagag gactacttca agaaaatcga gtgcttcgac
6361 tccgtggaaa tctccggcgt ggaagatcgg ttcaacgcct ccctgggcac ataccacgat
6421 ctgctgaaaa ttatcaagga caaggacttc ctggacaatg aggaaaacga ggacattctg
6481 gaagatatcg tgctgaccct gacactgttt gaggacagag agatgatcga ggaacggctg
6541 aaaacctatg cccacctgtt cgacgacaaa gtgatgaagc agctgaagcg gcggagatac
6601 accggctggg gcaggctgag ccggaagctg atcaacggca tccgggacaa gcagtccggc
6661 aagacaatcc tggatttcct gaagtccgac ggcttcgcca acagaaactt catgcagctg
6721 atccacgacg acagcctgac ctttaaagag gacatccaga aagcccaggt gtccggccag
6781 ggcgatagcc tgcacgagca cattgccaat ctggccggca gccccgccat taagaagggc
6841 atcctgcaga cagtgaaggt ggtggacgag ctcgtgaaag tgatgggccg gcacaagccc
6901 gagaacatcg tgatcgaaat ggccagagag aaccagacca cccagaaggg acagaagaac
6961 agccgcgaga gaatgaagcg gatcgaagag ggcatcaaag agctgggcag ccagatcctg
7021 aaagaacacc ccgtggaaaa cacccagctg cagaacgaga agctgtacct gtactacctg
7081 cagaatgggc gggatatgta cgtggaccag gaactggaca tcaaccggct gtccgactac
7141 gatgtggacc atatcgtgcc tcagagcttt ctgaaggacg actccatcga caacaaggtg
7201 ctgaccagaa gcgacaagaa ccggggcaag agcgacaacg tgccctccga agaggtcgtg
7261 aagaagatga agaactactg gcggcagctg ctgaacgcca agctgattac ccagagaaag
7321 ttcgacaatc tgaccaaggc cgagagaggc ggcctgagcg aactggataa ggccggcttc
7381 atcaagagac agctggtgga aacccggcag atcacaaagc acgtggcaca gatcctggac
7441 tcccggatga acactaagta cgacgagaat gacaagctga tccgggaagt gaaagtgatc
7501 accctgaagt ccaagctggt gtccgatttc cggaaggatt tccagtttta caaagtgcgc
7561 gagatcaaca actaccacca cgcccacgac gcctacctga acgccgtcgt gggaaccgcc
7621 ctgatcaaaa agtaccctaa gctggaaagc gagttcgtgt acggcgacta caaggtgtac
7681 gacgtgcgga agatgatcgc caagagcgag caggaaatcg gcaaggctac cgccaagtac
7741 ttcttctaca gcaacatcat gaactttttc aagaccgaga ttaccctggc caacggcgag
7801 atccggaagc ggcctctgat cgagacaaac ggcgaaaccg gggagatcgt gtgggataag
7861 ggccgggatt ttgccaccgt gcggaaagtg ctgagcatgc cccaagtgaa tatcgtgaaa
7921 aagaccgagg tgcagacagg cggcttcagc aaagagtcta tcctgcccaa gaggaacagc
7981 gataagctga tcgccagaaa gaaggactgg gaccctaaga agtacggcgg cttcgacagc
8041 cccaccgtgg cctattctgt gctggtggtg gccaaagtgg aaaagggcaa gtccaagaaa
8101 ctgaagagtg tgaaagagct gctggggatc accatcatgg aaagaagcag cttcgagaag
8161 aatcccatcg actttctgga agccaagggc tacaaagaag tgaaaaagga cctgatcatc
8221 aagctgccta agtactccct gttcgagctg gaaaacggcc ggaagagaat gctggcctct
8281 gccggcgaac tgcagaaggg aaacgaactg gccctgccct ccaaatatgt gaacttcctg
8341 tacctggcca gccactatga gaagctgaag ggctcccccg aggataatga gcagaaacag
8401 ctgtttgtgg aacagcacaa gcactacctg gacgagatca tcgagcagat cagcgagttc
8461 tccaagagag tgatcctggc cgacgctaat ctggacaaag tgctgtccgc ctacaacaag
8521 caccgggata agcccatcag agagcaggcc gagaatatca tccacctgtt taccctgacc
8581 aatctgggag cccctgccgc cttcaagtac tttgacacca ccatcgaccg gaagaggtac
8641 accagcacca aagaggtgct ggacgccacc ctgatccacc agagcatcac cggcctgtac
8701 gagacacgga tcgacctgtc tcagctggga ggcgacaagc gtcctgctgc tactaagaaa
8761 gctggtcaag ctaagaaaaa gaaagctagc ggcagcggcg ccaccaactt cagcctgctg
8821 aagcaggccg gcgacgtgga ggagaacccc ggccccatgg tgtctaaggg cgaagagctg
8881 ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc
8941 agcgtgtccg gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc
9001 tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gacctacggc
9061 gtgcagtgct tcagccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc
9121 atgcccgaag gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag
9181 acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc
9241 atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaacagc
9301 cacaacgtct atatcatggc cgacaagcag aagaacggca tcaaggtgaa cttcaagatc
9361 cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc
9421 atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagcaccca gtccgccctg
9481 agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc
9541 gggatcactc tcggcatgga cgagctgtac aagtaaacgc gttaagtcga caatcaacct
9601 ctggattaca aaatttgtga aagattgact ggtattctta actatgttgc tccttttacg
9661 ctatgtggat acgctgcttt aatgcctttg tatcatgcta ttgcttcccg tatggctttc
9721 attttctcct ccttgtataa atcctggttg ctgtctcttt atgaggagtt gtggcccgtt
9781 gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg caacccccac tggttggggc
9841 attgccacca cctgtcagct cctttccggg actttcgctt tccccctccc tattgccacg
9901 gcggaactca tcgccgcctg ccttgcccgc tgctggacag gggctcggct gttgggcact
9961 gacaattccg tggtgttgtc ggggaaatca tcgtcctttc cttggctgct cgcctgtgtt
10021 gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct caatccagcg
10081 gaccttcctt cccgcggcct gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc
10141 cctcagacga gtcggatctc cctttgggcc gcctccccgc gtcgacttta agaccaatga
10201 cttacaaggc agctgtagat cttagccact ttttaaaaga aaagggggga ctggaagggc
10261 taattcactc ccaacgaaga caagatctgc tttttgcttg tactgggtct ctctggttag
10321 accagatctg agcctgggag ctctctggct aactagggaa cccactgctt aagcctcaat
10381 aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac tctggtaact
10441 agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtacg tatagtagtt
10501 catgtcatct tattattcag tatttataac ttgcaaagaa atgaatatca gagagtgaga
10561 ggaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca
10621 caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat
10681 cttatcatgt ctggctctag ctatcccgcc cctaactccg cccatcccgc ccctaactcc
10741 gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc
10801 cgaggccgcc tcggcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct
10861 agggacgtac ccaattcgcc ctatagtgag tcgtattacg cgcgctcact ggccgtcgtt
10921 ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat
10981 ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag
11041 ttgcgcagcc tgaatggcga atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt
11101 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc
11161 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
11221 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
11281 tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg
11341 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct
11401 atctcggtct attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa
11461 aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt
11521 taggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
11581 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
11641 aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat
11701 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc
11761 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga
11821 gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg
11881 cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc
11941 agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag
12001 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc
12061 tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg
12121 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg
12181 acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac
12241 ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac
12301 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg
12361 agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg
12421 tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg
12481 agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac
12541 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg
12601 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
12661 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
12721 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
12781 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt
12841 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
12901 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
12961 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
13021 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
13081 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
13141 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
13201 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
13261 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
13321 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct
13381 ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg
13441 aggaagcgga agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt
13501 aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta
13561 atgtgagtta gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta
13621 tgttgtgtgg aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt
13681 acgccaagcg cgcaattaac cctcactaaa gggaacaaaa gctggagctg caagc
//
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