| 出品公司: | Clontech |
|---|---|
| 载体名称: | pIRES2-DsRed-Express2 |
| 质粒类型: | 哺乳细胞表达载体 ； 双瞬反子载体；荧光报告载体 |
| 高拷贝/低拷贝: | 高拷贝 |
| 克隆方法: | 多克隆位点，限制性内切酶 |
| 启动子: | CMV |
| 载体大小: | 5265 bp |
| 5' 测序引物及序列: | -- |
| 3' 测序引物及序列: | -- |
| 载体标签: | 红色荧光蛋白DsRed-Express2 |
| 载体抗性: | 卡那霉素 |
| 筛选标记: | 新霉素（Neomycin） |
| 克隆菌株: | DH5α, HB101 |
| 宿主细胞（系）: | 常规细胞系，293、CV-1、CHO等 |
| 备注: | -- |
| 产品目录号: | 632540 |
| 稳定性: | 稳表达 或 瞬表达 |
| 组成型/诱导型: | 组成型 |
| 病毒/非病毒: | 非病毒 |
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pIRES2-DsRed-Express2载体质粒基本信息
pIRES2-DsRed-Express2质粒图谱载体图谱和pIRES2-DsRed-Express2载体序列质粒序列多克隆位点信息
pIRES2-DsRed-Express2质粒载体简介
pIRES2-DsRed-Express2 is a bicistronic expression vector that allows the simultaneous expression of your protein of interest and DsRed-Express2 from the same mRNA transcript.The vector is designed to allow efficient flow cytometric detection of transiently transfected mammalian cells expressing either DsRed-Express2 and a protein of interest, or DsRedExpress2 alone (at lower signal intensity).
DsRed-Express2 is a variant of the Discosoma sp. red fluorescent protein, DsRed (1).It retains the fast maturation and high photostability characteristic of its predecessor,DsRed-Express (2), and has been engineered (through additional amino acid substitutions) for increased solubility (3). Although it most likely forms the same tetrameric structure as wild-type DsRed, DsRed-Express2 displays a greatly reduced tendency to aggregate,resulting in reduced cyto- and phototoxicity, and making DsRed-Express2 much better suited for in vivo applications involving sensitive cells, such as primary or stem cells.DsRed-Express2 also exhibits extremely low residual green fluorescence, which allows cells expressing the protein to be effectively separated from other fluorescently labeled cell populations by flow cytometry
Bicistronic expression of the protein of interest and DsRed-Express2 is facilitated by the encephalomyocarditis virus (EMCV) internal ribosome entry site 2 (IRES2), located between the multiple cloning site (MCS) and the DsRed-Express2 coding region. This IRES allows cap-independent translation of DsRed-Express2 from an internal start site at the IRES/DsRed-Express2 junction (4, 5). Expression of the bicistronic transcript is driven by the constitutively active human cytomegalovirus immediate early promoter (PCMV IE) located just upstream of the MCS.
SV40 polyadenylation signals downstream of the DsRed-Express2 coding sequence direct proper processing of the 3' ends of the bicistronic mRNA. The vector backbone also contains an SV40 origin for replication in mammalian cells expressing the SV40 large T antigen, a pUC origin of replication for propagation in E. coli,and an f1 origin for single-stranded DNA production. This vector also has a neomycin-resistance cassette (Neor) that allows G418 selection of stably transfected eukaryotic cells (6). This cassette consists of the SV40 early promoter, a Tn5 kanamycin/neomycin resistance gene, and herpes simplex virus thymidine kinase (HSV TK) polyadenylation signals. A bacterial promoter upstream of this cassette allows kanamycin resistance in E. coli.
Use
When cloning into the pIRES2 DsRed-Express2 MCS, your gene of interest must contain an initiation codon (ATG) and a stop codon. Cells expressing the gene of interest can be quickly identified by screening for DsRed-Express2 fluorescence.pIRES2 DsRed-Express2 and its derivatives can be introduced into mammalian cells using any standard transfection method. Cells expressing DsRed-Express2 (excitation and emission maxima: 554 and 591,respectively) can be detected by flow cytometry or microscopy 24 hr after transfection. However, in some cases, up to 48 hr may be required for detection. If required, stable transfectants can be selected using G418. Please refer to the Living Colors® User Manual provided with this vector for additional information on detection of DsRed-Express2.
pIRES2-DsRed-Express2质粒序列载体序列
LOCUS pIRES2 DsRed-Express2 5265 bp DNA SYN DEFINITION pIRES2 DsRed-Express2 ACCESSION KEYWORDS SOURCE ORGANISM other sequences; artificial sequences; vectors. FEATURES Location/Qualifiers source 1..5265 /organism="pIRES2 DsRed-Express2" /mol_type="other DNA" promoter 10..562 /label="CMV_immearly_promoter" misc_feature 65..352 /label="CAG_enhancer" misc_feature 519..539 /label="CMV_fwd_primer" promoter 520..589 /label="CMV_promoter" misc_feature 666..1250 /label="IRES" CDS 1242..1931 /label="ORF frame 3" /translation="MATTMDSTENVIKPFMRFKVHMEGSVNGHEFEIEGEGEGKPYEG TQTAKLQVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWERVMN FEDGGVVTVTQDSSLQDGTFIYHVKFIGVNFPSDGPVMQKKTLGWEPSTERLYPRDGV LKGEIHKALKLKGGGHYLVEFKSIYMAKKPVKLPGYYYVDSKLDITSHNEDYTVVEQY ERAEARHHLFQ*" gene 1257..1928 /label="E2_Crimson (variant)" /gene="E2_Crimson (variant)" /translation="MATTMDSTENVIKPFMRFKVHMEGSVNGHEFEIEGEGEGKPYEG TQTAKLQVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWERVMN FEDGGVVTVTQDSSLQDGTFIYHVKFIGVNFPSDGPVMQKKTLGWEPSTERLYPRDGV LKGEIHKALKLKGGGHYLVEFKSIYMAKKPVKLPGYYYVDSKLDITSHNEDYTVVEQY ERAEARHHLFQ*" misc_feature complement(1434..1454) /label="dsRed1_N_primer" /translation="MATTMDSTENVIKPFMRFKVHMEGSVNGHEFEIEGEGEGKPYEG TQTAKLQVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWERVMN FEDGGVVTVTQDSSLQDGTFIYHVKFIGVNFPSDGPVMQKKTLGWEPSTERLYPRDGV LKGEIHKALKLKGGGHYLVEFKSIYMAKKPVKLPGYYYVDSKLDITSHNEDYTVVEQY ERAEARHHLFQ*" misc_feature 1846..1869 /label="dsRed1_C_primer" /translation="MATTMDSTENVIKPFMRFKVHMEGSVNGHEFEIEGEGEGKPYEG TQTAKLQVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWERVMN FEDGGVVTVTQDSSLQDGTFIYHVKFIGVNFPSDGPVMQKKTLGWEPSTERLYPRDGV LKGEIHKALKLKGGGHYLVEFKSIYMAKKPVKLPGYYYVDSKLDITSHNEDYTVVEQY ERAEARHHLFQ*" misc_feature 2144..2163 /label="EBV_rev_primer" /translation="MATTMDSTENVIKPFMRFKVHMEGSVNGHEFEIEGEGEGKPYEG TQTAKLQVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWERVMN FEDGGVVTVTQDSSLQDGTFIYHVKFIGVNFPSDGPVMQKKTLGWEPSTERLYPRDGV LKGEIHKALKLKGGGHYLVEFKSIYMAKKPVKLPGYYYVDSKLDITSHNEDYTVVEQY ERAEARHHLFQ*" rep_origin complement(2313..2619) /label="f1_origin" /translation="MATTMDSTENVIKPFMRFKVHMEGSVNGHEFEIEGEGEGKPYEG TQTAKLQVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWERVMN FEDGGVVTVTQDSSLQDGTFIYHVKFIGVNFPSDGPVMQKKTLGWEPSTERLYPRDGV LKGEIHKALKLKGGGHYLVEFKSIYMAKKPVKLPGYYYVDSKLDITSHNEDYTVVEQY ERAEARHHLFQ*" promoter 2698..2726 /label="AmpR_promoter" /translation="MATTMDSTENVIKPFMRFKVHMEGSVNGHEFEIEGEGEGKPYEG TQTAKLQVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWERVMN FEDGGVVTVTQDSSLQDGTFIYHVKFIGVNFPSDGPVMQKKTLGWEPSTERLYPRDGV LKGEIHKALKLKGGGHYLVEFKSIYMAKKPVKLPGYYYVDSKLDITSHNEDYTVVEQY ERAEARHHLFQ*" misc_feature complement(2792..2812) /label="pBABE_3_primer" /translation="MATTMDSTENVIKPFMRFKVHMEGSVNGHEFEIEGEGEGKPYEG TQTAKLQVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWERVMN FEDGGVVTVTQDSSLQDGTFIYHVKFIGVNFPSDGPVMQKKTLGWEPSTERLYPRDGV LKGEIHKALKLKGGGHYLVEFKSIYMAKKPVKLPGYYYVDSKLDITSHNEDYTVVEQY ERAEARHHLFQ*" misc_feature complement(2798..3013) /label="SV40_enhancer" /translation="MATTMDSTENVIKPFMRFKVHMEGSVNGHEFEIEGEGEGKPYEG TQTAKLQVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWERVMN FEDGGVVTVTQDSSLQDGTFIYHVKFIGVNFPSDGPVMQKKTLGWEPSTERLYPRDGV LKGEIHKALKLKGGGHYLVEFKSIYMAKKPVKLPGYYYVDSKLDITSHNEDYTVVEQY ERAEARHHLFQ*" promoter 2810..3078 /label="SV40_promoter" /translation="MATTMDSTENVIKPFMRFKVHMEGSVNGHEFEIEGEGEGKPYEG TQTAKLQVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWERVMN FEDGGVVTVTQDSSLQDGTFIYHVKFIGVNFPSDGPVMQKKTLGWEPSTERLYPRDGV LKGEIHKALKLKGGGHYLVEFKSIYMAKKPVKLPGYYYVDSKLDITSHNEDYTVVEQY ERAEARHHLFQ*" rep_origin 2977..3054 /label="SV40_origin" /translation="MATTMDSTENVIKPFMRFKVHMEGSVNGHEFEIEGEGEGKPYEG TQTAKLQVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWERVMN FEDGGVVTVTQDSSLQDGTFIYHVKFIGVNFPSDGPVMQKKTLGWEPSTERLYPRDGV LKGEIHKALKLKGGGHYLVEFKSIYMAKKPVKLPGYYYVDSKLDITSHNEDYTVVEQY ERAEARHHLFQ*" misc_feature 3039..3058 /label="SV40pro_F_primer" /translation="MATTMDSTENVIKPFMRFKVHMEGSVNGHEFEIEGEGEGKPYEG TQTAKLQVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWERVMN FEDGGVVTVTQDSSLQDGTFIYHVKFIGVNFPSDGPVMQKKTLGWEPSTERLYPRDGV LKGEIHKALKLKGGGHYLVEFKSIYMAKKPVKLPGYYYVDSKLDITSHNEDYTVVEQY ERAEARHHLFQ*" CDS 3161..3955 /label="ORF frame 2" /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGR PVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDL LSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE EHQGLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRY QDIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF*" gene 3164..3952 /label="NeoR/KanR" /gene="NeoR/KanR" /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGR PVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDL LSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE EHQGLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRY QDIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF*" CDS complement(3470..4006) /label="ORF frame 3" /translation="MAGWASLGRSFRTPESRSEELVKKAIEGDALRIGSGDTVKHEEA VSPFAAKLFSNITGSQRYVLIAVRHTQPATVDESRKAAIFHHDIRQAGIAMGHDEILA VGHARLEPGEQFGWREPLMLFVQIILIDKTGFHPSTCSLDAMFRLVVEWAGSRIKRMQ PPHCISHDGYFLGRSKVR*" terminator 4130..4399 /label="TK_PA_terminator" /translation="MAGWASLGRSFRTPESRSEELVKKAIEGDALRIGSGDTVKHEEA VSPFAAKLFSNITGSQRYVLIAVRHTQPATVDESRKAAIFHHDIRQAGIAMGHDEILA VGHARLEPGEQFGWREPLMLFVQIILIDKTGFHPSTCSLDAMFRLVVEWAGSRIKRMQ PPHCISHDGYFLGRSKVR*" rep_origin 4547..5166 /label="pBR322_origin" /translation="MAGWASLGRSFRTPESRSEELVKKAIEGDALRIGSGDTVKHEEA VSPFAAKLFSNITGSQRYVLIAVRHTQPATVDESRKAAIFHHDIRQAGIAMGHDEILA VGHARLEPGEQFGWREPLMLFVQIILIDKTGFHPSTCSLDAMFRLVVEWAGSRIKRMQ PPHCISHDGYFLGRSKVR*" ORIGIN 1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg 61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt 121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca 181 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc 241 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta 301 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac 361 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg 421 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg 481 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt 541 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta 601 ccggactcag atctcgagct caagcttcga attctgcagt cgacggtacc gcgggcccgg 661 gatccgcccc tctccctccc ccccccctaa cgttactggc cgaagccgct tggaataagg 721 ccggtgtgcg tttgtctata tgttattttc caccatattg ccgtcttttg gcaatgtgag 781 ggcccggaaa cctggccctg tcttcttgac gagcattcct aggggtcttt cccctctcgc 841 caaaggaatg caaggtctgt tgaatgtcgt gaaggaagca gttcctctgg aagcttcttg 901 aagacaaaca acgtctgtag cgaccctttg caggcagcgg aaccccccac ctggcgacag 961 gtgcctctgc ggccaaaagc cacgtgtata agatacacct gcaaaggcgg cacaacccca 1021 gtgccacgtt gtgagttgga tagttgtgga aagagtcaaa tggctctcct caagcgtatt 1081 caacaagggg ctgaaggatg cccagaaggt accccattgt atgggatctg atctggggcc 1141 tcggtacaca tgctttacat gtgtttagtc gaggttaaaa aaacgtctag gccccccgaa 1201 ccacggggac gtggttttcc tttgaaaaac acgatgataa tatggccaca accatggata 1261 gcactgagaa cgtcatcaag cccttcatgc gcttcaaggt gcacatggag ggctccgtga 1321 acggccacga gttcgagatc gagggcgagg gcgagggcaa gccctacgag ggcacccaga 1381 ccgccaagct gcaggtgacc aagggcggcc ccctgccctt cgcctgggac atcctgtccc 1441 cccagttcca gtacggctcc aaggtgtacg tgaagcaccc cgccgacatc cccgactaca 1501 agaagctgtc cttccccgag ggcttcaagt gggagcgcgt gatgaacttc gaggacggcg 1561 gcgtggtgac cgtgacccag gactcctccc tgcaggacgg caccttcatc taccacgtga 1621 agttcatcgg cgtgaacttc ccctccgacg gccccgtaat gcagaagaag actctgggct 1681 gggagccctc caccgagcgc ctgtaccccc gcgacggcgt gctgaagggc gagatccaca 1741 aggcgctgaa gctgaagggc ggcggccact acctggtgga gttcaagtca atctacatgg 1801 ccaagaagcc cgtgaagctg cccggctact actacgtgga ctccaagctg gacatcacct 1861 cccacaacga ggactacacc gtggtggagc agtacgagcg cgccgaggcc cgccaccacc 1921 tgttccagta ggcggccgcg actctagatc ataatcagcc ataccacatt tgtagaggtt 1981 ttacttgctt taaaaaacct cccacacctc cccctgaacc tgaaacataa aatgaatgca 2041 attgttgttg ttaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc 2101 acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc 2161 atcaatgtat cttaaggcgt aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa 2221 tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa 2281 atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact 2341 attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc 2401 actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa 2461 tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc 2521 gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt 2581 cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtcagg 2641 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc 2701 aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag 2761 gaagagtcct gaggcggaaa gaaccagctg tggaatgtgt gtcagttagg gtgtggaaag 2821 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc 2881 aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat 2941 tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt 3001 tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc 3061 gcctcggcct ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt 3121 tgcaaagatc gatcaagaga caggatgagg atcgtttcgc atgattgaac aagatggatt 3181 gcacgcaggt tctccggccg cttgggtgga gaggctattc ggctatgact gggcacaaca 3241 gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct 3301 ttttgtcaag accgacctgt ccggtgccct gaatgaactg caagacgagg cagcgcggct 3361 atcgtggctg gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc 3421 gggaagggac tggctgctat tgggcgaagt gccggggcag gatctcctgt catctcacct 3481 tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg cggcggctgc atacgcttga 3541 tccggctacc tgcccattcg accaccaagc gaaacatcgc atcgagcgag cacgtactcg 3601 gatggaagcc ggtcttgtcg atcaggatga tctggacgaa gagcatcagg ggctcgcgcc 3661 agccgaactg ttcgccaggc tcaaggcgag catgcccgac ggcgaggatc tcgtcgtgac 3721 ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt ctggattcat 3781 cgactgtggc cggctgggtg tggcggaccg ctatcaggac atagcgttgg ctacccgtga 3841 tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc 3901 cgctcccgat tcgcagcgca tcgccttcta tcgccttctt gacgagttct tctgagcggg 3961 actctggggt tcgaaatgac cgaccaagcg acgcccaacc tgccatcacg agatttcgat 4021 tccaccgccg ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg 4081 atgatcctcc agcgcgggga tctcatgctg gagttcttcg cccaccctag ggggaggcta 4141 actgaaacac ggaaggagac aataccggaa ggaacccgcg ctatgacggc aataaaaaga 4201 cagaataaaa cgcacggtgt tgggtcgttt gttcataaac gcggggttcg gtcccagggc 4261 tggcactctg tcgatacccc accgagaccc cattggggcc aatacgcccg cgtttcttcc 4321 ttttccccac cccacccccc aagttcgggt gaaggcccag ggctcgcagc caacgtcggg 4381 gcggcaggcc ctgccatagc ctcaggttac tcatatatac tttagattga tttaaaactt 4441 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc 4501 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct 4561 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta 4621 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc 4681 ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac 4741 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct 4801 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat 4861 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg 4921 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa 4981 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg 5041 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga 5101 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc 5161 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct 5221 gcgttatccc ctgattctgt ggataaccgt attaccgcca tgcat //
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pIRES2-DsRed-Express2质粒载体应用举例
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