出品公司: | Clontech |
---|---|
载体名称: | pAcGFP1-N In-Fusion Ready |
质粒类型: | 哺乳动物细胞表达载体;荧光报告载体 |
高拷贝/低拷贝: | 高拷贝 |
克隆方法: | 限制性内切酶,多克隆位点 |
启动子: | CMV IE |
载体大小: | 4688 bp |
5' 测序引物及序列: | -- |
3' 测序引物及序列: | -- |
载体标签: | AcGFP1 (C-端) |
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: |
哺乳动物载体pAcGFP1-N In-Fusion Ready已经线性化,可以直接用于片段连接; AcGFP1与GFP相比,密码子经过了优化,更适合在哺乳动物细胞中高水平表达,同时也增强了亮度。 |
产品目录号: | 634306 |
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 |
买家导航
pAcGFP1-N In-Fusion Ready载体质粒基本信息
pAcGFP1-N In-Fusion Ready质粒图谱载体图谱和pAcGFP1-N In-Fusion Ready载体序列质粒序列多克隆位点信息
pAcGFP1-N In-Fusion Ready质粒载体简介
pAcGFP1-N In-Fusion Ready载体描述
pAcGFP1-N In-Fusion Ready vector is a linearized mammalian expression vector that encodes a Green Fluorescent Protein (GFP) from Aequorea coerulescens. This fluorescent protein coding sequence in this construct has been human codon-optimized for efficient expression and enhanced brightness. AcGFP1 protein has an excitation maximum at 475 nm and an emission maximum at 505 nm. The linearized vector allows direct cloning of PCR products without any need for restriction digestion when using Clontech's In-Fusion HD Cloning Plus (638909). This is accomplished by the use of a specific 15 nucleotide long sequence within the sense and antisense primers that overlap with the cut ends created by initial digestion of the vector with Sal I and Hind III.
The primers that will be used to amplify In-Fusion Ready PCR products require the following 15 nucleotides on their 5’ends:
Sense primer: 5’ AAGGCCTCTGTCGAC followed by sequence of amplification target 3’
Antisense primer: 5’ AGAATTCGCAAGCTT followed by sequence of amplification target 3’
If the sequence of the gene of interest is added in-frame immediately after the 15 nucleotides mentioned above, this sequence will automatically be in-frame with the AcGFP1 sequence upstream and therefore be expressed as a fusion protein to the N-terminus of AcGFP1.
SV40 polyadenylation signals downstream of the AcGFP1 gene direct proper processing of the 3’ end of the mRNA transcript. In addition, the vector also contains a SV40 origin of replication in mammalian cells expressing the SV40 T-antigen. A neomycin resistance cassette (Neor), containing an SV40 early promoter, the neomycin/kanamycin resistance gene of Tn5, and polyadenylation signals from the Herpes simplex virus thymidine kinase (HSV TK) gene, allows selection of stable transformants in eukaryotic cells using G418. A bacterial promoter upstream of the gene allows the plasmid to express kanamycin resistance in E. coli. The pAcGFP1-N In-Fusion Ready Vector also provides a pUC origin of replication for propagation in E. coli and an f1 origin for single-stranded DNA production.
Fusions to the N-terminus of AcGFP1 retain the fluorescent properties of the native protein and allow monitoring of fusion protein localization in vivo. The PCR-amplified gene of interest is directly cloned into pAcGFP1-N In-Fusion Ready vector so that it is in-frame with the AcGFP1 coding sequence with no intervening in-frame stop codons. This can be accomplished by using the suggested primers. The inserted gene must include the initiating ATG codon. The recombinant AcGFP1 vector can be transfected into mammalian cells using any standard transfection method. Stable transformants can be selected using G418 (2).
Propagation in E. coli
Suitable host strains: Stellar Competent Cells.
Selectable marker: plasmid confers resistance to kanamycin (50 μg/ml) in E. coli hosts.
E. coli replication origin: pUC
Copy number: ~ 500
Plasmid incompatibility group: pMB1/ColE1
pAcGFP1-N In-Fusion Ready质粒序列载体序列
LOCUS pAcGFP1-N In-Fusion Ready 4688 bp DNA SYN DEFINITION pAcGFP1-N In-Fusion Ready ACCESSION KEYWORDS SOURCE ORGANISM other sequences; artificial sequences; vectors. FEATURES Location/Qualifiers source 1..4688 /organism="pAcGFP1-N In-Fusion Ready" /mol_type="other DNA" gene 39..740 /label="AcGFP1" /gene="AcGFP1" CDS 258..743 /label="ORF frame 3" /translation="MKQHDFFKSAMPEGYIQERTIFFEDDGNYKSRAEVKFEGDTLVN RIELTGTDFKEDGNILGNKMEYNYNAHNVYIMTDKAKNGIKVNFKIRHNIEDGSVQLA DHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMIYFGFVTAAAITHGMDELY K*" misc_feature 956..975 /label="EBV_rev_primer" /translation="MKQHDFFKSAMPEGYIQERTIFFEDDGNYKSRAEVKFEGDTLVN RIELTGTDFKEDGNILGNKMEYNYNAHNVYIMTDKAKNGIKVNFKIRHNIEDGSVQLA DHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMIYFGFVTAAAITHGMDELY K*" rep_origin complement(1125..1431) /label="f1_origin" /translation="MKQHDFFKSAMPEGYIQERTIFFEDDGNYKSRAEVKFEGDTLVN RIELTGTDFKEDGNILGNKMEYNYNAHNVYIMTDKAKNGIKVNFKIRHNIEDGSVQLA DHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMIYFGFVTAAAITHGMDELY K*" promoter 1510..1538 /label="AmpR_promoter" /translation="MKQHDFFKSAMPEGYIQERTIFFEDDGNYKSRAEVKFEGDTLVN RIELTGTDFKEDGNILGNKMEYNYNAHNVYIMTDKAKNGIKVNFKIRHNIEDGSVQLA DHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMIYFGFVTAAAITHGMDELY K*" misc_feature complement(1604..1624) /label="pBABE_3_primer" /translation="MKQHDFFKSAMPEGYIQERTIFFEDDGNYKSRAEVKFEGDTLVN RIELTGTDFKEDGNILGNKMEYNYNAHNVYIMTDKAKNGIKVNFKIRHNIEDGSVQLA DHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMIYFGFVTAAAITHGMDELY K*" misc_feature complement(1610..1825) /label="SV40_enhancer" /translation="MKQHDFFKSAMPEGYIQERTIFFEDDGNYKSRAEVKFEGDTLVN RIELTGTDFKEDGNILGNKMEYNYNAHNVYIMTDKAKNGIKVNFKIRHNIEDGSVQLA DHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMIYFGFVTAAAITHGMDELY K*" promoter 1622..1890 /label="SV40_promoter" /translation="MKQHDFFKSAMPEGYIQERTIFFEDDGNYKSRAEVKFEGDTLVN RIELTGTDFKEDGNILGNKMEYNYNAHNVYIMTDKAKNGIKVNFKIRHNIEDGSVQLA DHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMIYFGFVTAAAITHGMDELY K*" rep_origin 1789..1866 /label="SV40_origin" /translation="MKQHDFFKSAMPEGYIQERTIFFEDDGNYKSRAEVKFEGDTLVN RIELTGTDFKEDGNILGNKMEYNYNAHNVYIMTDKAKNGIKVNFKIRHNIEDGSVQLA DHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMIYFGFVTAAAITHGMDELY K*" misc_feature 1851..1870 /label="SV40pro_F_primer" /translation="MKQHDFFKSAMPEGYIQERTIFFEDDGNYKSRAEVKFEGDTLVN RIELTGTDFKEDGNILGNKMEYNYNAHNVYIMTDKAKNGIKVNFKIRHNIEDGSVQLA DHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMIYFGFVTAAAITHGMDELY K*" CDS 1973..2767 /label="ORF frame 2" /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGR PVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDL LSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE EHQGLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRY QDIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF*" gene 1976..2764 /label="NeoR/KanR" /gene="NeoR/KanR" /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGR PVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDL LSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE EHQGLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRY QDIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF*" CDS complement(2282..2818) /label="ORF frame 2" /translation="MAGWASLGRSFRTPESRSEELVKKAIEGDALRIGSGDTVKHEEA VSPFAAKLFSNITGSQRYVLIAVRHTQPATVDESRKAAIFHHDIRQAGIAMGHDEILA VGHARLEPGEQFGWREPLMLFVQIILIDKTGFHPSTCSLDAMFRLVVEWAGSRIKRMQ PPHCISHDGYFLGRSKVR*" terminator 2942..3211 /label="TK_PA_terminator" /translation="MAGWASLGRSFRTPESRSEELVKKAIEGDALRIGSGDTVKHEEA VSPFAAKLFSNITGSQRYVLIAVRHTQPATVDESRKAAIFHHDIRQAGIAMGHDEILA VGHARLEPGEQFGWREPLMLFVQIILIDKTGFHPSTCSLDAMFRLVVEWAGSRIKRMQ PPHCISHDGYFLGRSKVR*" rep_origin 3359..3978 /label="pBR322_origin" /translation="MAGWASLGRSFRTPESRSEELVKKAIEGDALRIGSGDTVKHEEA VSPFAAKLFSNITGSQRYVLIAVRHTQPATVDESRKAAIFHHDIRQAGIAMGHDEILA VGHARLEPGEQFGWREPLMLFVQIILIDKTGFHPSTCSLDAMFRLVVEWAGSRIKRMQ PPHCISHDGYFLGRSKVR*" promoter 4087..4639 /label="CMV_immearly_promoter" /translation="MAGWASLGRSFRTPESRSEELVKKAIEGDALRIGSGDTVKHEEA VSPFAAKLFSNITGSQRYVLIAVRHTQPATVDESRKAAIFHHDIRQAGIAMGHDEILA VGHARLEPGEQFGWREPLMLFVQIILIDKTGFHPSTCSLDAMFRLVVEWAGSRIKRMQ PPHCISHDGYFLGRSKVR*" misc_feature 4142..4429 /label="CAG_enhancer" /translation="MAGWASLGRSFRTPESRSEELVKKAIEGDALRIGSGDTVKHEEA VSPFAAKLFSNITGSQRYVLIAVRHTQPATVDESRKAAIFHHDIRQAGIAMGHDEILA VGHARLEPGEQFGWREPLMLFVQIILIDKTGFHPSTCSLDAMFRLVVEWAGSRIKRMQ PPHCISHDGYFLGRSKVR*" misc_feature 4596..4616 /label="CMV_fwd_primer" /translation="MAGWASLGRSFRTPESRSEELVKKAIEGDALRIGSGDTVKHEEA VSPFAAKLFSNITGSQRYVLIAVRHTQPATVDESRKAAIFHHDIRQAGIAMGHDEILA VGHARLEPGEQFGWREPLMLFVQIILIDKTGFHPSTCSLDAMFRLVVEWAGSRIKRMQ PPHCISHDGYFLGRSKVR*" promoter 4597..4666 /label="CMV_promoter" /translation="MAGWASLGRSFRTPESRSEELVKKAIEGDALRIGSGDTVKHEEA VSPFAAKLFSNITGSQRYVLIAVRHTQPATVDESRKAAIFHHDIRQAGIAMGHDEILA VGHARLEPGEQFGWREPLMLFVQIILIDKTGFHPSTCSLDAMFRLVVEWAGSRIKRMQ PPHCISHDGYFLGRSKVR*" ORIGIN 1 AGCTTGCGAA TTCTGGCCTA CCGGTCGCGA GCAAGGGCGC CGAGCTGTTC ACCGGCATCG 61 TGCCCATCCT GATCGAGCTG AATGGCGATG TGAATGGCCA CAAGTTCAGC GTGAGCGGCG 121 AGGGCGAGGG CGATGCCACC TACGGCAAGC TGACCCTGAA GTTCATCTGC ACCACCGGCA 181 AGCTGCCTGT GCCCTGGCCC ACCCTGGTGA CCACCCTGAG CTACGGCGTG CAGTGCTTCT 241 CACGCTACCC CGATCACATG AAGCAGCACG ACTTCTTCAA GAGCGCCATG CCTGAGGGCT 301 ACATCCAGGA GCGCACCATC TTCTTCGAGG ATGACGGCAA CTACAAGTCG CGCGCCGAGG 361 TGAAGTTCGA GGGCGATACC CTGGTGAATC GCATCGAGCT GACCGGCACC GATTTCAAGG 421 AGGATGGCAA CATCCTGGGC AATAAGATGG AGTACAACTA CAACGCCCAC AATGTGTACA 481 TCATGACCGA CAAGGCCAAG AATGGCATCA AGGTGAACTT CAAGATCCGC CACAACATCG 541 AGGATGGCAG CGTGCAGCTG GCCGACCACT ACCAGCAGAA TACCCCCATC GGCGATGGCC 601 CTGTGCTGCT GCCCGATAAC CACTACCTGT CCACCCAGAG CGCCCTGTCC AAGGACCCCA 661 ACGAGAAGCG CGATCACATG ATCTACTTCG GCTTCGTGAC CGCCGCCGCC ATCACCCACG 721 GCATGGATGA GCTGTACAAG TGAGCGGCCG CGACTCTAGA TCATAATCAG CCATACCACA 781 TTTGTAGAGG TTTTACTTGC TTTAAAAAAC CTCCCACACC TCCCCCTGAA CCTGAAACAT 841 AAAATGAATG CAATTGTTGT TGTTAACTTG TTTATTGCAG CTTATAATGG TTACAAATAA 901 AGCAATAGCA TCACAAATTT CACAAATAAA GCATTTTTTT CACTGCATTC TAGTTGTGGT 961 TTGTCCAAAC TCATCAATGT ATCTTAAGGC GTAAATTGTA AGCGTTAATA TTTTGTTAAA 1021 ATTCGCGTTA AATTTTTGTT AAATCAGCTC ATTTTTTAAC CAATAGGCCG AAATCGGCAA 1081 AATCCCTTAT AAATCAAAAG AATAGACCGA GATAGGGTTG AGTGTTGTTC CAGTTTGGAA 1141 CAAGAGTCCA CTATTAAAGA ACGTGGACTC CAACGTCAAA GGGCGAAAAA CCGTCTATCA 1201 GGGCGATGGC CCACTACGTG AACCATCACC CTAATCAAGT TTTTTGGGGT CGAGGTGCCG 1261 TAAAGCACTA AATCGGAACC CTAAAGGGAG CCCCCGATTT AGAGCTTGAC GGGGAAAGCC 1321 GGCGAACGTG GCGAGAAAGG AAGGGAAGAA AGCGAAAGGA GCGGGCGCTA GGGCGCTGGC 1381 AAGTGTAGCG GTCACGCTGC GCGTAACCAC CACACCCGCC GCGCTTAATG CGCCGCTACA 1441 GGGCGCGTCA GGTGGCACTT TTCGGGGAAA TGTGCGCGGA ACCCCTATTT GTTTATTTTT 1501 CTAAATACAT TCAAATATGT ATCCGCTCAT GAGACAATAA CCCTGATAAA TGCTTCAATA 1561 ATATTGAAAA AGGAAGAGTC CTGAGGCGGA AAGAACCAGC TGTGGAATGT GTGTCAGTTA 1621 GGGTGTGGAA AGTCCCCAGG CTCCCCAGCA GGCAGAAGTA TGCAAAGCAT GCATCTCAAT 1681 TAGTCAGCAA CCAGGTGTGG AAAGTCCCCA GGCTCCCCAG CAGGCAGAAG TATGCAAAGC 1741 ATGCATCTCA ATTAGTCAGC AACCATAGTC CCGCCCCTAA CTCCGCCCAT CCCGCCCCTA 1801 ACTCCGCCCA GTTCCGCCCA TTCTCCGCCC CATGGCTGAC TAATTTTTTT TATTTATGCA 1861 GAGGCCGAGG CCGCCTCGGC CTCTGAGCTA TTCCAGAAGT AGTGAGGAGG CTTTTTTGGA 1921 GGCCTAGGCT TTTGCAAAGA TCGATCAAGA GACAGGATGA GGATCGTTTC GCATGATTGA 1981 ACAAGATGGA TTGCACGCAG GTTCTCCGGC CGCTTGGGTG GAGAGGCTAT TCGGCTATGA 2041 CTGGGCACAA CAGACAATCG GCTGCTCTGA TGCCGCCGTG TTCCGGCTGT CAGCGCAGGG 2101 GCGCCCGGTT CTTTTTGTCA AGACCGACCT GTCCGGTGCC CTGAATGAAC TGCAAGACGA 2161 GGCAGCGCGG CTATCGTGGC TGGCCACGAC GGGCGTTCCT TGCGCAGCTG TGCTCGACGT 2221 TGTCACTGAA GCGGGAAGGG ACTGGCTGCT ATTGGGCGAA GTGCCGGGGC AGGATCTCCT 2281 GTCATCTCAC CTTGCTCCTG CCGAGAAAGT ATCCATCATG GCTGATGCAA TGCGGCGGCT 2341 GCATACGCTT GATCCGGCTA CCTGCCCATT CGACCACCAA GCGAAACATC GCATCGAGCG 2401 AGCACGTACT CGGATGGAAG CCGGTCTTGT CGATCAGGAT GATCTGGACG AAGAGCATCA 2461 GGGGCTCGCG CCAGCCGAAC TGTTCGCCAG GCTCAAGGCG AGCATGCCCG ACGGCGAGGA 2521 TCTCGTCGTG ACCCATGGCG ATGCCTGCTT GCCGAATATC ATGGTGGAAA ATGGCCGCTT 2581 TTCTGGATTC ATCGACTGTG GCCGGCTGGG TGTGGCGGAC CGCTATCAGG ACATAGCGTT 2641 GGCTACCCGT GATATTGCTG AAGAGCTTGG CGGCGAATGG GCTGACCGCT TCCTCGTGCT 2701 TTACGGTATC GCCGCTCCCG ATTCGCAGCG CATCGCCTTC TATCGCCTTC TTGACGAGTT 2761 CTTCTGAGCG GGACTCTGGG GTTCGAAATG ACCGACCAAG CGACGCCCAA CCTGCCATCA 2821 CGAGATTTCG ATTCCACCGC CGCCTTCTAT GAAAGGTTGG GCTTCGGAAT CGTTTTCCGG 2881 GACGCCGGCT GGATGATCCT CCAGCGCGGG GATCTCATGC TGGAGTTCTT CGCCCACCCT 2941 AGGGGGAGGC TAACTGAAAC ACGGAAGGAG ACAATACCGG AAGGAACCCG CGCTATGACG 3001 GCAATAAAAA GACAGAATAA AACGCACGGT GTTGGGTCGT TTGTTCATAA ACGCGGGGTT 3061 CGGTCCCAGG GCTGGCACTC TGTCGATACC CCACCGAGAC CCCATTGGGG CCAATACGCC 3121 CGCGTTTCTT CCTTTTCCCC ACCCCACCCC CCAAGTTCGG GTGAAGGCCC AGGGCTCGCA 3181 GCCAACGTCG GGGCGGCAGG CCCTGCCATA GCCTCAGGTT ACTCATATAT ACTTTAGATT 3241 GATTTAAAAC TTCATTTTTA ATTTAAAAGG ATCTAGGTGA AGATCCTTTT TGATAATCTC 3301 ATGACCAAAA TCCCTTAACG TGAGTTTTCG TTCCACTGAG CGTCAGACCC CGTAGAAAAG 3361 ATCAAAGGAT CTTCTTGAGA TCCTTTTTTT CTGCGCGTAA TCTGCTGCTT GCAAACAAAA 3421 AAACCACCGC TACCAGCGGT GGTTTGTTTG CCGGATCAAG AGCTACCAAC TCTTTTTCCG 3481 AAGGTAACTG GCTTCAGCAG AGCGCAGATA CCAAATACTG TTCTTCTAGT GTAGCCGTAG 3541 TTAGGCCACC ACTTCAAGAA CTCTGTAGCA CCGCCTACAT ACCTCGCTCT GCTAATCCTG 3601 TTACCAGTGG CTGCTGCCAG TGGCGATAAG TCGTGTCTTA CCGGGTTGGA CTCAAGACGA 3661 TAGTTACCGG ATAAGGCGCA GCGGTCGGGC TGAACGGGGG GTTCGTGCAC ACAGCCCAGC 3721 TTGGAGCGAA CGACCTACAC CGAACTGAGA TACCTACAGC GTGAGCTATG AGAAAGCGCC 3781 ACGCTTCCCG AAGGGAGAAA GGCGGACAGG TATCCGGTAA GCGGCAGGGT CGGAACAGGA 3841 GAGCGCACGA GGGAGCTTCC AGGGGGAAAC GCCTGGTATC TTTATAGTCC TGTCGGGTTT 3901 CGCCACCTCT GACTTGAGCG TCGATTTTTG TGATGCTCGT CAGGGGGGCG GAGCCTATGG 3961 AAAAACGCCA GCAACGCGGC CTTTTTACGG TTCCTGGCCT TTTGCTGGCC TTTTGCTCAC 4021 ATGTTCTTTC CTGCGTTATC CCCTGATTCT GTGGATAACC GTATTACCGC CATGCATTAG 4081 TTATTAATAG TAATCAATTA CGGGGTCATT AGTTCATAGC CCATATATGG AGTTCCGCGT 4141 TACATAACTT ACGGTAAATG GCCCGCCTGG CTGACCGCCC AACGACCCCC GCCCATTGAC 4201 GTCAATAATG ACGTATGTTC CCATAGTAAC GCCAATAGGG ACTTTCCATT GACGTCAATG 4261 GGTGGAGTAT TTACGGTAAA CTGCCCACTT GGCAGTACAT CAAGTGTATC ATATGCCAAG 4321 TACGCCCCCT ATTGACGTCA ATGACGGTAA ATGGCCCGCC TGGCATTATG CCCAGTACAT 4381 GACCTTATGG GACTTTCCTA CTTGGCAGTA CATCTACGTA TTAGTCATCG CTATTACCAT 4441 GGTGATGCGG TTTTGGCAGT ACATCAATGG GCGTGGATAG CGGTTTGACT CACGGGGATT 4501 TCCAAGTCTC CACCCCATTG ACGTCAATGG GAGTTTGTTT TGGCACCAAA ATCAACGGGA 4561 CTTTCCAAAA TGTCGTAACA ACTCCGCCCC ATTGACGCAA ATGGGCGGTA GGCGTGTACG 4621 GTGGGAGGTC TATATAAGCA GAGCTGGTTT AGTGAACCGT CAGATCCGCT AGCTAAGGCC 4681 TCTGTCGA //
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pAcGFP1-N In-Fusion Ready质粒载体应用举例
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