质粒名称: | pRRL-MHCK7-GCaMP6 |
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出品公司: | Addgene |
目录编号: | 65042 |
存储实验室: | Nenad Bursac |
载体骨架基本信息 |
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载体名称: | pRRL |
空载体大小: | 6904 bp |
完整质粒大小: | 8257 bp |
载体类型: | 哺乳细胞表达, Lentiviral |
载体抗性: | 氨苄霉素 |
生长温度: | 37 ℃ |
宿主菌: | Stbl3 |
拷贝数: | 未知 |
插入基因信息 |
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插入基因名称: | GCaMP6 |
物种来源: | Synthetic |
插入大小: | 1353bp |
启动子: | MHCK7 |
标签/融合蛋白: |
6xHis (N terminal on insert)
T7 tag (N terminal on insert)
Xpress epitope (N terminal on insert)
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克隆方法: | 未知 |
5’测序引物 | gcctcgaccggtcgatctcg |
3’测序引物 | ttttgtaatccagaggttga |
文献引用方法 |
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材料和方法部分: | pRRL-MHCK7-GCaMP6 was a gift from Nenad Bursac (Addgene plasmid # 65042 ; http://n2t.net/addgene:65042 ; RRID:Addgene_65042) |
参考文献部分: | Bioengineered human myobundles mimic clinical responses of skeletal muscle to drugs. Madden L, Juhas M, Kraus WE, Truskey GA, Bursac N. Elife. 2015 Jan 9;4:e04885. doi: 10.7554/eLife.04885. 10.7554/eLife.04885 PubMed 25575180 |
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LOCUS pRRL-MHCK7-GCaMP6 8270 bp ds-DNA circular SYN 21-DEC-2023 DEFINITION Lentiviral vector expressing the calcium indicator GCaMP6 driven by the muscle-specific promoter MHCK7. ACCESSION . VERSION . KEYWORDS pRRL-MHCK7-GCaMP6 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8270) AUTHORS Madden L, Juhas M, Kraus WE, Truskey GA, Bursac N TITLE Bioengineered human myobundles mimic clinical responses of skeletal muscle to drugs. JOURNAL Elife. 2015 Jan 9;4:e04885. doi: 10.7554/eLife.04885. PUBMED 25575180 REFERENCE 2 (bases 1 to 8270) AUTHORS . TITLE Direct Submission JOURNAL Exported Dec 21, 2023 from SnapGene Server 1.1.58 http://www.biofeng.com FEATURES Location/Qualifiers source 1..8270 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 8..234 /label=RSV promoter /note="Rous sarcoma virus enhancer/promoter" LTR 235..415 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" misc_feature 462..587 /label=HIV-1 Psi /note="packaging signal of human immunodeficiency virus type 1" misc_feature 1080..1313 /label=RRE /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." CDS 1498..1542 /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" regulatory 2754..2763 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 2760..4112 /codon_start=1 /product="improved fluorescent protein-based calcium sensor (Chen et al., 2013)" /label=GCaMP6f /translation="MGSHHHHHHGMASMTGGQQMGRDLYDDDDKDLATMVDSSRRKWNK TGHAVRAIGRLSSLENVYIKADKQKNGIKANFKIRHNIEDGGVQLAYHYQQNTPIGDGP VLLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEE LFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLT YGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIE LKGIDFKEDGNILGHKLEYNLPDQLTEEQIAEFKEEFSLFDKDGDGTITTKELGTVMRS LGQNPTEAELQDMINEVDADGDGTIDFPEFLTMMARKMKYRDTEEEIREAFGVFDKDGN GYISAAELRHVMTNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVQMMTAK" regulatory 2856..2865 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" primer_bind 3146..3167 /label=EGFP-C /note="EGFP, forward primer" primer_bind complement(3273..3294) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(3534..3553) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" misc_feature 4121..4709 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(4174..4194) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(4592..4603) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" LTR 4796..5029 /label=3' LTR (Delta-U3) /note="self-inactivating 3' long terminal repeat (LTR) from HIV-1" polyA_signal 5101..5222 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(5138..5157) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 5192..5211 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" rep_origin 5262..5397 /label=SV40 ori /note="SV40 origin of replication" primer_bind 5324..5343 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" primer_bind complement(5417..5436) /label=T7 /note="T7 promoter, forward primer" promoter complement(5418..5436) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(5446..5463) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(5446..5462) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(5455..5477) /label=M13/pUC Forward /note="In lacZ gene" rep_origin 5604..6059 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(5691..5710) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 5901..5922 /label=F1ori-F /note="F1 origin, forward primer" promoter 6085..6189 /gene="bla" /label=AmpR promoter CDS 6190..7050 /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(6408..6427) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 7221..7809 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 7710..7729 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 7963..7980 /label=L4440 /note="L4440 vector, forward primer" protein_bind 8097..8118 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 8133..8163 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 8171..8187 /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 8176..8198 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 8195..8211 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 8195..8211 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind 8230..8250 /label=T3 /note="T3 promoter, forward primer" promoter 8232..8250 /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" ORIGIN 1 agcttaatgt agtcttatgc aatactcttg tagtcttgca acatggtaac gatgagttag 61 caacatgcct tacaaggaga gaaaaagcac cgtgcatgcc gattggtgga agtaaggtgg 121 tacgatcgtg ccttattagg aaggcaacag acgggtctga catggattgg acgaaccact 181 gaattgccgc attgcagaga tattgtattt aagtgcctag ctcgatacat aaacgggtct 241 ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt 301 aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac 361 tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtggc 421 gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc 481 ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa 541 ttttgactag cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg 601 ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata 661 aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc 721 tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga 781 caggatcaga agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc 841 aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca 901 aaagtaagac caccgcacag caagcggccg ctgatcttca gacctggagg aggagatatg 961 agggacaatt ggagaagtga attatataaa tataaagtag taaaaattga accattagga 1021 gtagcaccca ccaaggcaaa gagaagagtg gtgcagagag aaaaaagagc agtgggaata 1081 ggagctttgt tccttgggtt cttgggagca gcaggaagca ctatgggcgc agcgtcaatg 1141 acgctgacgg tacaggccag acaattattg tctggtatag tgcagcagca gaacaatttg 1201 ctgagggcta ttgaggcgca acagcatctg ttgcaactca cagtctgggg catcaagcag 1261 ctccaggcaa gaatcctggc tgtggaaaga tacctaaagg atcaacagct cctggggatt 1321 tggggttgct ctggaaaact catttgcacc actgctgtgc cttggaatgc tagttggagt 1381 aataaatctc tggaacagat ttggaatcac acgacctgga tggagtggga cagagaaatt 1441 aacaattaca caagcttaat acactcctta attgaagaat cgcaaaacca gcaagaaaag 1501 aatgaacaag aattattgga attagataaa tgggcaagtt tgtggaattg gtttaacata 1561 acaaattggc tgtggtatat aaaattattc ataatgatag taggaggctt ggtaggttta 1621 agaatagttt ttgctgtact ttctatagtg aatagagtta ggcagggata ttcaccatta 1681 tcgtttcaga cccacctccc aaccccgagg ggacccgaca ggcccgaagg aatagaagaa 1741 gaaggtggag agagagacag agacagatcc attcgattag tgaacggatc tcgacggtat 1801 cggttaactt ttaaaagaaa aggggggatt ggggggtaca gtgcagggga aagaatagta 1861 gacataatag caacagacat acaaactaaa gaattacaaa aacaaattac aaaattcaaa 1921 attctcgaca ttgattattg actagagctt gcatgtctaa gctagaccct tcagattaaa 1981 aataactgag gtaagggcct gggtagggga ggtggtgtga gacgctcctg tctctcctct 2041 atctgcccat cggccctttg gggaggagga atgtgcccaa ggactaaaaa aaggccatgg 2101 agccagaggg gcgagggcaa cagacctttc atgggcaaac cttggggccc tgctgtctag 2161 catgccccac tacgggtcta ggctgcccat gtaaggaggc aaggcctggg gacacccgag 2221 atgcctggtt ataattaacc cagacatgtg gctgcccccc ccccccaaca cctgctgcct 2281 ctaaaaataa ccctgtccct ggtggatccc ctgcatgcga agatcttcga acaaggctgt 2341 gggggactga gggcaggctg taacaggctt gggggccagg gcttatacgt gcctgggact 2401 cccaaagtat tactgttcca tgttcccggc gaagggccag ctgtcccccg ccagctagac 2461 tcagcactta gtttaggaac cagtgagcaa gtcagccctt ggggcagccc atacaaggcc 2521 atggggctgg gcaagctgca cgcctgggtc cggggtgggc acggtgcccg ggcaacgagc 2581 tgaaagctca tctgctctca ggggcccctc cctggggaca gcccctcctg gctagtcaca 2641 ccctgtaggc tcctctatat aacccagggg cacaggggct gccctcattc taccaccacc 2701 tccacagcac agacagacac tcaggagcca gccagcctcg accggtcgat ctcgccacca 2761 tgggttctca tcatcatcat catcatggta tggctagcat gactggtgga cagcaaatgg 2821 gtcgggatct gtacgacgat gacgataagg atctcgccac catggtcgac tcatcacgtc 2881 gtaagtggaa taagacaggt cacgcagtca gagctatagg tcggctgagc tcactcgaga 2941 acgtctatat caaggccgac aagcagaaga acggcatcaa ggcgaacttc aagatccgcc 3001 acaacatcga ggacggcggc gtgcagctcg cctaccacta ccagcagaac acccccatcg 3061 gcgacggccc cgtgctgctg cccgacaacc actacctgag cgtgcagtcc aaactttcga 3121 aagaccccaa cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc gccgccggga 3181 tcactctcgg catggacgag ctgtacaagg gcggtaccgg agggagcatg gtgagcaagg 3241 gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg 3301 gccacaagtt cagcgtgtcc ggcgagggtg agggcgatgc cacctacggc aagctgaccc 3361 tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc 3421 tgacctacgg cgtgcagtgc ttcagccgct accccgacca catgaagcag cacgacttct 3481 tcaagtccgc catgcccgaa ggctacatcc aggagcgcac catcttcttc aaggacgacg 3541 gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg 3601 agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca 3661 acctgccgga ccaactgact gaagagcaga tcgcagaatt taaagaggaa ttctccctat 3721 ttgacaagga cggggatggg acaataacaa ccaaggagct ggggacggtg atgcggtctc 3781 tggggcagaa ccccacagaa gcagagctgc aggacatgat caatgaagta gatgccgacg 3841 gtgacggcac aatcgacttc cctgagttcc tgacaatgat ggcaagaaaa atgaaataca 3901 gggacacgga agaagaaatt agagaagcgt tcggtgtgtt tgataaggat ggcaatggct 3961 acatcagtgc agcagagctt cgccacgtga tgacaaacct tggagagaag ttaacagatg 4021 aagaggttga tgaaatgatc agggaagcag acatcgatgg ggatggtcag gtaaactacg 4081 aagagtttgt acaaatgatg acagcgaagt gagcgtcgac aatcaacctc tggattacaa 4141 aatttgtgaa agattgactg gtattcttaa ctatgttgct ccttttacgc tatgtggata 4201 cgctgcttta atgcctttgt atcatgctat tgcttcccgt atggctttca ttttctcctc 4261 cttgtataaa tcctggttgc tgtctcttta tgaggagttg tggcccgttg tcaggcaacg 4321 tggcgtggtg tgcactgtgt ttgctgacgc aacccccact ggttggggca ttgccaccac 4381 ctgtcagctc ctttccggga ctttcgcttt ccccctccct attgccacgg cggaactcat 4441 cgccgcctgc cttgcccgct gctggacagg ggctcggctg ttgggcactg acaattccgt 4501 ggtgttgtcg gggaagctga cgtcctttcc atggctgctc gcctgtgttg ccacctggat 4561 tctgcgcggg acgtccttct gctacgtccc ttcggccctc aatccagcgg accttccttc 4621 ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt cgccttcgcc ctcagacgag 4681 tcggatctcc ctttgggccg cctccccgcc tggaattcga gctcggtacc tttaagacca 4741 atgacttaca aggcagctgt agatcttagc cactttttaa aagaaaaggg gggactggaa 4801 gggctaattc actcccaacg aagacaagat ctgctttttg cttgtactgg gtctctctgg 4861 ttagaccaga tctgagcctg ggagctctct ggctaactag ggaacccact gcttaagcct 4921 caataaagct tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt 4981 aactagagat ccctcagacc cttttagtca gtgtggaaaa tctctagcag tagtagttca 5041 tgtcatctta ttattcagta tttataactt gcaaagaaat gaatatcaga gagtgagagg 5101 aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca 5161 aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct 5221 tatcatgtct ggctctagct atcccgcccc taactccgcc catcccgccc ctaactccgc 5281 ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg 5341 aggccgcctc ggcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag 5401 ggacgtaccc aattcgccct atagtgagtc gtattacgcg cgctcactgg ccgtcgtttt 5461 acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc 5521 ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt 5581 gcgcagcctg aatggcgaat gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt 5641 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc 5701 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg 5761 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta 5821 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt 5881 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat 5941 ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa 6001 tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc ttacaattta 6061 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat 6121 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa 6181 aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt 6241 tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag 6301 ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt 6361 tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg 6421 gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag 6481 aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta 6541 agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg 6601 acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta 6661 actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac 6721 accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt 6781 actctagctt cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca 6841 cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag 6901 cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta 6961 gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag 7021 ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt 7081 tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat 7141 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta 7201 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa 7261 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt 7321 tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag 7381 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta 7441 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca 7501 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag 7561 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa 7621 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga 7681 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc 7741 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc 7801 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt 7861 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt 7921 gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag 7981 gaagcggaag agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa 8041 tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat 8101 gtgagttagc tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg 8161 ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac 8221 gccaagcgcg caattaaccc tcactaaagg gaacaaaagc tggagctgca //
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