| 质粒名称: | BPK764 |
|---|---|
| 出品公司: | 爱迪基因 |
| 目录编号: | 65767 |
| 存储实验室: | Keith Joung |
|
载体骨架基本信息 |
|
| 载体名称: | pACYCDuet-1 |
| 载体出品公司: | EMD Millipore |
| 载体抗性: | 氯霉素 |
| 载体类型: | Bacterial Expression, CRISPR |
| 高拷贝/低拷贝: | 低拷贝 |
| 生长条件: | 37 ℃ |
|
插入基因信息 |
|
| 插入基因名称: | mammalian codon-optimized Streptococcus pyogenes Cas9-NLS-3XFlag, and SpCas9 gRNA |
| 启动子 | T7 (x2) |
| 标签/融合蛋白: |
NLS (C terminal on insert)
3x FLAG (C terminal on insert)
|
| 克隆方法: | Gibson Cloning |
| 5' 测序引物 | ACYC-DuetUP1 |
| 3' 测序引物 | oBK390 (5'-GCAAATTCGACCCGGTCGTCG-3') |
|
文献引用方法 |
|
| 材料和方法部分: | BPK764 was a gift from Keith Joung (爱迪基因 plasmid # 65767) |
| 参考文献部分: | Engineered CRISPR-Cas9 nucleases with altered PAM specificities. Kleinstiver BP, Prew MS, Tsai SQ, Topkar VV, Nguyen NT, Zheng Z, Gonzales AP, Li Z, Peterson RT, Yeh JJ, Aryee MJ, Joung JK. Nature. 2015 Jun 22. doi: 10.1038/nature14592. 10.1038/nature14592 PubMed 26098369 |
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 BPK764 8055 bp ds-DNA circular SYN 10-JAN-2018
DEFINITION Bacterial expression plasmid for SpCas9.
ACCESSION .
VERSION .
KEYWORDS BPK764
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 8055)
AUTHORS Kleinstiver BP, Prew MS, Tsai SQ, Topkar VV, Nguyen NT, Zheng Z,
Gonzales AP, Li Z, Peterson RT, Yeh JJ, Aryee MJ, Joung JK
TITLE Engineered CRISPR-Cas9 nucleases with altered PAM specificities.
JOURNAL Nature. 2015 Jun 22. doi: 10.1038/nature14592.
PUBMED 26098369
COMMENT This file is created by Vector NTI
http://www.biofeng.com/
COMMENT ORIGDB|GenBank
COMMENT VNTAUTHORNAME|biofeng.com|
COMMENT VNTNAME|BPK764|
FEATURES Location/Qualifiers
source 1..8055
/organism="synthetic DNA construct"
/mol_type="other DNA"
protein_bind 3..27
/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)."
CDS 71..4174
/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="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
DATLIHQSITGLYETRIDLSQLGGD"
CDS 4181..4201
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 4208..4273
/codon_start=1
/product="three tandem FLAG(R) epitope tags, followed by an
enterokinase cleavage site"
/label=3xFLAG
/translation="DYKDHDGDYKDHDIDYKDDDDK"
promoter 4343..4361
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
misc_RNA 4417..4492
/label=gRNA scaffold
/note="guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system"
primer_bind complement(4494..4512)
/label=T7 Term
/note="T7 terminator, reverse primer"
terminator 4508..4555
/label=T7 terminator
/note="transcription terminator for bacteriophage T7 RNA
polymerase"
CDS complement(4776..5435)
/codon_start=1
/gene="cat"
/product="chloramphenicol acetyltransferase"
/label=CmR
/note="confers resistance to chloramphenicol"
/translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
primer_bind 5350..5369
/label=CAT-R
/note="Chloramphenicol resistance gene, reverse primer"
promoter complement(5436..5538)
/label=cat promoter
/note="promoter of the E. coli cat gene encoding
chloramphenicol acetyltransferase"
primer_bind complement(5930..5947)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(6064..6608)
/direction=LEFT
/label=p15A ori
/note="Plasmids containing the medium-copy-number p15A
origin of replication can be propagated in E. coli cells
that contain a second plasmid with the ColE1 origin.
"
primer_bind complement(6144..6163)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS complement(6833..7915)
/codon_start=1
/gene="lacI"
/product="lac repressor"
/label=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)."
/translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
ALADSLMQLARQVSRLESGQ"
primer_bind 7877..7896
/label=LacI-R
/note="LacI, reverse primer"
promoter complement(7916..7993)
/gene="lacI"
/label=lacI promoter
promoter 8039..2
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
1 ggggaattgt gagcggataa caattcccct gtagaaataa ttttgtttaa ctttaataag
61 gagatatacc atggataaaa agtattctat tggtttagac atcggcacta attccgttgg
121 atgggctgtc ataaccgatg aatacaaagt accttcaaag aaatttaagg tgttggggaa
181 cacagaccgt cattcgatta aaaagaatct tatcggtgcc ctcctattcg atagtggcga
241 aacggcagag gcgactcgcc tgaaacgaac cgctcggaga aggtatacac gtcgcaagaa
301 ccgaatatgt tacttacaag aaatttttag caatgagatg gccaaagttg acgattcttt
361 ctttcaccgt ttggaagagt ccttccttgt cgaagaggac aagaaacatg aacggcaccc
421 catctttgga aacatagtag atgaggtggc atatcatgaa aagtacccaa cgatttatca
481 cctcagaaaa aagctagttg actcaactga taaagcggac ctgaggttaa tctacttggc
541 tcttgcccat atgataaagt tccgtgggca ctttctcatt gagggtgatc taaatccgga
601 caactcggat gtcgacaaac tgttcatcca gttagtacaa acctataatc agttgtttga
661 agagaaccct ataaatgcaa gtggcgtgga tgcgaaggct attcttagcg cccgcctctc
721 taaatcccga cggctagaaa acctgatcgc acaattaccc ggagagaaga aaaatgggtt
781 gttcggtaac cttatagcgc tctcactagg cctgacacca aattttaagt cgaacttcga
841 cttagctgaa gatgccaaat tgcagcttag taaggacacg tacgatgacg atctcgacaa
901 tctactggca caaattggag atcagtatgc ggacttattt ttggctgcca aaaaccttag
961 cgatgcaatc ctcctatctg acatactgag agttaatact gagattacca aggcgccgtt
1021 atccgcttca atgatcaaaa ggtacgatga acatcaccaa gacttgacac ttctcaaggc
1081 cctagtccgt cagcaactgc ctgagaaata taaggaaata ttctttgatc agtcgaaaaa
1141 cgggtacgca ggttatattg acggcggagc gagtcaagag gaattctaca agtttatcaa
1201 acccatatta gagaagatgg atgggacgga agagttgctt gtaaaactca atcgcgaaga
1261 tctactgcga aagcagcgga ctttcgacaa cggtagcatt ccacatcaaa tccacttagg
1321 cgaattgcat gctatactta gaaggcagga ggatttttat ccgttcctca aagacaatcg
1381 tgaaaagatt gagaaaatcc taacctttcg cataccttac tatgtgggac ccctggcccg
1441 agggaactct cggttcgcat ggatgacaag aaagtccgaa gaaacgatta ctccctggaa
1501 ttttgaggaa gttgtcgata aaggtgcgtc agctcaatcg ttcatcgaga ggatgaccaa
1561 ctttgacaag aatttaccga acgaaaaagt attgcctaag cacagtttac tttacgagta
1621 tttcacagtg tacaatgaac tcacgaaagt taagtatgtc actgagggca tgcgtaaacc
1681 cgcctttcta agcggagaac agaagaaagc aatagtagat ctgttattca agaccaaccg
1741 caaagtgaca gttaagcaat tgaaagagga ctactttaag aaaattgaat gcttcgattc
1801 tgtcgagatc tccggggtag aagatcgatt taatgcgtca cttggtacgt atcatgacct
1861 cctaaagata attaaagata aggacttcct ggataacgaa gagaatgaag atatcttaga
1921 agatatagtg ttgactctta ccctctttga agatcgggaa atgattgagg aaagactaaa
1981 aacatacgct cacctgttcg acgataaggt tatgaaacag ttaaagaggc gtcgctatac
2041 gggctgggga cgattgtcgc ggaaacttat caacgggata agagacaagc aaagtggtaa
2101 aactattctc gattttctaa agagcgacgg cttcgccaat aggaacttta tgcagctgat
2161 ccatgatgac tctttaacct tcaaagagga tatacaaaag gcacaggttt ccggacaagg
2221 ggactcattg cacgaacata ttgcgaatct tgctggttcg ccagccatca aaaagggcat
2281 actccagaca gtcaaagtag tggatgagct agttaaggtc atgggacgtc acaaaccgga
2341 aaacattgta atcgagatgg cacgcgaaaa tcaaacgact cagaaggggc aaaaaaacag
2401 tcgagagcgg atgaagagaa tagaagaggg tattaaagaa ctgggcagcc agatcttaaa
2461 ggagcatcct gtggaaaata cccaattgca gaacgagaaa ctttacctct attacctaca
2521 aaatggaagg gacatgtatg ttgatcagga actggacata aaccgtttat ctgattacga
2581 cgtcgatcac attgtacccc aatccttttt gaaggacgat tcaatcgaca ataaagtgct
2641 tacacgctcg gataagaacc gagggaaaag tgacaatgtt ccaagcgagg aagtcgtaaa
2701 gaaaatgaag aactattggc ggcagctcct aaatgcgaaa ctgataacgc aaagaaagtt
2761 cgataactta actaaagctg agaggggtgg cttgtctgaa cttgacaagg ccggatttat
2821 taaacgtcag ctcgtggaaa cccgccaaat cacaaagcat gttgcacaga tactagattc
2881 ccgaatgaat acgaaatacg acgagaacga taagctgatt cgggaagtca aagtaatcac
2941 tttaaagtca aaattggtgt cggacttcag aaaggatttt caattctata aagttaggga
3001 gataaataac taccaccatg cgcacgacgc ttatcttaat gccgtcgtag ggaccgcact
3061 cattaagaaa tacccgaagc tagaaagtga gtttgtgtat ggtgattaca aagtttatga
3121 cgtccgtaag atgatcgcga aaagcgaaca ggagataggc aaggctacag ccaaatactt
3181 cttttattct aacattatga atttctttaa gacggaaatc actctggcaa acggagagat
3241 acgcaaacga cctttaattg aaaccaatgg ggagacaggt gaaatcgtat gggataaggg
3301 ccgggacttc gcgacggtga gaaaagtttt gtccatgccc caagtcaaca tagtaaagaa
3361 aactgaggtg cagaccggag ggttttcaaa ggaatcgatt cttccaaaaa ggaatagtga
3421 taagctcatc gctcgtaaaa aggactggga cccgaaaaag tacggtggct tcgatagccc
3481 tacagttgcc tattctgtcc tagtagtggc aaaagttgag aagggaaaat ccaagaaact
3541 gaagtcagtc aaagaattat tggggataac gattatggag cgctcgtctt ttgaaaagaa
3601 ccccatcgac ttccttgagg cgaaaggtta caaggaagta aaaaaggatc tcataattaa
3661 actaccaaag tatagtctgt ttgagttaga aaatggccga aaacggatgt tggctagcgc
3721 cggagagctt caaaagggga acgaactcgc actaccgtct aaatacgtga atttcctgta
3781 tttagcgtcc cattacgaga agttgaaagg ttcacctgaa gataacgaac agaagcaact
3841 ttttgttgag cagcacaaac attatctcga cgaaatcata gagcaaattt cggaattcag
3901 taagagagtc atcctagctg atgccaatct ggacaaagta ttaagcgcat acaacaagca
3961 cagggataaa cccatacgtg agcaggcgga aaatattatc catttgttta ctcttaccaa
4021 cctcggcgct ccagccgcat tcaagtattt tgacacaacg atagatcgca aacgatacac
4081 ttctaccaag gaggtgctag acgcgacact gattcaccaa tccatcacgg gattatatga
4141 aactcggata gatttgtcac agcttggggg tgacggatcc cccaagaaga agaggaaagt
4201 ctcgagcgac tacaaagacc atgacggtga ttataaagat catgacatcg attacaagga
4261 tgacgatgac aagtgaagcg gccgcataat gcttaagtcg aacagaaagt aatcgtattg
4321 tacacggccg cataatcgaa attaatacga ctcactatag ggagacccat gccatagcgt
4381 tgttcggaac agattcacca acacctagtg gtctccgttt tagagctaga aatagcaagt
4441 taaaataagg ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gctccgctga
4501 gcaataacta gcataacccc ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa
4561 acctcaggca tttgagaagc acacggtcac actgcttccg gtagtcaata aaccggtaaa
4621 ccagcaatag acataagcgg ctatttaacg accctgccct gaaccgacga ccgggtcgaa
4681 tttgctttcg aatttctgcc attcatccgc ttattatcac ttattcaggc gtagcaacca
4741 ggcgtttaag ggcaccaata actgccttaa aaaaattacg ccccgccctg ccactcatcg
4801 cagtactgtt gtaattcatt aagcattctg ccgacatgga agccatcaca aacggcatga
4861 tgaacctgaa tcgccagcgg catcagcacc ttgtcgcctt gcgtataata tttgcccata
4921 gtgaaaacgg gggcgaagaa gttgtccata ttggccacgt ttaaatcaaa actggtgaaa
4981 ctcacccagg gattggctga gacgaaaaac atattctcaa taaacccttt agggaaatag
5041 gccaggtttt caccgtaaca cgccacatct tgcgaatata tgtgtagaaa ctgccggaaa
5101 tcgtcgtggt attcactcca gagcgatgaa aacgtttcag tttgctcatg gaaaacggtg
5161 taacaagggt gaacactatc ccatatcacc agctcaccgt ctttcattgc catacggaac
5221 tccggatgag cattcatcag gcgggcaaga atgtgaataa aggccggata aaacttgtgc
5281 ttatttttct ttacggtctt taaaaaggcc gtaatatcca gctgaacggt ctggttatag
5341 gtacattgag caactgactg aaatgcctca aaatgttctt tacgatgcca ttgggatata
5401 tcaacggtgg tatatccagt gatttttttc tccattttag cttccttagc tcctgaaaat
5461 ctcgataact caaaaaatac gcccggtagt gatcttattt cattatggtg aaagttggaa
5521 cctcttacgt gccgatcaac gtctcatttt cgccaaaagt tggcccaggg cttcccggta
5581 tcaacaggga caccaggatt tatttattct gcgaagtgat cttccgtcac aggtatttat
5641 tcggcgcaaa gtgcgtcggg tgatgctgcc aacttactga tttagtgtat gatggtgttt
5701 ttgaggtgct ccagtggctt ctgtttctat cagctgtccc tcctgttcag ctactgacgg
5761 ggtggtgcgt aacggcaaaa gcaccgccgg acatcagcgc tagcggagtg tatactggct
5821 tactatgttg gcactgatga gggtgtcagt gaagtgcttc atgtggcagg agaaaaaagg
5881 ctgcaccggt gcgtcagcag aatatgtgat acaggatata ttccgcttcc tcgctcactg
5941 actcgctacg ctcggtcgtt cgactgcggc gagcggaaat ggcttacgaa cggggcggag
6001 atttcctgga agatgccagg aagatactta acagggaagt gagagggccg cggcaaagcc
6061 gtttttccat aggctccgcc cccctgacaa gcatcacgaa atctgacgct caaatcagtg
6121 gtggcgaaac ccgacaggac tataaagata ccaggcgttt cccctggcgg ctccctcgtg
6181 cgctctcctg ttcctgcctt tcggtttacc ggtgtcattc cgctgttatg gccgcgtttg
6241 tctcattcca cgcctgacac tcagttccgg gtaggcagtt cgctccaagc tggactgtat
6301 gcacgaaccc cccgttcagt ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
6361 caacccggaa agacatgcaa aagcaccact ggcagcagcc actggtaatt gatttagagg
6421 agttagtctt gaagtcatgc gccggttaag gctaaactga aaggacaagt tttggtgact
6481 gcgctcctcc aagccagtta cctcggttca aagagttggt agctcagaga accttcgaaa
6541 aaccgccctg caaggcggtt ttttcgtttt cagagcaaga gattacgcgc agaccaaaac
6601 gatctcaaga agatcatctt attaatcaga taaaatattt ctagatttca gtgcaattta
6661 tctcttcaaa tgtagcacct gaagtcagcc ccatacgata taagttgtaa ttctcatgtt
6721 agtcatgccc cgcgcccacc ggaaggagct gactgggttg aaggctctca agggcatcgg
6781 tcgagatccc ggtgcctaat gagtgagcta acttacatta attgcgttgc gctcactgcc
6841 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
6901 gagaggcggt ttgcgtattg ggcgccaggg tggtttttct tttcaccagt gagacgggca
6961 acagctgatt gcccttcacc gcctggccct gagagagttg cagcaagcgg tccacgctgg
7021 tttgccccag caggcgaaaa tcctgtttga tggtggttaa cggcgggata taacatgagc
7081 tgtcttcggt atcgtcgtat cccactaccg agatgtccgc accaacgcgc agcccggact
7141 cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc atcgcagtgg
7201 gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa accggacatg gcactccagt
7261 cgccttcccg ttccgctatc ggctgaattt gattgcgagt gagatattta tgccagccag
7321 ccagacgcag acgcgccgag acagaactta atgggcccgc taacagcgcg atttgctggt
7381 gacccaatgc gaccagatgc tccacgccca gtcgcgtacc gtcttcatgg gagaaaataa
7441 tactgttgat gggtgtctgg tcagagacat caagaaataa cgccggaaca ttagtgcagg
7501 cagcttccac agcaatggca tcctggtcat ccagcggata gttaatgatc agcccactga
7561 cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta
7621 ccatcgacac caccacgctg gcacccagtt gatcggcgcg agatttaatc gccgcgacaa
7681 tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac gccaatcagc aacgactgtt
7741 tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc atcgccgctt
7801 ccactttttc ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg cgggaaacgg
7861 tctgataaga gacaccggca tactctgcga catcgtataa cgttactggt ttcacattca
7921 ccaccctgaa ttgactctct tccgggcgct atcatgccat accgcgaaag gttttgcgcc
7981 attcgatggt gtccgggatc tcgacgctct cccttatgcg actcctgcat taggaaatta
8041 atacgactca ctata
//
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