| 质粒名称: | PB-NEO-GFP-RFP |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 157952 |
| 存储实验室: | Yan-zhen Bi |
|
载体骨架基本信息 |
|
| 载体名称: | pUC19 |
| 载体出品公司: | -- |
| 空载体大小: | 7500 bp |
| 完整质粒大小: | -- |
| 载体修饰: | -- |
| 载体类型: | 哺乳动物表达，小鼠靶向；基因打靶 |
| 筛选标记: | 新霉素（G418） |
| 载体抗性: | 氨苄青霉素 |
| 生长温度: | 37 ℃ |
| 宿主菌: | DH5a |
| 拷贝数: | 低拷贝 |
|
插入基因信息 |
|
| 插入基因名称: | -- |
| 物种来源: | -- |
| 插入大小: | -- |
| 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: | PB-NEO-GFP-RFP was a gift from Yan-zhen Bi (Addgene plasmid # 157952 ; http://n2t.net/addgene:157952 ; RRID:Addgene_157952) |
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 PB-NEO-GFP-RFP 7599 bp ds-DNA circular SYN 12-MAY-2021
DEFINITION seamless gene editing and component of Piggybac transposase system.
ACCESSION .
VERSION .
KEYWORDS PB-NEO-GFP-RFP
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7599)
TITLE Bi Lab mammalian expression constructs
REFERENCE 2 (bases 1 to 7599)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..7599
/organism="synthetic DNA construct"
/mol_type="other DNA"
primer_bind complement(29..51)
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
primer_bind 151..170
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 364..386
/label=M13/pUC Forward
/note="In lacZ gene"
primer_bind 378..395
/label=M13 Forward
/note="In lacZ gene. Also called M13-F20 or M13 (-21)
Forward"
primer_bind 379..395
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
repeat_region 440..474
/label=piggyBac left (5') inverted repeat
/note="piggyBac transposon-specific inverted terminal
repeat sequence (ITR)"
protein_bind complement(768..801)
/label=loxP
/bound_moiety="Cre recombinase"
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (GCATACAT)."
enhancer 868..1171
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 1172..1375
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
CDS 1404..2195
/codon_start=1
/gene="aph(3')-II (or nptII)"
/product="aminoglycoside phosphotransferase from Tn5"
/label=NeoR/KanR
/note="confers resistance to neomycin, kanamycin, and G418
(Geneticin(R))"
/translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
primer_bind complement(1458..1477)
/label=Neo-R
/note="Neomycin resistance gene, reverse primer"
primer_bind 2068..2087
/label=Neo-F
/note="Neomycin resistance gene, forward primer"
CDS 2217..2282
/codon_start=1
/product="2A peptide from foot-and-mouth disease virus
polyprotein"
/label=F2A
/note="Eukaryotic ribosomes fail to insert a peptide bond
between the Gly and Pro residues, yielding separate
polypeptides."
/translation="VKQTLNFDLLKLAGDVESNPGP"
CDS 2289..3008
/codon_start=1
/product="the original enhanced GFP (Yang et al., 1996)"
/label=EGFP
/note="mammalian codon-optimized"
/translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
EFVTAAGITLGMDELYK"
CDS 2289..3005
/codon_start=1
/product="enhanced GFP"
/label=EGFP
/translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
EFVTAAGITLGMDELYK"
primer_bind complement(2334..2355)
/label=EGFP-N
/note="EGFP, reverse primer"
primer_bind complement(2595..2614)
/label=EXFP-R
/note="For distinguishing EGFP variants, reverse primer"
primer_bind 2942..2963
/label=EGFP-C
/note="EGFP, forward primer"
polyA_signal 3131..3212
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
protein_bind complement(3219..3252)
/label=loxP
/bound_moiety="Cre recombinase"
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (GCATACAT)."
repeat_region 3443..3505
/label=piggyBac right (3') inverted repeat
/note="piggyBac transposon-specific inverted terminal
repeat sequence (ITR)"
enhancer 3594..3897
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 3898..4101
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
regulatory 4128..4137
/regulatory_class="other"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
CDS 4134..4811
/codon_start=1
/product="monomeric derivative of DsRed fluorescent protein
(Strongin et al., 2007)"
/label=DsRed-Monomer
/note="mammalian codon-optimized"
/translation="MDNTEDVIKEFMQFKVRMEGSVNGHYFEIEGEGEGKPYEGTQTAK
LQVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYMKLSFPEGFTWERSMNFEDGGV
VEVQQDSSLQDGTFIYKVKFKGVNFPADGPVMQKKTAGWEPSTEKLYPQDGVLKGEISH
ALKLKDGGHYTCDFKTVYKAKKPVQLPGNHYVDSKLDITNHNEDYTVVEQYEHAEARHS
GSQ"
primer_bind complement(4314..4334)
/label=DsRed1-N
/note="DsRed1, reverse primer"
primer_bind 4532..4551
/label=mCherry-F
/note="mCherry, forward primer"
polyA_signal 4934..5055
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind 5025..5044
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
primer_bind complement(5199..5220)
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind complement(5378..5394)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(5378..5394)
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind complement(5391..5413)
/label=M13/pUC Reverse
/note="In lacZ gene"
protein_bind 5402..5418
/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)."
promoter complement(5426..5456)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 5471..5492
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(5609..5626)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(5780..6368)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(5860..5879)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS complement(6539..7399)
/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 7162..7181
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
promoter complement(7400..7504)
/gene="bla"
/label=AmpR promoter
primer_bind 7572..7590
/label=pBRforEco
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
ORIGIN
1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
361 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgagctcggc gcgcctcatg
421 attaattaaa tcgatttaac cctagaaaga tagtctgcgt aaaattgacg catgcattct
481 tgaaatattg ctctctcttt ctaaatagcg cgaatccgtc gctgtgcatt taggacatct
541 cagtcgccgc ttggagctcc cgtgaggcgt gcttgtcaat gcggtaagtg tcactgattt
601 tgaactataa cgaccgcgtg agtcaaaatg acgcatgatt atcttttacg tgacttttaa
661 gatttaactc atacgataat tatattgtta tttcatgttc tacttacgtg ataacttatt
721 atatatatat tttcttgtta tagatatcat cgatgcgatc gcagctcata acttcgtata
781 atgtatgcta tacgaagtta tctcgactag ttattaatag taatcaatta cggggtcatt
841 agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg
901 ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac
961 gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt
1021 ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa
1081 atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta
1141 catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt acatcaatgg
1201 gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg acgtcaatgg
1261 gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca actccgcccc
1321 attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca gagctggttt
1381 agtgaaccgt cagatccgct agcatgattg aacaagatgg attgcacgca ggttctccgg
1441 ccgcttgggt ggagaggcta ttcggctatg actgggcaca acagacaatc ggctgctctg
1501 atgccgccgt gttccggctg tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc
1561 tgtccggtgc cctgaatgaa ctgcaagacg aggcagcgcg gctatcgtgg ctggccacga
1621 cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg gactggctgc
1681 tattgggcga agtgccgggg caggatctcc tgtcatctca ccttgctcct gccgagaaag
1741 tatccatcat ggctgatgca atgcggcggc tgcatacgct tgatccggct acctgcccat
1801 tcgaccacca agcgaaacat cgcatcgagc gagcacgtac tcggatggaa gccggtcttg
1861 tcgatcagga tgatctggac gaagagcatc aggggctcgc gccagccgaa ctgttcgcca
1921 ggctcaaggc gagcatgccc gacggcgagg atctcgtcgt gacccatggc gatgcctgct
1981 tgccgaatat catggtggaa aatggccgct tttctggatt catcgactgt ggccggctgg
2041 gtgtggcgga ccgctatcag gacatagcgt tggctacccg tgatattgct gaagagcttg
2101 gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc gattcgcagc
2161 gcatcgcctt ctatcgcctt cttgacgagt tcttcctcga gaaaattgtc gctcctgtca
2221 aacaaactct taactttgat ttactcaaac tggctgggga tgtagaaagc aatccaggtc
2281 caggatccat ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg
2341 agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg
2401 ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct
2461 ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc taccccgacc
2521 acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca
2581 ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg
2641 acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc
2701 tggggcacaa gctggagtac aactacaaca gccacaacgt ctatatcatg gccgacaagc
2761 agaagaacgg catcaaggtg aacttcaaga tccgccacaa catcgaggac ggcagcgtgc
2821 agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg
2881 acaaccacta cctgagcacc cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc
2941 acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg gacgagctgt
3001 acaagtaaag cggccgcgac tctagatcat aatcagccat accacatttg tagaggtttt
3061 acttgcttta aaaaacctcc cacacctccc cctgaacctg aaacataaaa tgaatgcaat
3121 tgttgttgtt aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac
3181 aaatttcaca aataaagcat ttttttcact gcagatccat aacttcgtat aatgtatgct
3241 atacgaagtt atcgacctgc aggcatgcgt cgactttgtt actttataga agaaattttg
3301 agtttttgtt tttttttaat aaataaataa acataaataa attgtttgtt gaatttatta
3361 ttagtatgta agtgtaaata taataaaact taatatctat tcaaattaat aaataaacct
3421 cgatatacag accgataaaa cacatgcgtc aattttacgc atgattatct ttaacgtacg
3481 tcacaatatg attatctttc tagggttaac ctaggaccgg tggtaccaag ctttagttat
3541 taatagtaat caattacggg gtcattagtt catagcccat atatggagtt ccgcgttaca
3601 taacttacgg taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca
3661 ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg
3721 gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg
3781 ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc
3841 ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtg
3901 atgcggtttt ggcagtacat caatgggcgt ggatagcggt ttgactcacg gggatttcca
3961 agtctccacc ccattgacgt caatgggagt ttgttttggc accaaaatca acgggacttt
4021 ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg
4081 gaggtctata taagcagagc tggtttagtg aaccgtcaga tccgagcgcc accatggaca
4141 acaccgagga cgtcatcaag gagttcatgc agttcaaggt gcgcatggag ggctccgtga
4201 acggccacta cttcgagatc gagggcgagg gcgagggcaa gccctacgag ggcacccaga
4261 ccgccaagct gcaggtgacc aagggcggcc ccctgccctt cgcctgggac atcctgtccc
4321 cccagttcca gtacggctcc aaggcctacg tgaagcaccc cgccgacatc cccgactaca
4381 tgaagctgtc cttccccgag ggcttcacct gggagcgctc catgaacttc gaggacggcg
4441 gcgtggtgga ggtgcagcag gactcctccc tgcaggacgg caccttcatc tacaaggtga
4501 agttcaaggg cgtgaacttc cccgccgacg gccccgtaat gcagaagaag actgccggct
4561 gggagccctc caccgagaag ctgtaccccc aggacggcgt gctgaagggc gagatctccc
4621 acgccctgaa gctgaaggac ggcggccact acacctgcga cttcaagacc gtgtacaagg
4681 ccaagaagcc cgtgcagctg cccggcaacc actacgtgga ctccaagctg gacatcacca
4741 accacaacga ggactacacc gtggtggagc agtacgagca cgccgaggcc cgccactccg
4801 gctcccagta gagcggccgc gactctagat cataatcagc cataccacat ttgtagaggt
4861 tttacttgct ttaaaaaacc tcccacacct ccccctgaac ctgaaacata aaatgaatgc
4921 aattgttgtt gttaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat
4981 cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact
5041 catcaatgta tcttaaggcg taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa
5101 atttttgtta aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata
5161 aatcaaaaga atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac
5221 tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc
5281 cactacgtga accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa
5341 atcggaaccc taaagggaga agcttggcgt aatcatggtc atagctgttt cctgtgtgaa
5401 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
5461 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
5521 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
5581 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
5641 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
5701 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
5761 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
5821 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
5881 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
5941 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
6001 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
6061 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
6121 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
6181 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
6241 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
6301 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
6361 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
6421 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
6481 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
6541 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
6601 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
6661 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
6721 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
6781 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
6841 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
6901 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
6961 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
7021 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
7081 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
7141 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
7201 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
7261 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
7321 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
7381 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
7441 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
7501 cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga aaccattatt atcatgacat
7561 taacctataa aaataggcgt atcacgaggc cctttcgtc
//
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