| 质粒名称: | pFX-Tom20-EGFP |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 174186 |
| 存储实验室: | Yusuke Ohba |
|
载体骨架基本信息 |
|
| 载体名称: | pFX |
| 空载体大小: | 4000bp |
| 完整质粒大小: | 4800 bp |
| 载体类型: | 哺乳细胞表达 |
| 筛选标记: | 新霉素（G418） |
| 载体抗性: | Kanamycin |
| 生长温度: | 37 ℃ |
| 宿主菌: | DH5a |
| 拷贝数: | 高拷贝 |
|
插入基因信息 |
|
| 插入基因名称: | EGFP with mitchondria-targeting sequence |
| 插入大小: | 800 bp |
| 启动子: | CMV |
| 标签/融合蛋白: | the mitochondria-targeting sequence of TOM20 (N terminal on insert) |
| 克隆方法: | 限制性内切酶 |
| 5’克隆位点: | XhoI(未破坏) |
| 3’克隆位点: | BglII(未破坏) |
| 5’测序引物 | 无 |
| 3’测序引物 | 无 |
|
文献引用方法 |
|
| 材料和方法部分: | pFX-Tom20-EGFP was a gift from Yusuke Ohba (Addgene plasmid # 174186 ; http://n2t.net/addgene:174186 ; RRID:Addgene_174186) |
| 参考文献部分: | Folding Latency of Fluorescent Proteins Affects the Mitochondrial Localization of Fusion Proteins. Kashiwagi S, Fujioka Y, Satoh AO, Yoshida A, Fujioka M, Nepal P, Tsuzuki A, Aoki O, Paudel S, Sasajima H, Ohba Y. Cell Struct Funct. 2019 Dec 26;44(2):183-194. doi: 10.1247/csf.19028. Epub 2019 Nov 15. 10.1247/csf.19028 PubMed 31735 |
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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 pFX-Tom20-EGFP 5254 bp ds-DNA circular SYN 18-FEB-2022
DEFINITION Express EGFP with mitochondria-targeting sequence in mammalian
cells.
ACCESSION .
VERSION .
KEYWORDS pFX-Tom20-EGFP
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 5254)
AUTHORS Kashiwagi S, Fujioka Y, Satoh AO, Yoshida A, Fujioka M, Nepal P,
Tsuzuki A, Aoki O, Paudel S, Sasajima H, Ohba Y
TITLE Folding Latency of Fluorescent Proteins Affects the Mitochondrial
Localization of Fusion Proteins.
JOURNAL Cell Struct Funct. 2019 Dec 26;44(2):183-194. doi:
10.1247/csf.19028. Epub 2019 Nov 15.
PUBMED 31735740
REFERENCE 2 (bases 1 to 5254)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Feb 18, 2022 from SnapGene Server 1.1.58
http://www.biofeng.com
FEATURES Location/Qualifiers
source 1..5254
/organism="synthetic DNA construct"
/mol_type="other DNA"
enhancer 61..364
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 365..568
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
primer_bind 519..539
/label=CMV-F
/note="Human CMV immediate early promoter, forward primer"
CDS 1098..1814
/codon_start=1
/product="enhanced GFP"
/label=EGFP
/translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
EFVTAAGITLGMDELYK"
CDS 1098..1814
/codon_start=1
/product="the original enhanced GFP (Yang et al., 1996)"
/label=EGFP
/note="mammalian codon-optimized"
/translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
EFVTAAGITLGMDELYK"
primer_bind complement(1143..1164)
/label=EGFP-N
/note="EGFP, reverse primer"
primer_bind complement(1404..1423)
/label=EXFP-R
/note="For distinguishing EGFP variants, reverse primer"
primer_bind 1751..1772
/label=EGFP-C
/note="EGFP, forward primer"
polyA_signal 2042..2163
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(2079..2098)
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
primer_bind 2133..2152
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
rep_origin complement(2170..2625)
/direction=LEFT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(2307..2328)
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind 2519..2538
/label=F1ori-R
/note="F1 origin, reverse primer"
promoter 2652..2756
/gene="bla"
/label=AmpR promoter
promoter 2758..3115
/label=SV40 promoter
/note="SV40 enhancer and early promoter"
primer_bind complement(2781..2801)
/label=pBABE 3'
/note="SV40 enhancer, reverse primer for pBABE vectors"
rep_origin 2966..3101
/label=SV40 ori
/note="SV40 origin of replication"
primer_bind 3028..3047
/label=SV40pro-F
/note="SV40 promoter/origin, forward primer"
CDS 3150..3944
/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(3204..3223)
/label=Neo-R
/note="Neomycin resistance gene, reverse primer"
primer_bind 3814..3833
/label=Neo-F
/note="Neomycin resistance gene, forward primer"
primer_bind complement(4132..4151)
/label=TK-pA-R
/note="Thymidine kinase polyA, reverse primer"
polyA_signal 4176..4223
/label=HSV TK poly(A) signal
/note="herpes simplex virus thymidine kinase
polyadenylation signal (Cole and Stacy, 1985)"
rep_origin 4552..5140
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 5041..5060
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
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 ccggttctag aagtactctc gagcccgcca tggtgggtcg gaacagcgcc atcgccgccg
661 gtgtatgcgg ggcccttttc attgggtact gcatctactt cgaccgcaaa agacgaagtg
721 accccaactt caagaacagg cttcgagaac gaagaaagaa acagaagctt gccaaggaga
781 gagctgggct ttccaagtta cctgacctta aagatgctga agctgttcag aagttcttcc
841 ttgaagaaat acagcttggt gaagagttac tagctcaagg tgaatatgag aagggcgtag
901 accatctgac aaatgcaatt gctgtgtgtg gacagccaca gcagttactg caggtcttac
961 agcaaactct tccaccacca gtgttccaga tgcttctgac taagctccca acaattagtc
1021 agagaattgt aagtgctcag agcttggctg aagatgatgt ggaaggcggc cgcggaggat
1081 ccggaggaga attcaccatg gtgagcaagg gcgaggagct gttcaccggg gtggtgccca
1141 tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg
1201 agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc
1261 ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct
1321 accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc
1381 aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt
1441 tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg
1501 gcaacatcct ggggcacaag ctggagtaca actacaacag ccacaacgtc tatatcatgg
1561 ccgacaagca gaagaacggc atcaaggtga acttcaagat ccgccacaac atcgaggacg
1621 gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc
1681 tgctgcccga caaccactac ctgagcaccc agtccgccct gagcaaagac cccaacgaga
1741 agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg
1801 acgagctgta caagagatct ctgactgact gagatgcgcg gatgtcgaac ggcagttcgg
1861 ccgaataggc cgacagccgg ccgtcggccg acgacacggc cagggccggc ggggatccac
1921 cggatctaga taactgatca taatcagcca taccacattt gtagaggttt tacttgcttt
1981 aaaaaacctc ccacacctcc ccctgaacct gaaacataaa atgaatgcaa ttgttgttgt
2041 taacttgttt attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac
2101 aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc
2161 ttaaggcgta aattgtaagc gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa
2221 tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat
2281 agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg
2341 tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac
2401 catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat cggaacccta
2461 aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg agaaaggaag
2521 ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg
2581 taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtcaggt ggcacttttc
2641 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc
2701 cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtcctg
2761 aggcggaaag aaccagctgt ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc
2821 cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca ggtgtggaaa
2881 gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac
2941 catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc
3001 tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg cctcggcctc
3061 tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaagatcg
3121 atcaagagac aggatgagga tcgtttcgca tgattgaaca agatggattg cacgcaggtt
3181 ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag acaatcggct
3241 gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt tttgtcaaga
3301 ccgacctgtc cggtgccctg aatgaactgc aagacgaggc agcgcggcta tcgtggctgg
3361 ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg ggaagggact
3421 ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt gctcctgccg
3481 agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat ccggctacct
3541 gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg atggaagccg
3601 gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca gccgaactgt
3661 tcgccaggct caaggcgagc atgcccgacg gcgaggatct cgtcgtgacc catggcgatg
3721 cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc gactgtggcc
3781 ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat attgctgaag
3841 agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt
3901 cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgagcggga ctctggggtt
3961 cgaaatgacc gaccaagcga cgcccaacct gccatcacga gatttcgatt ccaccgccgc
4021 cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga tgatcctcca
4081 gcgcggggat ctcatgctgg agttcttcgc ccaccctagg gggaggctaa ctgaaacacg
4141 gaaggagaca ataccggaag gaacccgcgc tatgacggca ataaaaagac agaataaaac
4201 gcacggtgtt gggtcgtttg ttcataaacg cggggttcgg tcccagggct ggcactctgt
4261 cgatacccca ccgagacccc attggggcca atacgcccgc gtttcttcct tttccccacc
4321 ccacccccca agttcgggtg aaggcccagg gctcgcagcc aacgtcgggg cggcaggccc
4381 tgccatagcc tcaggttact catatatact ttagattgat ttaaaacttc atttttaatt
4441 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga
4501 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
4561 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
4621 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
4681 gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc
4741 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
4801 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
4861 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
4921 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
4981 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
5041 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
5101 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
5161 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
5221 tgattctgtg gataaccgta ttaccgccat gcat
//
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