| 质粒名称: | NES-EGFP-cPHx1 |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 116853 |
| 存储实验室: | Gerry Hammond |
|
载体骨架基本信息 |
|
| 载体名称: | pEGFP-1 |
| 载体出品公司: | Clontech |
| 空载体大小: | -- |
| 完整质粒大小: | 5259 bp |
| 载体修饰: | -- |
| 载体类型: | 哺乳动物表达 |
| 筛选标记: | 新霉素（G418筛选） |
| 载体抗性: | 卡那霉素，50μg/mL |
| 生长温度: | 37 ℃ |
| 宿主菌: | NEB Stable |
| 拷贝数: | 高拷贝 |
|
插入基因信息 |
|
| 插入基因名称: | PLEKHA1 |
| 物种来源: | -- |
| 插入大小: | 483 bp |
| 基因突变: | 氨基酸169.329 |
| GenBank编号 | -- |
| 启动子: | -- |
| 标签/融合蛋白: | EGFP（插入物上的N末端）X.leavis映射2k1.L（32-44）（插入时的N端子） |
| 克隆方法: | Gibson Cloning |
| 5’克隆位点: | -- |
| 3’克隆位点: | -- |
| 5’测序引物 | EGFP-C1 |
| 3’测序引物 | SV40-pArev |
|
文献引用方法 |
|
| 材料和方法部分: | NES-EGFP-cPHx1 was a gift from Gerry Hammond (Addgene plasmid # 116853 ; http://n2t.net/addgene:116853 ; RRID:Addgene_116853) |
| 参考文献部分: | A high-avidity biosensor reveals plasma membrane PI(3,4)P2 is predominantly a class I PI3K signaling product. Goulden BD, Pacheco J, Dull A, Zewe JP, Deiters A, Hammond GRV. J Cell Biol. 2019 Mar 4;218(3):1066-1079. doi: 10.1083/jcb.201809026. Epub 2018 Dec 27. 10.1083/jcb.201809026 PubMed 30591513 |
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 NES-EGFP-cPHx1 5259 bp ds-DNA circular SYN 12-MAY-2021
DEFINITION PI(3,4)P2 biosensor.
ACCESSION .
VERSION .
KEYWORDS NES-EGFP-cPHx1
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 5259)
AUTHORS Goulden BD, Pacheco J, Dull A, Zewe JP, Deiters A, Hammond GRV
TITLE A high-avidity biosensor reveals plasma membrane PI(3,4)P2 is
predominantly a class I PI3K signaling product.
JOURNAL J Cell Biol. 2019 Mar 4;218(3):1066-1079. doi:
10.1083/jcb.201809026. Epub 2018 Dec 27.
PUBMED 30591513
REFERENCE 2 (bases 1 to 5259)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58
http://www.biofeng.com
FEATURES Location/Qualifiers
source 1..5259
/organism="synthetic DNA construct"
/mol_type="other DNA"
enhancer 1..304
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 305..508
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
primer_bind 459..479
/label=CMV-F
/note="Human CMV immediate early promoter, forward primer"
regulatory 535..544
/regulatory_class="other"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
regulatory 592..601
/regulatory_class="other"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
CDS 598..1314
/codon_start=1
/product="enhanced GFP"
/label=EGFP
/translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
EFVTAAGITLGMDELYK"
CDS 598..1314
/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(643..664)
/label=EGFP-N
/note="EGFP, reverse primer"
primer_bind complement(904..923)
/label=EXFP-R
/note="For distinguishing EGFP variants, reverse primer"
primer_bind 1251..1272
/label=EGFP-C
/note="EGFP, forward primer"
polyA_signal 1987..2108
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(2024..2043)
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
primer_bind 2078..2097
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
rep_origin complement(2115..2570)
/direction=LEFT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(2252..2273)
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind 2464..2483
/label=F1ori-R
/note="F1 origin, reverse primer"
promoter 2597..2701
/gene="bla"
/label=AmpR promoter
promoter 2703..3060
/label=SV40 promoter
/note="SV40 enhancer and early promoter"
primer_bind complement(2726..2746)
/label=pBABE 3'
/note="SV40 enhancer, reverse primer for pBABE vectors"
rep_origin 2911..3046
/label=SV40 ori
/note="SV40 origin of replication"
primer_bind 2973..2992
/label=SV40pro-F
/note="SV40 promoter/origin, forward primer"
CDS 3095..3889
/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(3149..3168)
/label=Neo-R
/note="Neomycin resistance gene, reverse primer"
primer_bind 3759..3778
/label=Neo-F
/note="Neomycin resistance gene, forward primer"
primer_bind complement(4077..4096)
/label=TK-pA-R
/note="Thymidine kinase polyA, reverse primer"
polyA_signal 4121..4168
/label=HSV TK poly(A) signal
/note="herpes simplex virus thymidine kinase
polyadenylation signal (Cole and Stacy, 1985)"
rep_origin 4497..5085
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 4986..5005
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
ORIGIN
1 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
61 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
121 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
181 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
241 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
301 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
361 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
421 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
481 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagccacc
541 atggctctgc agaaaaagtt ggaagagctt gagctggatg aggcaccggt cgccaccatg
601 gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc
661 gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc
721 aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc
781 gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct accccgacca catgaagcag
841 cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc
901 aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg
961 aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag
1021 ctggagtaca actacaacag ccacaacgtc tatatcatgg ccgacaagca gaagaacggc
1081 atcaaggtga acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac
1141 cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac
1201 ctgagcaccc agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg
1261 ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagtccgga
1321 ctcagatctc gagctcaagc ttcgaattct agaaataatc tgaaacggtc acaaagccat
1381 cttccttact ttactcctaa accacctcaa gatagtgcgg ttatcaaagc tggatattgt
1441 gtaaaacaag gagcagtgat gaaaaactgg aagagaagat attttcaatt ggatgaaaac
1501 acaataggct acttcaaatc tgaactggaa aaggaacctc ttcgtgtaat accacttaaa
1561 gaggttcata aagtccagga atgtaagcaa agcgacataa tgatgaggga caacctcttt
1621 gaaattgtaa caacgtctcg aactttctat gtgcaggctg atagccctga agagatgcac
1681 agttggatta aagcagtctc tggcgccatt gtagcacagc ggggtcccgg cagatctgcg
1741 tcttctgagc atccccccgg tccttcagaa tccaaacacg ctttccgtcc taccaacgca
1801 gccaccgcca cctcacattc cacagcctct cgctgagtcg acggtaccgc gggcccggga
1861 tccaccggat ctagataact gatcataatc agccatacca catttgtaga ggttttactt
1921 gctttaaaaa acctcccaca cctccccctg aacctgaaac ataaaatgaa tgcaattgtt
1981 gttgttaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat
2041 ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat
2101 gtatcttaac gcgtaaattg taagcgttaa tattttgtta aaattcgcgt taaatttttg
2161 ttaaatcagc tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa
2221 agaatagacc gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa
2281 gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg
2341 tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa
2401 ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa
2461 ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct
2521 gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt caggtggcac
2581 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
2641 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
2701 tcctgaggcg gaaagaacca gctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca
2761 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaggtgt
2821 ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca
2881 gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc
2941 cattctccgc cccatggctg actaattttt tttatttatg cagaggccga ggccgcctcg
3001 gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa
3061 gatcgatcaa gagacaggat gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc
3121 aggttctccg gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat
3181 cggctgctct gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt
3241 caagaccgac ctgtccggtg ccctgaatga actgcaagac gaggcagcgc ggctatcgtg
3301 gctggccacg acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag
3361 ggactggctg ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc
3421 tgccgagaaa gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc
3481 tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga
3541 agccggtctt gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga
3601 actgttcgcc aggctcaagg cgagcatgcc cgacggcgag gatctcgtcg tgacccatgg
3661 cgatgcctgc ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg
3721 tggccggctg ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc
3781 tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc
3841 cgattcgcag cgcatcgcct tctatcgcct tcttgacgag ttcttctgag cgggactctg
3901 gggttcgaaa tgaccgacca agcgacgccc aacctgccat cacgagattt cgattccacc
3961 gccgccttct atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc
4021 ctccagcgcg gggatctcat gctggagttc ttcgcccacc ctagggggag gctaactgaa
4081 acacggaagg agacaatacc ggaaggaacc cgcgctatga cggcaataaa aagacagaat
4141 aaaacgcacg gtgttgggtc gtttgttcat aaacgcgggg ttcggtccca gggctggcac
4201 tctgtcgata ccccaccgag accccattgg ggccaatacg cccgcgtttc ttccttttcc
4261 ccaccccacc ccccaagttc gggtgaaggc ccagggctcg cagccaacgt cggggcggca
4321 ggccctgcca tagcctcagg ttactcatat atactttaga ttgatttaaa acttcatttt
4381 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa
4441 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
4501 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
4561 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
4621 agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag
4681 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
4741 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
4801 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
4861 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
4921 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
4981 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
5041 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
5101 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta
5161 tcccctgatt ctgtggataa ccgtattacc gccatgcatt agttattaat agtaatcaat
5221 tacggggtca ttagttcata gcccatatat ggagttccg
//
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