| 质粒名称: | EGFP-IpgD |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 183666 |
| 存储实验室: | Sergio Grinstein |
|
载体骨架基本信息 |
|
| 载体名称: | pEGFP-C1 |
| 载体出品公司: | Clontech |
| 空载体大小: | -- |
| 完整质粒大小: | 6351 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: | DH5a |
| 拷贝数: | 高拷贝 |
|
插入基因信息 |
|
| 插入基因名称: | IpgD |
| 物种来源: | Shigella flexneri 5a strain M90T plasmid pWR100 |
| 插入大小: | -- |
| 基因突变: | -- |
| GenBank编号 | -- |
| 启动子: | CMV |
| 标签/融合蛋白: | EGFP（插入物上的N末端） |
| 克隆方法: | Gibson Cloning |
| 5’克隆位点: | -- |
| 3’克隆位点: | -- |
| 5’测序引物 | CMV-Fwd, EGFP-Fwd |
| 3’测序引物 | SV40pA-R |
|
文献引用方法 |
|
| 材料和方法部分: | EGFP-IpgD was a gift from Sergio Grinstein (Addgene plasmid # 183666 ; http://n2t.net/addgene:183666 ; RRID:Addgene_183666) |
| 参考文献部分: | Kinase-independent synthesis of 3-phosphorylated phosphoinositides by a phosphotransferase. Walpole GFW, Pacheco J, Chauhan N, Clark J, Anderson KE, Abbas YM, Brabant-Kirwan D, Montano-Rendon F, Liu Z, Zhu H, Brumell JH, Deiters A, Stephens LR, Hawkins PT, Hammond GRV, Grinstein S, Fairn GD. Nat Cell Biol. 2022 Apr 28. pii: 10.1038/s41556-022-00895-y. doi: 10.1038/s41556-022-00895-y. 10.1038/s41556-022-00895-y PubMed 35484249 |
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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 EGFP-IpgD 6351 bp ds-DNA circular SYN 25-APR-2022
DEFINITION N-terminally tagged Shigella flexneri IpgD for mammalian expression.
ACCESSION .
VERSION .
KEYWORDS EGFP-IpgD
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 6351)
TITLE Kinase-independent synthesis of 3-phosphorylated phosphoinositides
by a phosphotransferase
REFERENCE 2 (bases 1 to 6351)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Apr 25, 2022 from SnapGene Server 1.1.58
http://www.biofeng.com
FEATURES Location/Qualifiers
source 1..6351
/organism="synthetic DNA construct"
/mol_type="other DNA"
enhancer 70..373
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 374..577
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
primer_bind 528..548
/label=CMV-F
/note="Human CMV immediate early promoter, forward primer"
regulatory 616..625
/regulatory_class="other"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
CDS 622..1338
/codon_start=1
/product="enhanced GFP"
/label=EGFP
/translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
EFVTAAGITLGMDELYK"
CDS 622..1338
/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(667..688)
/label=EGFP-N
/note="EGFP, reverse primer"
primer_bind complement(928..947)
/label=EXFP-R
/note="For distinguishing EGFP variants, reverse primer"
primer_bind 1275..1296
/label=EGFP-C
/note="EGFP, forward primer"
polyA_signal 3148..3269
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(3185..3204)
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
primer_bind 3239..3258
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
rep_origin complement(3276..3731)
/direction=LEFT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(3413..3434)
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind 3625..3644
/label=F1ori-R
/note="F1 origin, reverse primer"
promoter 3758..3862
/gene="bla"
/label=AmpR promoter
promoter 3864..4221
/label=SV40 promoter
/note="SV40 enhancer and early promoter"
primer_bind complement(3887..3907)
/label=pBABE 3'
/note="SV40 enhancer, reverse primer for pBABE vectors"
rep_origin 4072..4207
/label=SV40 ori
/note="SV40 origin of replication"
primer_bind 4134..4153
/label=SV40pro-F
/note="SV40 promoter/origin, forward primer"
CDS 4256..5050
/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(4310..4329)
/label=Neo-R
/note="Neomycin resistance gene, reverse primer"
primer_bind 4920..4939
/label=Neo-F
/note="Neomycin resistance gene, forward primer"
primer_bind complement(5238..5257)
/label=TK-pA-R
/note="Thymidine kinase polyA, reverse primer"
polyA_signal 5282..5329
/label=HSV TK poly(A) signal
/note="herpes simplex virus thymidine kinase
polyadenylation signal (Cole and Stacy, 1985)"
rep_origin 5658..6246
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 6147..6166
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
ORIGIN
1 gccatgcatt agttattaat agtaatcaat tacggggtca ttagttcata gcccatatat
61 ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc
121 ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca
181 ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta
241 tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta
301 tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat
361 cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat agcggtttga
421 ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt tttggcacca
481 aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg
541 taggcgtgta cggtgggagg tctatataag cagagctggt ttagtgaacc gtcagatccg
601 ctagcgctac cggtcgccac catggtgagc aagggcgagg agctgttcac cggggtggtg
661 cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt gtccggcgag
721 ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac caccggcaag
781 ctgcccgtgc cctggcccac cctcgtgacc accctgacct acggcgtgca gtgcttcagc
841 cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac
901 gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg cgccgaggtg
961 aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag
1021 gacggcaaca tcctggggca caagctggag tacaactaca acagccacaa cgtctatatc
1081 atggccgaca agcagaagaa cggcatcaag gtgaacttca agatccgcca caacatcgag
1141 gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg cgacggcccc
1201 gtgctgctgc ccgacaacca ctacctgagc acccagtccg ccctgagcaa agaccccaac
1261 gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat cactctcggc
1321 atggacgagc tgtacaagtc cggactcaga tctcgagctc aagcttcgaa ttctgcagtc
1381 gacggtggta gtgcttccgg aatgcacata actaatttgg gattgcatca ggtttcattt
1441 caaagcggag attcctataa aggcgcagaa gaaaccggaa agcacaaagg tgtaagcgtg
1501 atttcatatc aaagagttaa aaatggagaa agaaataagg ggatcgaagc gcttaatcga
1561 ttatatttac agaaccaaac aagcttaaca ggaaaaagcc ttttgtttgc gcgtgacaaa
1621 gctgaagttt tttgcgaggc aataaagttg gcaggtggtg atacgtcaaa aattaaagcc
1681 atgatggaac gattagatac ctataaactt ggtgaagtta ataaaagaca tattaatgag
1741 cttaataaag taataagtga agagataaga gcacagctag gcattaaaaa taaaaaggaa
1801 ttgcaaacga aaataaaaca aatatttact gattatttaa ataataagaa ttggggaccg
1861 gtcaacaaga atattagtca tcatgggaaa aattatagtt ttcaattaac tcctgcctct
1921 catatgaaga tagggaataa aaatatattt gtcaaagagt ataatggaaa aggaatttgt
1981 tgtgcttcta cgagagagcg tgatcatatc gcgaatatgt ggctttcaaa agtggtggat
2041 gacgagggga aggaaatttt ttcgggtatc agacatggag ttatatcagc ctatggattg
2101 aaaaaaaact catctgaaag agctgttgct gctcgtaata aagctgagga gttagtaagc
2161 gcagcattat atagcagacc agagttatta tcacaggctt tgtctggtaa aacagtagat
2221 ttaaagattg tttcgacttc tcttctgacg ccgaccagtt taaccggggg agaggagagt
2281 atgttaaaag atcaggtgag cgcattaaaa gggttaaact ctaaaagggg ggggccaacg
2341 aaattattaa ttcggaatag tgatggtctt ctgaaagaag taagcgttaa tctaaaagtg
2401 gtgacattta actttggtgt aaatgaatta gcgctaaaaa tgggcttagg ctggaggaat
2461 gttgacaagc ttaatgatga atcaatatgt tctttgttgg gggataattt cctcaaaaat
2521 ggtgtgattg gaggctgggc tgcagaagca atagaaaaaa atccaccatg taagaatgat
2581 gtgatatatc tggctaacca gataaaagag attgtaaaca ataagttaca aaaaaatgat
2641 aatggagagc cgtataaatt gtcacaaaga gtgactctat tggcttatac tataggtgct
2701 gtaccttgct ggaattgtaa gagtgggaag gacagaacag gtatgcagga tgctgaaatt
2761 aaaagagaaa taataagaaa acatgaaaca ggtcagtttt ctcaattaaa tagtaaatta
2821 tcctcagaag agaaaagatt attttctact attctaatga atagtggtaa tatggaaatc
2881 caagagatga atactggtgt gcccggaaat aaagtcatga aaaaattgcc gctatcttcg
2941 ttagagctat cttattctga aagaataggg gacccaaaaa tatggaatat ggtgaaaggg
3001 tattcgtcat ttgtataagg atccaccgga tctagataac tgatcataat cagccatacc
3061 acatttgtag aggttttact tgctttaaaa aacctcccac acctccccct gaacctgaaa
3121 cataaaatga atgcaattgt tgttgttaac ttgtttattg cagcttataa tggttacaaa
3181 taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt
3241 ggtttgtcca aactcatcaa tgtatcttaa cgcgtaaatt gtaagcgtta atattttgtt
3301 aaaattcgcg ttaaattttt gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg
3361 caaaatccct tataaatcaa aagaatagac cgagataggg ttgagtgttg ttccagtttg
3421 gaacaagagt ccactattaa agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta
3481 tcagggcgat ggcccactac gtgaaccatc accctaatca agttttttgg ggtcgaggtg
3541 ccgtaaagca ctaaatcgga accctaaagg gagcccccga tttagagctt gacggggaaa
3601 gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct
3661 ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta atgcgccgct
3721 acagggcgcg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt
3781 tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca
3841 ataatattga aaaaggaaga gtcctgaggc ggaaagaacc agctgtggaa tgtgtgtcag
3901 ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
3961 aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
4021 agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc
4081 ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat
4141 gcagaggccg aggccgcctc ggcctctgag ctattccaga agtagtgagg aggctttttt
4201 ggaggcctag gcttttgcaa agatcgatca agagacagga tgaggatcgt ttcgcatgat
4261 tgaacaagat ggattgcacg caggttctcc ggccgcttgg gtggagaggc tattcggcta
4321 tgactgggca caacagacaa tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca
4381 ggggcgcccg gttctttttg tcaagaccga cctgtccggt gccctgaatg aactgcaaga
4441 cgaggcagcg cggctatcgt ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga
4501 cgttgtcact gaagcgggaa gggactggct gctattgggc gaagtgccgg ggcaggatct
4561 cctgtcatct caccttgctc ctgccgagaa agtatccatc atggctgatg caatgcggcg
4621 gctgcatacg cttgatccgg ctacctgccc attcgaccac caagcgaaac atcgcatcga
4681 gcgagcacgt actcggatgg aagccggtct tgtcgatcag gatgatctgg acgaagagca
4741 tcaggggctc gcgccagccg aactgttcgc caggctcaag gcgagcatgc ccgacggcga
4801 ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat atcatggtgg aaaatggccg
4861 cttttctgga ttcatcgact gtggccggct gggtgtggcg gaccgctatc aggacatagc
4921 gttggctacc cgtgatattg ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt
4981 gctttacggt atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga
5041 gttcttctga gcgggactct ggggttcgaa atgaccgacc aagcgacgcc caacctgcca
5101 tcacgagatt tcgattccac cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc
5161 cgggacgccg gctggatgat cctccagcgc ggggatctca tgctggagtt cttcgcccac
5221 cctaggggga ggctaactga aacacggaag gagacaatac cggaaggaac ccgcgctatg
5281 acggcaataa aaagacagaa taaaacgcac ggtgttgggt cgtttgttca taaacgcggg
5341 gttcggtccc agggctggca ctctgtcgat accccaccga gaccccattg gggccaatac
5401 gcccgcgttt cttccttttc cccaccccac cccccaagtt cgggtgaagg cccagggctc
5461 gcagccaacg tcggggcggc aggccctgcc atagcctcag gttactcata tatactttag
5521 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat
5581 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
5641 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
5701 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
5761 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg
5821 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
5881 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
5941 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
6001 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
6061 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
6121 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
6181 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
6241 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
6301 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac c
//
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