| 质粒名称: | EGFP-Rab5A S34N |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 28045 |
| 存储实验室: | Qing Zhong |
|
载体骨架基本信息 |
|
| 载体名称: | pcDNA3 |
| 载体出品公司: | Invitrogen |
| 空载体大小: | 45446bp |
| 完整质粒大小: | 6748bp |
| 载体类型: | 哺乳细胞表达 |
| 筛选标记: | 新霉素（G418） |
| 载体抗性: | 氨苄霉素 |
| 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: | Rab5A |
| 物种来源: | H. sapiens (human) |
| 插入大小: | 647bp |
| 基因突变: | S34N |
| 标签/融合蛋白: | EGFP on N-term |
| 克隆方法: | 限制性内切酶 |
| 5’测序引物 | CMV-F |
| 3’测序引物 | BGH-rev |
|
文献引用方法 |
|
| 材料和方法部分: | EGFP-Rab5A S34N was a gift from Qing Zhong (Addgene plasmid # 28045 ; http://n2t.net/addgene:28045 ; RRID:Addgene_28045) |
| 参考文献部分: | Rubicon controls endosome maturation as a Rab7 effector. Sun Q, Westphal W, Wong KN, Tan I, Zhong Q. Proc Natl Acad Sci U S A. 2010 Nov 9. 107(45):19338-43. 10.1073/pnas.1010554107 PubMed 20974968 |
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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-Rab5A S34N 6748 bp ds-DNA circular SYN 15-JUN-2022
DEFINITION synthetic circular DNA
ACCESSION .
VERSION .
KEYWORDS EGFP-Rab5A S34N
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 6748)
AUTHORS Sun Q, Westphal W, Wong KN, Tan I, Zhong Q
TITLE Rubicon controls endosome maturation as a Rab7 effector.
JOURNAL Proc Natl Acad Sci U S A. 2010 Nov 9. 107(45):19338-43.
PUBMED 20974968
REFERENCE 2 (bases 1 to 6748)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Jun 15, 2022 from SnapGene Server 1.1.58
http://www.biofeng.com
FEATURES Location/Qualifiers
source 1..6748
/organism="synthetic DNA construct"
/mol_type="other DNA"
primer_bind complement(13..33)
/label=pBABE 3'
/note="SV40 enhancer, reverse primer for pBABE vectors"
promoter 18..347
/label=SV40 promoter
/note="SV40 enhancer and early promoter"
rep_origin 198..333
/label=SV40 ori
/note="SV40 origin of replication"
primer_bind 260..279
/label=SV40pro-F
/note="SV40 promoter/origin, forward primer"
CDS 414..1208
/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
GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
primer_bind complement(468..487)
/label=Neo-R
/note="Neomycin resistance gene, reverse primer"
primer_bind 1078..1097
/label=Neo-F
/note="Neomycin resistance gene, forward primer"
polyA_signal 1382..1503
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(1419..1438)
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
primer_bind 1473..1492
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
primer_bind complement(1552..1568)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(1552..1568)
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind complement(1565..1587)
/label=M13/pUC Reverse
/note="In lacZ gene"
protein_bind 1576..1592
/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(1600..1630)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 1645..1666
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(1783..1800)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(1954..2542)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(2034..2053)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS complement(2713..3573)
/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 3336..3355
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
promoter complement(3574..3678)
/gene="bla"
/label=AmpR promoter
primer_bind complement(3753..3772)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
enhancer 3944..4323
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 4324..4527
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
primer_bind 4478..4498
/label=CMV-F
/note="Human CMV immediate early promoter, forward primer"
primer_bind 4572..4591
/label=T7
/note="T7 promoter, forward primer"
promoter 4572..4590
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
CDS 4604..5320
/codon_start=1
/product="enhanced GFP"
/label=EGFP
/translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
EFVTAAGITLGMDELYK"
CDS 4604..5320
/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(4649..4670)
/label=EGFP-N
/note="EGFP, reverse primer"
primer_bind complement(4910..4929)
/label=EXFP-R
/note="For distinguishing EGFP variants, reverse primer"
primer_bind 5257..5278
/label=EGFP-C
/note="EGFP, forward primer"
promoter complement(6010..6028)
/label=SP6 promoter
/note="promoter for bacteriophage SP6 RNA polymerase"
primer_bind complement(6011..6028)
/label=SP6
/note="SP6 promoter, forward primer"
primer_bind complement(6048..6065)
/label=BGH-rev
/note="Bovine growth hormone terminator, reverse primer.
Also called BGH reverse"
polyA_signal 6054..6278
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
rep_origin 6324..4
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(6411..6430)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 6621..6642
/label=F1ori-F
/note="F1 origin, forward primer"
ORIGIN
1 aattaattct gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag
61 gcagaagtat gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag
121 gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc
181 cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc
241 atggctgact aatttttttt atttatgcag aggccgaggc cgcctctgcc tctgagctat
301 tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag ctcccgggag
361 cttgtatatc cattttcgga tctgatcaag agacaggatg aggatcgttt cgcatgattg
421 aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg
481 actgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg
541 ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaggacg
601 aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg
661 ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc
721 tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc
781 tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc
841 gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc
901 aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gcgcatgccc gacggcgagg
961 atctcgtcgt gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct
1021 tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt
1081 tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc
1141 tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt
1201 tcttctgagc gggactctgg ggttcgaaat gaccgaccaa gcgacgccca acctgccatc
1261 acgagatttc gattccaccg ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg
1321 ggacgccggc tggatgatcc tccagcgcgg ggatctcatg ctggagttct tcgcccaccc
1381 caacttgttt attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac
1441 aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc
1501 ttatcatgtc tgtataccgt cgacctctag ctagagcttg gcgtaatcat ggtcatagct
1561 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat
1621 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc
1681 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
1741 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct
1801 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
1861 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
1921 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
1981 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
2041 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
2101 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
2161 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
2221 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
2281 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
2341 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
2401 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
2461 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
2521 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
2581 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
2641 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
2701 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
2761 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
2821 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
2881 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
2941 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
3001 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
3061 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
3121 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
3181 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
3241 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
3301 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
3361 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
3421 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
3481 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
3541 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
3601 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
3661 acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtcg acggatcggg
3721 agatctcccg atcccctatg gtgcactctc agtacaatct gctctgatgc cgcatagtta
3781 agccagtatc tgctccctgc ttgtgtgttg gaggtcgctg agtagtgcgc gagcaaaatt
3841 taagctacaa caaggcaagg cttgaccgac aattgcatga agaatctgct tagggttagg
3901 cgttttgcgc tgcttcgcga tgtacgggcc agatatacgc gttgacattg attattgact
3961 agttattaat agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc
4021 gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg
4081 acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa
4141 tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca
4201 agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac
4261 atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc
4321 atggtgatgc ggttttggca gtacatcaat gggcgtggat agcggtttga ctcacgggga
4381 tttccaagtc tccaccccat tgacgtcaat gggagtttgt tttggcacca aaatcaacgg
4441 gactttccaa aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg taggcgtgta
4501 cggtgggagg tctatataag cagagctctc tggctaacta gagaacccac tgcttactgg
4561 cttatcgaaa ttaatacgac tcactatagg gagacccaag cttatggtga gcaagggcga
4621 ggagctgttc accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca
4681 caagttcagc gtgtccggcg agggcgaggg cgatgccacc tacggcaagc tgaccctgaa
4741 gttcatctgc accaccggca agctgcccgt gccctggccc accctcgtga ccaccctgac
4801 ctacggcgtg cagtgcttca gccgctaccc cgaccacatg aagcagcacg acttcttcaa
4861 gtccgccatg cccgaaggct acgtccagga gcgcaccatc ttcttcaagg acgacggcaa
4921 ctacaagacc cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct
4981 gaagggcatc gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaacta
5041 caacagccac aacgtctata tcatggccga caagcagaag aacggcatca aggtgaactt
5101 caagatccgc cacaacatcg aggacggcag cgtgcagctc gccgaccact accagcagaa
5161 cacccccatc ggcgacggcc ccgtgctgct gcccgacaac cactacctga gcacccagtc
5221 cgccctgagc aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac
5281 cgccgccggg atcactctcg gcatggacga gctgtacaag ggatccatgg ctagtcgagg
5341 cgcaacaaga cccaacgggc caaatactgg aaataaaata tgccagttca aactagtact
5401 tctgggagag tctgctgttg gcaaaaatag cctagtgctt cgttttgtga aaggccaatt
5461 tcatgaattt caagagagta ccattggggc tgcttttcta acccaaactg tatgtcttga
5521 tgacactaca gtaaagtttg aaatatggga tacagctggt caagaacgat accatagcct
5581 agcaccaatg tactacagag gagcacaagc agccatagtt gtatatgata tcacaaatga
5641 ggagtccttt gcaagagcaa aaaattgggt taaagaactt cagaggcaag caagtcctaa
5701 cattgtaata gctttatcgg gaaacaaggc cgacctagca aataaaagag cagtagattt
5761 ccaggaagca cagtcctatg cagatgacaa tagtttatta ttcatggaga catccgctaa
5821 aacatcaatg aatgtaaatg aaatattcat ggcaatagct aaaaaattgc caaagaatga
5881 accacaaaat ccaggagcaa attctgccag aggaagagga gtagacctta ccgaacccac
5941 acaaccaacc aggaatcagt gttgtagtaa ctgaggcggc cgctcgagca tgcatctaga
6001 gggccctatt ctatagtgtc acctaaatgc tagagctcgc tgatcagcct cgactgtgcc
6061 ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg
6121 tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag
6181 gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga
6241 caatagcagg catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag
6301 ctggggctct agggggtatc cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
6361 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
6421 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
6481 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta
6541 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
6601 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat
6661 ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa
6721 tgagctgatt taacaaaaat ttaacgcg
//
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