| 质粒名称: | pcDNA3/Flag-METTL3 |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 53739 |
| 存储实验室: | Chuan He |
|
载体骨架基本信息 |
|
| 载体名称: | pcDNA3 |
| 载体出品公司: | Invitrogen |
| 载体抗性: | 氨苄青霉素 |
| 空载体大小: | 5446 bp |
| 完整质粒大小: | 7125 bp |
| 载体修饰: | -- |
| 载体类型: | 哺乳细胞表达 |
| 筛选标记: | -- |
| 高拷贝/低拷贝: | -- |
| 生长条件: | 37 ℃ |
|
插入基因信息 |
|
| 插入基因名称: | Methyltransferase-like 3,METTL3,MTA-70 |
| 物种来源: | H. sapiens (human) |
| 插入大小: | 1743 bp |
| 基因突变: | -- |
| GenBank编号 | NM_019852.3 |
| 启动子 | T7 |
| 标签/融合蛋白: | FLAG tag（插入N端子） |
| 克隆方法: | 限制性内切酶 |
| 5' 克隆位点: | Hind III(未破坏) |
| 3' 克隆位点: | XbaI(未破坏) |
| 5' 测序引物 | 5' TAATACGACTCACTATAGGG 3' |
| 3' 测序引物 | 5' ATTTAGGTGACACTATAG 3' |
|
文献引用方法 |
|
| 材料和方法部分: | pcDNA3/Flag-METTL3 was a gift from Chuan He (Addgene plasmid # 53739 ; http://n2t.net/addgene:53739 ; RRID:Addgene_53739) |
| 参考文献部分: | A METTL3-METTL14 complex mediates mammalian nuclear RNA N6-adenosine methylation. Liu J, Yue Y, Han D, Wang X, Fu Y, Zhang L, Jia G, Yu M, Lu Z, Deng X, Dai Q, Chen W, He C. Nat Chem Biol. 2014 Feb;10(2):93-5. doi: 10.1038/nchembio.1432. Epub 2013 Dec 6. 10.1038/nchembio.1432 PubMed 24316715 |
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 pcDNA3/Flag-METTL3 7052 bp ds-DNA circular SYN 06-AUG-2018
DEFINITION For expression of METTL3 (methyltransferase-like 3) in human cells.
ACCESSION .
VERSION .
KEYWORDS pcDNA3/Flag-METTL3
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7052)
AUTHORS Liu J, Yue Y, Han D, Wang X, Fu Y, Zhang L, Jia G, Yu M, Lu Z, Deng
X, Dai Q, Chen W, He C
TITLE A METTL3-METTL14 complex mediates mammalian nuclear RNA N6-adenosine
methylation.
JOURNAL Nat Chem Biol. 2014 Feb;10(2):93-5. doi: 10.1038/nchembio.1432. Epub
2013 Dec 6.
PUBMED 24316715
REFERENCE 2 (bases 1 to 7052)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Aug 6, 2018 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..7052
/organism="synthetic DNA construct"
/mol_type="other DNA"
primer_bind complement(32..51)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
enhancer 223..602
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 603..806
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
primer_bind 757..777
/label=CMV-F
/note="Human CMV immediate early promoter, forward primer"
primer_bind 851..870
/label=T7
/note="T7 promoter, forward primer"
promoter 851..869
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
CDS 886..909
/codon_start=1
/product="FLAG(R) epitope tag, followed by an enterokinase
cleavage site"
/label=FLAG
/translation="DYKDDDDK"
promoter complement(2665..2683)
/label=SP6 promoter
/note="promoter for bacteriophage SP6 RNA polymerase"
primer_bind complement(2666..2683)
/label=SP6
/note="SP6 promoter, forward primer"
primer_bind complement(2703..2720)
/label=BGH-rev
/note="Bovine growth hormone terminator, reverse primer.
Also called BGH reverse"
polyA_signal 2709..2933
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
rep_origin 2979..3407
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(3066..3085)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 3276..3297
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind complement(3416..3436)
/label=pBABE 3'
/note="SV40 enhancer, reverse primer for pBABE vectors"
promoter 3460..3670
/label=SV40 promoter
/note="SV40 early promoter"
promoter 3482..3678
/label=SV40 promoter
/note="SV40 early promoter"
rep_origin 3529..3664
/label=SV40 ori
/note="SV40 origin of replication"
primer_bind 3591..3610
/label=SV40pro-F
/note="SV40 promoter/origin, forward primer"
CDS 3745..4539
/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(3799..3818)
/label=Neo-R
/note="Neomycin resistance gene, reverse primer"
primer_bind 4409..4428
/label=Neo-F
/note="Neomycin resistance gene, forward primer"
polyA_signal 4713..4834
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(4750..4769)
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
primer_bind 4804..4823
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
primer_bind complement(4883..4899)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(4883..4899)
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind complement(4896..4918)
/label=M13/pUC Reverse
/note="In lacZ gene"
protein_bind 4907..4923
/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(4931..4961)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 4976..4997
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(5114..5131)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(5285..5873)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(5365..5384)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS complement(6044..6904)
/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 6667..6686
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
promoter complement(6905..7009)
/gene="bla"
/label=AmpR promoter
ORIGIN
1 gatctcccga tcccctatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa
61 gccagtatct gctccctgct tgtgtgttgg aggtcgctga gtagtgcgcg agcaaaattt
121 aagctacaac aaggcaaggc ttgaccgaca attgcatgaa gaatctgctt agggttaggc
181 gttttgcgct gcttcgcgat gtacgggcca gatatacgcg ttgacattga ttattgacta
241 gttattaata gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg
301 ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga
361 cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat
421 gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa
481 gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca
541 tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc gctattacca
601 tggtgatgcg gttttggcag tacatcaatg ggcgtggata gcggtttgac tcacggggat
661 ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg
721 actttccaaa atgtcgtaac aactccgccc cattgacgca aatgggcggt aggcgtgtac
781 ggtgggaggt ctatataagc agagctctct ggctaactag agaacccact gcttactggc
841 ttatcgaaat taatacgact cactataggg agacccaagc ttatggatta caaggatgac
901 gatgacaagt cggacacgtg gagctctatc caggcccaca agaagcagct ggactctctg
961 cgggagaggc tgcagcggag gcggaagcag gactcggggc acttggatct acggaatcca
1021 gaggcagcat tgtctccaac cttccgtagt gacagcccag tgcctactgc acccacctct
1081 ggtggcccta agcccagcac agcttcagca gttcctgaat tagctacaga tcctgagtta
1141 gagaagaagt tgctacacca cctctctgat ctggccttaa cattgcccac tgatgctgtg
1201 tccatctgtc ttgccatctc cacgccagat gctcctgcca ctcaagatgg ggtagaaagc
1261 ctcctgcaga agtttgcagc tcaggagttg attgaggtaa agcgaggtct cctacaagat
1321 gatgcacatc ctactcttgt aacctatgct gaccattcca agctctctgc catgatgggt
1381 gctgtggcag aaaagaaggg ccctggggag gtagcaggga ctgtcacagg gcagaagcgg
1441 cgtgcagaac aggactcgac tacagtagct gcctttgcca gttcgttagt ctctggtctg
1501 aactcttcag catcggaacc agcaaaggag ccagccaaga aatcaaggaa acatgctgcc
1561 tcagatgttg atctggagat agagagcctt ctgaaccaac agtccactaa ggaacaacag
1621 agcaagaagg tcagtcagga gatcctagag ctattaaata ctacaacagc caaggaacaa
1681 tccattgttg aaaaatttcg ctctcgaggt cgggcccaag tgcaagaatt ctgtgactat
1741 ggaaccaagg aggagtgcat gaaagccagt gatgctgatc gaccctgtcg caagctgcac
1801 ttcagacgaa ttatcaataa acacactgat gagtctttag gtgactgctc tttccttaat
1861 acatgtttcc acatggatac ctgcaagtat gttcactatg aaattgatgc ttgcatggat
1921 tctgaggccc ctggcagcaa agaccacacg ccaagccagg agcttgctct tacacagagt
1981 gtcggaggtg attccagtgc agaccgactc ttcccacctc agtggatctg ttgtgatatc
2041 cgctacctgg acgtcagtat cttgggcaag tttgcagttg tgatggctga cccaccctgg
2101 gatattcaca tggaactgcc ctatgggacc ctgacagatg atgagatgcg caggctcaac
2161 atacccgtac tacaggatga tggctttctc ttcctctggg tcacaggcag ggccatggag
2221 ttggggagag aatgtctaaa tctctggggg tatgaacggg tagatgaaat tatttgggtg
2281 aagacaaatc aactgcaacg catcattcgg acaggccgta caggtcactg gttgaaccat
2341 gggaaggaac actgcttggt tggtgtcaaa ggaaatcccc aaggcttcaa ccagggtctg
2401 gattgtgatg tgatcgtagc tgaggttcgt tccaccagtc ataaaccaga tgaaatctat
2461 ggcatgattg aaagactatc tcctggcact cgcaagattg agttatttgg acgaccacac
2521 aatgtgcaac ccaactggat cacccttgga aaccaactgg atgggatcca cctactagac
2581 ccagatgtgg ttgcacggtt caagcaaagg tacccagatg gtatcatctc taaacctaag
2641 aatttatagt ctagagggcc ctattctata gtgtcaccta aatgctagag ctcgctgatc
2701 agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc
2761 cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc
2821 gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg
2881 ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga
2941 ggcggaaaga accagctggg gctctagggg gtatccccac gcgccctgta gcggcgcatt
3001 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
3061 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
3121 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
3181 caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
3241 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
3301 aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc
3361 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaattaat tctgtggaat
3421 gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
3481 atgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta
3541 actccgccca gttccgccca ttctccgccc catggctgac taattttttt tatttatgca
3601 gaggccgagg ccgcctctgc ctctgagcta ttccagaagt agtgaggagg cttttttgga
3661 ggcctaggct tttgcaaaaa gctcccggga gcttgtatat ccattttcgg atctgatcaa
3721 gagacaggat gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg
3781 gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat cggctgctct
3841 gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac
3901 ctgtccggtg ccctgaatga actgcaggac gaggcagcgc ggctatcgtg gctggccacg
3961 acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg
4021 ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa
4081 gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca
4141 ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt
4201 gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc
4261 aggctcaagg cgcgcatgcc cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc
4321 ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg
4381 ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt
4441 ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag
4501 cgcatcgcct tctatcgcct tcttgacgag ttcttctgag cgggactctg gggttcgaaa
4561 tgaccgacca agcgacgccc aacctgccat cacgagattt cgattccacc gccgccttct
4621 atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg
4681 gggatctcat gctggagttc ttcgcccacc ccaacttgtt tattgcagct tataatggtt
4741 acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta
4801 gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctgtataccg tcgacctcta
4861 gctagagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca
4921 caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag
4981 tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
5041 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
5101 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg
5161 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
5221 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
5281 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
5341 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
5401 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
5461 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
5521 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
5581 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
5641 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
5701 ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag
5761 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
5821 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
5881 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
5941 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
6001 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
6061 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
6121 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
6181 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
6241 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
6301 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta
6361 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
6421 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
6481 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
6541 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
6601 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
6661 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
6721 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
6781 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
6841 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
6901 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
6961 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
7021 gaaaagtgcc acctgacgtc gacggatcgg ga
//
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