| 质粒名称: | pcDNA3-ExRai-AMPKAR |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 192446 |
| 存储实验室: | Jin Zhang |
|
载体骨架基本信息 |
|
| 载体名称: | pcDNA3 |
| 载体出品公司: | Clontech |
| 空载体大小: | 5517bp |
| 完整质粒大小: | 6738bp |
| 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: | ExRai-AMPKAR、AMPKsubstrate-cpEGFP-FHA1 |
| 物种来源: | Synthetic |
| 插入大小: | 1221bp |
| 启动子: | CMV |
| 标签/融合蛋白: | 6xHIS - T7 tag (gene 10 leader) - Xpress (TM) tag (N terminal on backbone) |
| 克隆方法: | 限制性内切酶 |
| 5’克隆位点: | BamHI (not destroyed) |
| 3’克隆位点: | EcoRI (not destroyed) |
| 5’测序引物 | CMV-F |
| 3’测序引物 | BGH reverse |
|
文献引用方法 |
|
| 材料和方法部分: | pcDNA3-ExRai-AMPKAR was a gift from Jin Zhang (Addgene plasmid # 192446 ; http://n2t.net/addgene:192446 ; RRID:Addgene_192446) |
| 参考文献部分: | Spatial regulation of AMPK signaling revealed by a sensitive kinase activity reporter. Schmitt DL, Curtis SD, Lyons AC, Zhang JF, Chen M, He CY, Mehta S, Shaw RJ, Zhang J. Nat Commun. 2022 Jul 5;13(1):3856. doi: 10.1038/s41467-022-31190-x. 10.1038/s41467-022-31190-x PubMed 35790710 |
买家导航
pcDNA3-ExRai-AMPKAR载体质粒基本信息
pcDNA3-ExRai-AMPKAR质粒图谱载体图谱和pcDNA3-ExRai-AMPKAR载体序列质粒序列多克隆位点信息
pcDNA3-ExRai-AMPKAR质粒载体简介
pcDNA3-ExRai-AMPKAR质粒序列载体序列
LOCUS pcDNA3-ExRai-AMPKAR 6737 bp DNA circular SYN 14-APR-2025
DEFINITION Genetically encoded excitation-ratiometric fluorescent biosensor for
monitoring AMPK activity in living cells..
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 6737)
AUTHORS Schmitt DL, Curtis SD, Lyons AC, Zhang JF, Chen M, He CY, Mehta S,
Shaw RJ, Zhang J
TITLE Spatial regulation of AMPK signaling revealed by a sensitive kinase
activity reporter.
JOURNAL Nat Commun. 2022 Jul 5;13(1):3856. doi: 10.1038/s41467-022-31190-x.
PUBMED 35790710
REFERENCE 2 (bases 1 to 6737)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Apr 14, 2025 from SnapGene Server 8.0.1
https://www.biofeng.com
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Nat
Commun."; date: "2022-07-5"; volume: "13(1):3856. doi"; pages: "
10.1038/s41467-022-31190-x"
COMMENT Sequence Label: pcDNA3-ExRai-AMPKAR
FEATURES Location/Qualifiers
source 1..6737
/mol_type="other DNA"
/organism="synthetic DNA construct"
primer_bind complement(44..63)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
enhancer 235..614
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 615..818
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
primer_bind 769..789
/label=CMV-F
/note="Human CMV immediate early promoter, forward primer"
primer_bind 863..882
/label=T7
/note="T7 promoter, forward primer"
promoter 863..881
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
regulatory 901..910
/label=Kozak sequence
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
/regulatory_class="other"
CDS 922..939
/codon_start=1
/product="6xHis affinity tag"
/label=6xHis
/translation="HHHHHH"
CDS 943..975
/codon_start=1
/product="leader peptide from bacteriophage T7 gene 10"
/label=T7 tag (gene 10 leader)
/note="promotes efficient translation in E. coli"
/translation="MASMTGGQQMG"
CDS join(979..987,988..1002)
/codon_start=1
/product="Xpress(TM) epitope tag, including an enterokinase
recognition and cleavage site"
/label=Xpress(TM) tag
/translation="DLYDDDDK"
primer_bind 1278..1299
/label=EGFP-C
/note="EGFP, forward primer"
primer_bind complement(1405..1426)
/label=EGFP-N
/note="EGFP, reverse primer"
primer_bind complement(1666..1685)
/label=EXFP-R
/note="For distinguishing EGFP variants, reverse primer"
promoter complement(2290..2308)
/label=SP6 promoter
/note="promoter for bacteriophage SP6 RNA polymerase"
primer_bind complement(2291..2308)
/label=SP6
/note="SP6 promoter, forward primer"
primer_bind complement(2328..2345)
/label=BGH-rev
/note="Bovine growth hormone terminator, reverse primer.
Also called BGH reverse"
polyA_signal 2334..2558
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
rep_origin 2604..3032
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(2691..2710)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 2901..2922
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind complement(3041..3061)
/label=pBABE 3'
/note="SV40 enhancer, reverse primer for pBABE vectors"
promoter 3046..3375
/label=SV40 promoter
/note="SV40 enhancer and early promoter"
rep_origin 3226..3361
/label=SV40 ori
/note="SV40 origin of replication"
primer_bind 3288..3307
/label=SV40pro-F
/note="SV40 promoter/origin, forward primer"
CDS 3442..4236
/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(3496..3515)
/label=Neo-R
/note="Neomycin resistance gene, reverse primer"
primer_bind 4106..4125
/label=Neo-F
/note="Neomycin resistance gene, forward primer"
polyA_signal 4410..4531
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(4447..4466)
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
primer_bind 4501..4520
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
primer_bind complement(4580..4596)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(4580..4596)
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind complement(4593..4615)
/label=M13/pUC Reverse
/note="In lacZ gene"
protein_bind 4604..4620
/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(join(4628..4634,4635..4652,4653..4658))
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 4673..4694
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(4811..4828)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(4982..5570)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(5062..5081)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS complement(join(5741..6532,6533..6601))
/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 6364..6383
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
promoter complement(6602..6706)
/gene="bla"
/label=AmpR promoter
ORIGIN
1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttgcggcc
901 gccaccatgg tgcggggttc tcatcatcat catcatcatg gtatggctag catgactggt
961 ggacagcaaa tgggtcggga tctgtacgac gatgacgata aggatcccat gaggagagtg
1021 gctaccctgg tggatctggg cggcaccggc ggcagcgagc tcagcttctg caacgtctat
1081 atcaaggccg acaagcagaa gaacggcatc aaggcgaact tcaagatccg ccacaacatc
1141 gaggacggcg gcgtgcagct cgcctaccac taccagcaga acacccccat cggcgacggc
1201 cccgtgctgc tgcccgacaa ccactacctg agcgtgcagt ccaaactttc gaaagacccc
1261 aacgagaagc gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc
1321 ggcatggacg agctgtacaa gggcggtacc ggagggagca tggtgagcaa gggcgaggag
1381 ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag
1441 ttcagcgtgt ccggcgaggg tgagggcgat gccacctacg gcaagctgac cctgaagttc
1501 atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctac
1561 ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc
1621 gccatgcccg aaggctacat ccaggagcgc accatcttct tcaaggacga cggcaactac
1681 aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag
1741 ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta caacttgctg
1801 cataagtttt ctcaagaaca gatcggcgaa aacattgtgt gcagggtcat ttgtaccacg
1861 ggtcaaattc ccatccgaga tttgtcagct gatatttcac aagtgcttaa ggaaaaacga
1921 tccataaaga aagtttggac atttggtaga aacccagcct gtgactatca tttaggaaac
1981 atttcaagac tgtcaaataa gcatttccaa atactactag gagaagacgg taacctttta
2041 ttgaatgaca tttccactaa tgggacctgg ttaaatgggc aaaaagtcga gaagaacagc
2101 aatcagttac tgtctcaagg tgatgaaata accgttggtg taggcgtgga atcagatatt
2161 ttatctctgg tcattttcat aaacgacaaa tttaagcagt gcctcgagca gaacaaagtt
2221 gatcgctaag aattctgcag atatccatca cactggcggc cgctcgagca tgcatctaga
2281 gggccctatt ctatagtgtc acctaaatgc tagagctcgc tgatcagcct cgactgtgcc
2341 ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg
2401 tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag
2461 gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga
2521 caatagcagg catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag
2581 ctggggctct agggggtatc cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
2641 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
2701 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
2761 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta
2821 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
2881 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat
2941 ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa
3001 tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg
3061 tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag
3121 tcagcaacca ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg
3181 catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact
3241 ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag
3301 gccgaggccg cctctgcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc
3361 ctaggctttt gcaaaaagct cccgggagct tgtatatcca ttttcggatc tgatcaagag
3421 acaggatgag gatcgtttcg catgattgaa caagatggat tgcacgcagg ttctccggcc
3481 gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat
3541 gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg
3601 tccggtgccc tgaatgaact gcaggacgag gcagcgcggc tatcgtggct ggccacgacg
3661 ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta
3721 ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta
3781 tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc
3841 gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc
3901 gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg
3961 ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg
4021 ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt
4081 gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc
4141 ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc
4201 atcgccttct atcgccttct tgacgagttc ttctgagcgg gactctgggg ttcgaaatga
4261 ccgaccaagc gacgcccaac ctgccatcac gagatttcga ttccaccgcc gccttctatg
4321 aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg
4381 atctcatgct ggagttcttc gcccacccca acttgtttat tgcagcttat aatggttaca
4441 aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt
4501 gtggtttgtc caaactcatc aatgtatctt atcatgtctg tataccgtcg acctctagct
4561 agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa
4621 ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga
4681 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
4741 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct
4801 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
4861 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
4921 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
4981 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
5041 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
5101 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
5161 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
5221 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
5281 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
5341 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
5401 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
5461 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
5521 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
5581 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
5641 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
5701 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
5761 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
5821 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
5881 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
5941 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
6001 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
6061 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
6121 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
6181 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
6241 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
6301 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
6361 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
6421 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
6481 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
6541 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
6601 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
6661 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
6721 aagtgccacc tgacgtc
//
