| 质粒名称: | pcDNA3-TORCAR |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 64927 |
| 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: | Invitrogen |
| 空载体大小: | 5446 bp |
| 完整质粒大小: | 7225 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: | 氨苄青霉素，100μg/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: | TORCAR |
| 物种来源: | H. sapiens (human), Synthetic |
| 插入大小: | 1803 bp |
| 基因突变: | -- |
| 启动子: | CMV |
| 标签/融合蛋白: | Cerulean（插入件上的N端子）YPET（插入式C端子） |
| 克隆方法: | 限制性内切酶 |
| 5’克隆位点: | BamHI(未破坏) |
| 3’克隆位点: | EcoRI (未破坏) |
| 5’测序引物 | T7 forward |
| 3’测序引物 | BGH reverse |
|
文献引用方法 |
|
| 材料和方法部分: | pcDNA3-TORCAR was a gift from Jin Zhang (Addgene plasmid # 64927 ; http://n2t.net/addgene:64927 ; RRID:Addgene_64927) |
| 参考文献部分: | Dynamic Visualization of mTORC1 Activity in Living Cells. Zhou X, Clister TL, Lowry PR, Seldin MM, Wong GW, Zhang J. Cell Rep. 2015 Mar 11. pii: S2211-1247(15)00177-1. doi: 10.1016/j.celrep.2015.02.031. 10.1016/j.celrep.2015.02.031 PubMed 25772363 |
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-TORCAR 7224 bp ds-DNA circular SYN 12-MAY-2021
DEFINITION FRET-based biosensor for monitoring mTORC1 kinase activity..
ACCESSION .
VERSION .
KEYWORDS pcDNA3-TORCAR
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7224)
AUTHORS Zhou X, Clister TL, Lowry PR, Seldin MM, Wong GW, Zhang J
TITLE Dynamic Visualization of mTORC1 Activity in Living Cells.
JOURNAL Cell Rep. 2015 Mar 11. pii: S2211-1247(15)00177-1. doi:
10.1016/j.celrep.2015.02.031.
PUBMED 25772363
REFERENCE 2 (bases 1 to 7224)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58
http://www.biofeng.com
FEATURES Location/Qualifiers
source 1..7224
/organism="synthetic DNA construct"
/mol_type="other DNA"
primer_bind complement(54..73)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
enhancer 245..624
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 625..828
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
primer_bind 779..799
/label=CMV-F
/note="Human CMV immediate early promoter, forward primer"
primer_bind 873..892
/label=T7
/note="T7 promoter, forward primer"
promoter 873..891
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
CDS 924..1640
/codon_start=1
/product="enhanced monomeric variant of CFP (Rizzo et al.,
2004)"
/label=mCerulean
/note=" mammalian codon-optimized"
/translation="MVSKGEELFTGVVPILVELDGDVNGHRFSVSGEGEGDATYGKLTL
KFICTTGKLPVPWPTLVTTLTWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNAISDNVYITADKQKNGIK
AHFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
EFVTAAGITLGMDELYK"
primer_bind complement(969..990)
/label=EGFP-N
/note="EGFP, reverse primer"
primer_bind complement(1230..1249)
/label=EXFP-R
/note="For distinguishing EGFP variants, reverse primer"
primer_bind 1577..1598
/label=EGFP-C
/note="EGFP, forward primer"
CDS 2010..2720
/codon_start=1
/product="yellow fluorescent protein optimized for FRET
with CyPet (Nguyen and Daugherty, 2005)"
/label=YPet
/translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKL
LCTTGKLPVPWPTLVTTLGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGN
YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIKAN
FKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALFKDPNEKRDHMVLLEF
LTAAGITEGMNELYK"
promoter complement(2787..2805)
/label=SP6 promoter
/note="promoter for bacteriophage SP6 RNA polymerase"
primer_bind complement(2788..2805)
/label=SP6
/note="SP6 promoter, forward primer"
primer_bind complement(2825..2842)
/label=BGH-rev
/note="Bovine growth hormone terminator, reverse primer.
Also called BGH reverse"
polyA_signal 2831..3055
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
rep_origin 3101..3529
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(3188..3207)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 3398..3419
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind complement(3538..3558)
/label=pBABE 3'
/note="SV40 enhancer, reverse primer for pBABE vectors"
promoter 3543..3872
/label=SV40 promoter
/note="SV40 enhancer and early promoter"
rep_origin 3723..3858
/label=SV40 ori
/note="SV40 origin of replication"
primer_bind 3785..3804
/label=SV40pro-F
/note="SV40 promoter/origin, forward primer"
CDS 3939..4733
/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(3993..4012)
/label=Neo-R
/note="Neomycin resistance gene, reverse primer"
primer_bind 4603..4622
/label=Neo-F
/note="Neomycin resistance gene, forward primer"
polyA_signal 4907..5028
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(4944..4963)
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
primer_bind 4998..5017
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
primer_bind complement(5077..5093)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(5077..5093)
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind complement(5090..5112)
/label=M13/pUC Reverse
/note="In lacZ gene"
protein_bind 5101..5117
/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(5125..5155)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 5170..5191
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(5308..5325)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(5479..6067)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(5559..5578)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS complement(6238..7098)
/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 6861..6880
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
promoter complement(7099..7203)
/gene="bla"
/label=AmpR promoter
ORIGIN
1 acctgacgtc gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc
61 tgctctgatg ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct
121 gagtagtgcg cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg
181 aagaatctgc ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg
241 cgttgacatt gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat
301 agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg
361 cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata
421 gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta
481 catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc
541 gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca gtacatctac
601 gtattagtca tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga
661 tagcggtttg actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg
721 ttttggcacc aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg
781 caaatgggcg gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact
841 agagaaccca ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa
901 gcttggtacc gagctcggat cccatggtga gcaagggcga ggagctgttc accggggtgg
961 tgcccatcct ggtcgagctg gacggcgacg taaacggcca caggttcagc gtgtccggcg
1021 agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca
1081 agctgcccgt gccctggccc accctcgtga ccaccctgac ctggggcgtg cagtgcttcg
1141 cccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct
1201 acgtccagga gcgtaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg
1261 tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg
1321 aggacggcaa catcctgggg cacaagctgg agtacaacgc catcagtgac aacgtctata
1381 tcaccgccga caagcagaag aacggcatca aggcccactt caagatccgc cacaacatcg
1441 aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc
1501 ccgtgctgct gcccgacaac cactacctga gcacccagtc cgccctgagc aaagacccca
1561 acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg atcactctcg
1621 gcatggacga gctgtacaag cgcatgcata tgtccggggg cagcagctgc agccagaccc
1681 caagccgggc catccccgcc actcgccggg tggtgctcgg cgacggcgtg cagctcccgc
1741 ccggggacta cagcacgacc cccggcggca cgctcttcag caccaccccg ggaggtacca
1801 ggatcatcta tgaccggaaa ttcctgatgg agtgtcggaa ctcacctgtg accaaaacac
1861 ccccaaggga tctgcccacc attccggggg tcaccagccc ttccagtgat gagcccccca
1921 tggaagccag ccagagccac ctgcgcaata gcccagaaga taagcgggcg ggcggtgaag
1981 agtcacagtt tgagatggac attgagctct ctaaaggtga agaattattc actggtgttg
2041 tcccaatttt ggttgaatta gatggtgatg ttaatggtca caaattttct gtctccggtg
2101 aaggtgaagg tgatgctacg tacggtaaat tgaccttaaa attactctgt actactggta
2161 aattgccagt tccatggcca accttagtca ctactttagg ttatggtgtt caatgttttg
2221 ctagataccc agatcatatg aaacaacatg actttttcaa gtctgccatg ccagaaggtt
2281 atgttcaaga aagaactatt tttttcaaag atgacggtaa ctacaagacc agagctgaag
2341 tcaagtttga aggtgatacc ttagttaata gaatcgaatt aaaaggtatt gattttaaag
2401 aagatggtaa cattttaggt cacaaattgg aatacaacta taactctcac aatgtttaca
2461 tcactgctga caaacaaaag aatggtatca aagctaactt caaaattaga cacaacattg
2521 aagatggtgg tgttcaatta gctgaccatt atcaacaaaa tactccaatt ggtgatggtc
2581 cagtcttgtt accagacaac cattacttat cctatcaatc tgccttattc aaagatccaa
2641 acgaaaagag agaccacatg gtcttgttag aatttttgac tgctgctggt attaccgagg
2701 gtatgaatga attgtacaaa tagtaagaat tctgcagata tccatcacac tggcggccgc
2761 tcgagcatgc atctagaggg ccctattcta tagtgtcacc taaatgctag agctcgctga
2821 tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct
2881 tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca
2941 tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag
3001 ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc tatggcttct
3061 gaggcggaaa gaaccagctg gggctctagg gggtatcccc acgcgccctg tagcggcgca
3121 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta
3181 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
3241 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
3301 cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt
3361 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga
3421 acaacactca accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg
3481 gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaatta attctgtgga
3541 atgtgtgtca gttagggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa
3601 gcatgcatct caattagtca gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca
3661 gaagtatgca aagcatgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc
3721 ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt
3781 tttttattta tgcagaggcc gaggccgcct ctgcctctga gctattccag aagtagtgag
3841 gaggcttttt tggaggccta ggcttttgca aaaagctccc gggagcttgt atatccattt
3901 tcggatctga tcaagagaca ggatgaggat cgtttcgcat gattgaacaa gatggattgc
3961 acgcaggttc tccggccgct tgggtggaga ggctattcgg ctatgactgg gcacaacaga
4021 caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt
4081 ttgtcaagac cgacctgtcc ggtgccctga atgaactgca ggacgaggca gcgcggctat
4141 cgtggctggc cacgacgggc gttccttgcg cagctgtgct cgacgttgtc actgaagcgg
4201 gaagggactg gctgctattg ggcgaagtgc cggggcagga tctcctgtca tctcaccttg
4261 ctcctgccga gaaagtatcc atcatggctg atgcaatgcg gcggctgcat acgcttgatc
4321 cggctacctg cccattcgac caccaagcga aacatcgcat cgagcgagca cgtactcgga
4381 tggaagccgg tcttgtcgat caggatgatc tggacgaaga gcatcagggg ctcgcgccag
4441 ccgaactgtt cgccaggctc aaggcgcgca tgcccgacgg cgaggatctc gtcgtgaccc
4501 atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct ggattcatcg
4561 actgtggccg gctgggtgtg gcggaccgct atcaggacat agcgttggct acccgtgata
4621 ttgctgaaga gcttggcggc gaatgggctg accgcttcct cgtgctttac ggtatcgccg
4681 ctcccgattc gcagcgcatc gccttctatc gccttcttga cgagttcttc tgagcgggac
4741 tctggggttc gaaatgaccg accaagcgac gcccaacctg ccatcacgag atttcgattc
4801 caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg ccggctggat
4861 gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccccaact tgtttattgc
4921 agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt
4981 ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctgtat
5041 accgtcgacc tctagctaga gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa
5101 ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg
5161 gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca
5221 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
5281 tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg
5341 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
5401 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
5461 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
5521 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
5581 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
5641 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
5701 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
5761 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
5821 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
5881 gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc
5941 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
6001 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
6061 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
6121 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
6181 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
6241 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
6301 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
6361 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca
6421 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
6481 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
6541 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
6601 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
6661 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
6721 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
6781 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
6841 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
6901 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
6961 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
7021 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
7081 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta
7141 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
7201 gcgcacattt ccccgaaaag tgcc
//
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