| 质粒名称: | NLSx3-CRY2PHR-p65 |
|---|---|
| 出品公司: | 爱迪基因 |
| 目录编号: | 64124 |
| 存储实验室: | Moritoshi Sato |
|
载体骨架基本信息 |
|
| 载体名称: | pcDNA3.1/V5-His A |
| 载体出品公司: | Invitrogen |
| 载体抗性: | 氨苄青霉素 |
| 空载体大小: | 5500 bp |
| 完整质粒大小: | -- |
| 载体修饰: | -- |
| 载体类型: | 哺乳细胞表达 |
| 筛选标记: | 新霉素（G418） |
| 高拷贝/低拷贝: | 高拷贝 |
| 生长条件: | 37 ℃ |
|
插入基因信息 |
|
| 插入基因名称: | CRY2PHR-p65 |
| 物种来源: | Synthetic |
| 插入大小: | 2544 bp |
| 基因突变: | -- |
| GenBank编号 | -- |
| 启动子 | CMV |
| 标签/融合蛋白: |
NLSx3（插入件上的N端子）
V5（主干上的C终端）
His（主干上的C终端）
|
| 克隆方法: | 限制性内切酶 |
| 5' 克隆位点: | HindIII (未破坏) |
| 3' 克隆位点: | EcoRI(未破坏) |
| 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: | NLSx3-CRY2PHR-p65 was a gift from Moritoshi Sato (Addgene plasmid # 64124 ; http://n2t.net/addgene:64124 ; RRID:Addgene_64124) |
| 参考文献部分: | CRISPR-Cas9-based Photoactivatable Transcription System. Nihongaki Y, Yamamoto S, Kawano F, Suzuki H, Sato M. Chem Biol. 2015 Feb 19;22(2):169-74. doi: 10.1016/j.chembiol.2014.12.011. Epub 2015 Jan 22. 10.1016/j.chembiol.2014.12.011 PubMed 25619936 |
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 NLSx3-CRY2PHR-p65 8026 bp ds-DNA circular SYN 06-AUG-2018
DEFINITION Photoactivatable transcription system. Expression of activator
probe, which includes the photolyase homology region of CRY2
(CRY2PHR) and the transcriptional activator domain..
ACCESSION .
VERSION .
KEYWORDS NLSx3-CRY2PHR-p65
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 8026)
AUTHORS Nihongaki Y, Yamamoto S, Kawano F, Suzuki H, Sato M
TITLE CRISPR-Cas9-based Photoactivatable Transcription System.
JOURNAL Chem Biol. 2015 Feb 19;22(2):169-74. doi:
10.1016/j.chembiol.2014.12.011. Epub 2015 Jan 22.
PUBMED 25619936
REFERENCE 2 (bases 1 to 8026)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Aug 6, 2018 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..8026
/organism="synthetic DNA construct"
/mol_type="other DNA"
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 908..917
/regulatory_class="other"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
CDS 923..943
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 959..979
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 2636..2662
/codon_start=1
/product="HA (human influenza hemagglutinin) epitope tag"
/label=HA
/translation="YPYDVPDYA"
CDS 3092..3463
/codon_start=1
/product="transcriptional activation domain of human RelA,
also known as p65 (O'Shea and Perkins, 2008)"
/label=RelA (p65) AD
/translation="PTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNS
EFQQLLNQGIPVAPHTTEPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGD
EDFSSIADMDFSALLSQISS"
CDS 3524..3565
/codon_start=1
/product="epitope tag from simian virus 5"
/label=V5 tag
/translation="GKPIPNPLLGLDST"
CDS 3575..3592
/codon_start=1
/product="6xHis affinity tag"
/label=6xHis
/translation="HHHHHH"
primer_bind complement(3615..3632)
/label=BGH-rev
/note="Bovine growth hormone terminator, reverse primer.
Also called BGH reverse"
polyA_signal 3621..3845
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
rep_origin 3891..4319
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(3978..3997)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 4188..4209
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind complement(4328..4348)
/label=pBABE 3'
/note="SV40 enhancer, reverse primer for pBABE vectors"
promoter 4333..4662
/label=SV40 promoter
/note="SV40 enhancer and early promoter"
rep_origin 4513..4648
/label=SV40 ori
/note="SV40 origin of replication"
primer_bind 4575..4594
/label=SV40pro-F
/note="SV40 promoter/origin, forward primer"
CDS 4729..5523
/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(4783..4802)
/label=Neo-R
/note="Neomycin resistance gene, reverse primer"
primer_bind 5393..5412
/label=Neo-F
/note="Neomycin resistance gene, forward primer"
polyA_signal 5699..5820
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(5736..5755)
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
primer_bind 5790..5809
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
primer_bind complement(5869..5885)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(5869..5885)
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind complement(5882..5904)
/label=M13/pUC Reverse
/note="In lacZ gene"
protein_bind 5893..5909
/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(5917..5947)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 5962..5983
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(6100..6117)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(6271..6859)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(6351..6370)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS complement(7030..7890)
/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 7653..7672
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
promoter complement(7891..7995)
/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 gctggctagt
901 taagcttgcc accatggcca gccccaagaa gaagcgcaag gtggaagcta gcgcttcccc
961 gaagaaaaag cggaaagtcg aggcctccgc atctccaaaa aaaaaaagca aggttgaagc
1021 atctggatcc ggtaccggag gaagtggcag ctctggcggc agtggagggt ctggtggcag
1081 cggaatgaag atggacaaaa agactatagt ttggtttaga agagacctaa ggattgagga
1141 taatcctgca ttagcagcag ctgctcacga aggatctgtt tttcctgtct tcatttggtg
1201 tcctgaagaa gaaggacagt tttatcctgg aagagcttca agatggtgga tgaaacaatc
1261 acttgctcac ttatctcaat ccttgaaggc tcttggatct gacctcactt taatcaaaac
1321 ccacaacacg atttcagcga tcttggattg tatccgcgtt accggtgcta caaaagtcgt
1381 ctttaaccac ctctatgatc ctgtttcgtt agttcgggac cataccgtaa aggagaagct
1441 ggtggaacgt gggatctctg tgcaaagcta caatggagat ctattgtatg aaccgtggga
1501 gatatactgc gaaaagggca aaccttttac gagtttcaat tcttactgga agaaatgctt
1561 agatatgtcg attgaatccg ttatgcttcc tcctccttgg cggttgatgc caataactgc
1621 agcggctgaa gcgatttggg cgtgttcgat tgaagaacta gggctggaga atgaggccga
1681 gaaaccgagc aatgcgttgt taactagagc ttggtctcca ggatggagca atgctgataa
1741 gttactaaat gagttcatcg agaagcagtt gatagattat gcaaagaaca gcaagaaagt
1801 tgttgggaat tctacttcac tactttctcc gtatctccat ttcggggaaa taagcgtcag
1861 acacgttttc cagtgtgccc ggatgaaaca aattatatgg gcaagagata agaacagtga
1921 aggagaagaa agtgcagatc tttttcttag gggaatcggt ttaagagagt attctcggta
1981 tatatgtttc aacttcccgt ttactcacga gcaatcgttg ttgagtcatc ttcggttttt
2041 cccttgggat gctgatgttg ataagttcaa ggcctggaga caaggcagga ccggttatcc
2101 gttggtggat gccggaatga gagagctttg ggctaccgga tggatgcata acagaataag
2161 agtgattgtt tcaagctttg ctgtgaagtt tcttctcctt ccatggaaat ggggaatgaa
2221 gtatttctgg gatacacttt tggatgctga tttggaatgt gacatccttg gctggcagta
2281 tatctctggg agtatccccg atggccacga gcttgatcgc ttggacaatc ccgcgttaca
2341 aggcgccaaa tatgacccag aaggtgagta cataaggcaa tggcttcccg agcttgcgag
2401 attgccaact gaatggatcc atcatccatg ggacgctcct ttaaccgtac tcaaagcttc
2461 tggtgtggaa ctcggaacaa actatgcgaa acccattgta gacatcgaca cagctcgtga
2521 gctactagct aaagctattt caagaacccg tgaagcacag atcatgatcg gagcagcagg
2581 gggttctggg gggagcggcg ggggatctgg aagtggcagc ggaggcctcg agcagtaccc
2641 ctacgacgtg cccgattacg ccgagttcca gtacctgcca gatacagacg atcgtcaccg
2701 gattgaggag aaacgtaaaa ggacatatga gaccttcaag agcatcatga agaagagtcc
2761 tttcagcgga cccaccgacc cccggcctcc acctcgacgc attgctgtgc cttcccgcag
2821 ctcagcttct gtccccaagc cagcacccca gccctatccc tttacgtcat ccctgagcac
2881 catcaactat gatgagtttc ccaccatggt gtttccttct gggcagatca gccaggcctc
2941 ggccttggcc ccggcccctc cccaagtcct gccccaggct ccagcccctg cccctgctcc
3001 agccatggta tcagctctgg cccaggcccc agcccctgtc ccagtcctag ccccaggccc
3061 tcctcaggct gtggccccac ctgcccccaa gcccacccag gctggggaag gaacgctgtc
3121 agaggccctg ctgcagctgc agtttgatga tgaagacctg ggggccttgc ttggcaacag
3181 cacagaccca gctgtgttca cagacctggc atccgtcgac aactccgagt ttcagcagct
3241 gctgaaccag ggcatacctg tggcccccca cacaactgag cccatgctga tggagtaccc
3301 tgaggctata actcgcctag tgacaggggc ccagaggccc cccgacccag ctcctgctcc
3361 actgggggcc ccggggctcc ccaatggcct cctttcagga gatgaagact tctcctccat
3421 tgcggacatg gacttctcag ccctgctgag tcagatcagc tcctaagaat tctgcagata
3481 tccagcacag tggcggccgc tcgagtctag agggcccttc gaaggtaagc ctatccctaa
3541 ccctctcctc ggtctcgatt ctacgcgtac cggtcatcat caccatcacc attgagttta
3601 aacccgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc
3661 ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga
3721 ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca
3781 ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc
3841 tatggcttct gaggcggaaa gaaccagctg gggctctagg gggtatcccc acgcgccctg
3901 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc
3961 cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg
4021 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg
4081 gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg
4141 atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt
4201 ccaaactgga acaacactca accctatctc ggtctattct tttgatttat aagggatttt
4261 gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaatta
4321 attctgtgga atgtgtgtca gttagggtgt ggaaagtccc caggctcccc agcaggcaga
4381 agtatgcaaa gcatgcatct caattagtca gcaaccaggt gtggaaagtc cccaggctcc
4441 ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat agtcccgccc
4501 ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc
4561 tgactaattt tttttattta tgcagaggcc gaggccgcct ctgcctctga gctattccag
4621 aagtagtgag gaggcttttt tggaggccta ggcttttgca aaaagctccc gggagcttgt
4681 atatccattt tcggatctga tcaagagaca ggatgaggat cgtttcgcat gattgaacaa
4741 gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg ctatgactgg
4801 gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc
4861 ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca ggacgaggca
4921 gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct cgacgttgtc
4981 actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga tctcctgtca
5041 tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg gcggctgcat
5101 acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat cgagcgagca
5161 cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga gcatcagggg
5221 ctcgcgccag ccgaactgtt cgccaggctc aaggcgcgca tgcccgacgg cgaggatctc
5281 gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct
5341 ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat agcgttggct
5401 acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct cgtgctttac
5461 ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga cgagttcttc
5521 tgagcgggac tctggggttc gcgaaatgac cgaccaagcg acgcccaacc tgccatcacg
5581 agatttcgat tccaccgccg ccttctatga aaggttgggc ttcggaatcg ttttccggga
5641 cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg cccaccccaa
5701 cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa
5761 taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta
5821 tcatgtctgt ataccgtcga cctctagcta gagcttggcg taatcatggt catagctgtt
5881 tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa
5941 gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact
6001 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc
6061 ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg
6121 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
6181 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
6241 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
6301 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
6361 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
6421 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
6481 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
6541 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
6601 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
6661 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt
6721 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
6781 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
6841 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
6901 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
6961 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
7021 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
7081 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
7141 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
7201 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
7261 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
7321 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
7381 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
7441 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
7501 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
7561 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
7621 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
7681 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
7741 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
7801 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
7861 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
7921 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
7981 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtc
//
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