| 质粒名称: | pcDNA3.1 iTEV |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 64277 |
| 存储实验室: | Xiaokun Shu |
|
载体骨架基本信息 |
|
| 载体名称: | pcDNA3.1 |
| 空载体大小: | 5360 bp |
| 完整质粒大小: | 7000 bp |
| 载体类型: | 哺乳细胞表达 |
| 载体抗性: | 氨苄霉素,100 μg/mL |
| 生长温度: | 37 ℃ |
| 宿主菌: | Top10 |
|
插入基因信息 |
|
| 插入基因名称: | Infrared fluorescent tobacco etch virus protease sensor (iTEV)，iTEV |
| 物种来源: | Synthetic |
| 插入大小: | 1635 bp |
| 启动子: | CMV |
| 克隆方法: | 限制性内切酶 |
| 5’克隆位点: | HindIII (未破坏) |
| 3’克隆位点: | XbaI (未破坏) |
| 5’测序引物 | TAATACGACTCACTATAGGG |
| 3’测序引物 | TAGAAGGCACAGTCGAGG |
|
文献引用方法 |
|
| 材料和方法部分: | pcDNA3.1 iTEV was a gift from Xiaokun Shu (Addgene plasmid # 64277 ; http://n2t.net/addgene:64277 ; RRID:Addgene_64277) |
| 参考文献部分: | Rationally designed fluorogenic protease reporter visualizes spatiotemporal dynamics of apoptosis in vivo. To TL, Piggott BJ, Makhijani K, Yu D, Jan YN, Shu X. Proc Natl Acad Sci U S A. 2015 Mar 17;112(11):3338-43. doi: 10.1073/pnas.1502857112. Epub 2015 Mar 2. 10.1073/pnas.1502857112 PubMed 25733847 |
买家导航
pcDNA3.1iTEV载体基本信息
pcDNA3.1iTEV载体图谱质粒图谱和多克隆位点信息
pcDNA3.1iTEV载体简介
pcDNA3.1iTEV载体序列
LOCUS pcDNA3.1 iTEV 6998 bp ds-DNA circular SYN 12-MAY-2021
DEFINITION Expresses infrared fluorescent tobacco etch virus protease sensor
(iTEV).
ACCESSION .
VERSION .
KEYWORDS pcDNA3.1 iTEV
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 6998)
AUTHORS To TL, Piggott BJ, Makhijani K, Yu D, Jan YN, Shu X
TITLE Rationally designed fluorogenic protease reporter visualizes
spatiotemporal dynamics of apoptosis in vivo.
JOURNAL Proc Natl Acad Sci U S A. 2015 Mar 17;112(11):3338-43. doi:
10.1073/pnas.1502857112. Epub 2015 Mar 2.
PUBMED 25733847
REFERENCE 2 (bases 1 to 6998)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..6998
/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"
CDS 923..1564
/codon_start=1
/product="fragment of superfolder GFP consisting of the
first 10 beta-strands"
/label=GFP(1-10)
/note="associates with GFP11, the 11th beta-strand, to
reconstitute fluorescent GFP (Cabantous et al., 2005;
Kamiyama et al., 2016)"
/translation="MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATIGKLTLK
FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDG
KYKTRAVVKFEGDTLVNRIELKGTDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
NFTVRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQTVLSKDPNEK"
CDS 2147..2167
/codon_start=1
/product="tobacco etch virus (TEV) protease recognition and
cleavage site"
/label=TEV site
/translation="ENLYFQS"
CDS 2510..2557
/codon_start=1
/product="11th beta-strand of superfolder GFP"
/label=GFP11
/note="associates with GFP(1-10), consisting of the first
10 beta-strands, to reconstitute fluorescent GFP (Cabantous
et al., 2005; Kamiyama et al., 2016)"
/translation="RDHMVLHEYVNAAGIT"
primer_bind complement(2592..2609)
/label=BGH-rev
/note="Bovine growth hormone terminator, reverse primer.
Also called BGH reverse"
polyA_signal 2598..2822
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
rep_origin 2868..3296
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(2955..2974)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 3165..3186
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind complement(3305..3325)
/label=pBABE 3'
/note="SV40 enhancer, reverse primer for pBABE vectors"
promoter 3310..3639
/label=SV40 promoter
/note="SV40 enhancer and early promoter"
rep_origin 3490..3625
/label=SV40 ori
/note="SV40 origin of replication"
primer_bind 3552..3571
/label=SV40pro-F
/note="SV40 promoter/origin, forward primer"
CDS 3706..4500
/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(3760..3779)
/label=Neo-R
/note="Neomycin resistance gene, reverse primer"
primer_bind 4370..4389
/label=Neo-F
/note="Neomycin resistance gene, forward primer"
polyA_signal 4674..4795
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(4711..4730)
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
primer_bind 4765..4784
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
primer_bind complement(4844..4860)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(4844..4860)
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind complement(4857..4879)
/label=M13/pUC Reverse
/note="In lacZ gene"
protein_bind 4868..4884
/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(4892..4922)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 4937..4958
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(5075..5092)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(5246..5831)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(5326..5345)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS complement(6002..6862)
/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 6625..6644
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
promoter complement(6863..6967)
/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 gctggctagc
901 gtttaaactt aagcttgcca ccatgtccaa aggagaagaa ctgtttaccg gtgttgtgcc
961 aattttggtt gaactcgatg gtgatgtcaa cggacataag ttctcagtga gaggcgaagg
1021 agaaggtgac gccaccattg gaaaattgac tcttaaattc atctgtacta ctggtaaact
1081 tcctgtacca tggccgactc tcgtaacaac gcttacgtac ggagttcagt gcttttcgag
1141 atacccagac catatgaaaa gacatgactt ttttaagtcg gctatgcctg aaggttacgt
1201 gcaagaaaga acaatttcgt tcaaagatga tggaaaatat aaaactagag cagttgttaa
1261 atttgaagga gatactttgg ttaaccgcat tgaactgaaa ggaacagatt ttaaagaaga
1321 tggtaatatt cttggacaca aactcgaata caattttaat agtcataacg tatacatcac
1381 tgctgataag caaaagaacg gaattaaagc gaatttcaca gtacgccata atgtagaaga
1441 tggcagtgtt caacttgccg accattacca acaaaacacc cctattggag acggtccggt
1501 acttcttcct gataatcact acctctcaac acaaacagtc ctgagcaaag atccaaatga
1561 aaaaggaggc agcggatcca ccactaacat tgcgccggct ctggacggtg cgtttcatcg
1621 tatcacttct tcatcctccc tgatgggcct gtgtgacgaa accgcgacta ttatccgtga
1681 gattactggc tacgaccgtg tgatggtagt acgtttcgat gaagagggta atgccgaaat
1741 tctgtccgaa cgtcgtcgtg cggacctgga agcgttcctg ggtaaccgct acccggcgtc
1801 tactattccg cagatcgctc gtcgcctgta cgaacataac cgtgttcgcc tgctggtaga
1861 tgtgaactat actccggttc cgctacagcc gcgcatcagc ccgctgaacg gtcgtgatct
1921 ggatatgtcc ctgtcttgcc tgcgctctat gtccccggtc caccagaaat acatgcagga
1981 catgggcgtt ggcgcgaccc tggtttgctc tctgatggtg tctggtcgtt tgtggggtct
2041 gatcgcttgc caccactacg aaccgcgctt cgttccgttc cacattcgcg ctgctggcga
2101 agcgctggcg gaaacttgtg cgaaccgcat cgcgacgctg gagagcgaaa atctctactt
2161 ccagagtgaa ttctgtgaac gcgagcagat ccacctggcg ggctccattc agccgcacgg
2221 tatcctgctg gctgtgaaag agccagacaa cgtggtgatc caggcttcta tcaacgctgc
2281 ggagttcctg aacaccaact ctgttgttgg ccgtccgctg cgtgacctgg gcggcgatct
2341 gcctttgcag atcctgccgc acctgaacgg cccgctgcac ctggctccgg tgaccctgcg
2401 ttgtaccgtg ggttctccgc cgcgccgtgt ggactgtacc attcaccgtc cgtctaacgg
2461 cggcctgatc gtagaactgg aaccagcaac caagctcgag ggaggcagcc gggaccacat
2521 ggtgctgcac gagtacgtga acgccgccgg catcacataa tctagagggc ccgtttaaac
2581 ccgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc
2641 cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga
2701 aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga
2761 cagcaagggg gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat
2821 ggcttctgag gcggaaagaa ccagctgggg ctctaggggg tatccccacg cgccctgtag
2881 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
2941 cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
3001 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
3061 cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata
3121 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca
3181 aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc
3241 gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattaatt
3301 ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag gctccccagc aggcagaagt
3361 atgcaaagca tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca
3421 gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta
3481 actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga
3541 ctaatttttt ttatttatgc agaggccgag gccgcctctg cctctgagct attccagaag
3601 tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa agctcccggg agcttgtata
3661 tccattttcg gatctgatca agagacagga tgaggatcgt ttcgcatgat tgaacaagat
3721 ggattgcacg caggttctcc ggccgcttgg gtggagaggc tattcggcta tgactgggca
3781 caacagacaa tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg
3841 gttctttttg tcaagaccga cctgtccggt gccctgaatg aactgcagga cgaggcagcg
3901 cggctatcgt ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact
3961 gaagcgggaa gggactggct gctattgggc gaagtgccgg ggcaggatct cctgtcatct
4021 caccttgctc ctgccgagaa agtatccatc atggctgatg caatgcggcg gctgcatacg
4081 cttgatccgg ctacctgccc attcgaccac caagcgaaac atcgcatcga gcgagcacgt
4141 actcggatgg aagccggtct tgtcgatcag gatgatctgg acgaagagca tcaggggctc
4201 gcgccagccg aactgttcgc caggctcaag gcgcgcatgc ccgacggcga ggatctcgtc
4261 gtgacccatg gcgatgcctg cttgccgaat atcatggtgg aaaatggccg cttttctgga
4321 ttcatcgact gtggccggct gggtgtggcg gaccgctatc aggacatagc gttggctacc
4381 cgtgatattg ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt gctttacggt
4441 atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga
4501 gcgggactct ggggttcgaa atgaccgacc aagcgacgcc caacctgcca tcacgagatt
4561 tcgattccac cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg
4621 gctggatgat cctccagcgc ggggatctca tgctggagtt cttcgcccac cccaacttgt
4681 ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag
4741 catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg
4801 tctgtatacc gtcgacctct agctagagct tggcgtaatc atggtcatag ctgtttcctg
4861 tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta
4921 aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg
4981 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
5041 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
5101 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
5161 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
5221 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
5281 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
5341 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
5401 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
5461 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
5521 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
5581 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
5641 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta
5701 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
5761 aacaaaccac cgctggtagc ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
5821 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
5881 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
5941 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
6001 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
6061 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
6121 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa
6181 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
6241 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
6301 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
6361 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
6421 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
6481 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
6541 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
6601 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc
6661 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
6721 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
6781 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
6841 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
6901 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
6961 ttccgcgcac atttccccga aaagtgccac ctgacgtc
//
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