pGL4.47[luc2P/SIE/Hygro]

价格:1500元

联系方式:I47-825O-882O

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pGL4.47载体基本信息

出品公司: Promega
载体名称: pGL4.47; pGL4.47[luc2P/SIE/Hygro]
质粒类型: 信号通路报告载体;哺乳动物载体;萤火虫荧光素酶报告载体
高拷贝/低拷贝: --
克隆方法: 限制性内切酶,多克隆位点
启动子: Minimal Promotor
载体大小: 6129 bp
5' 测序引物及序列: --
3' 测序引物及序列: --
载体标签: luc2P
载体抗性: 氨苄青霉素
筛选标记: 潮霉素(Hygromycin)
克隆菌株: TOP10等常规菌株
宿主细胞(系): HEK293等
备注: pGL4.47[luc2P/SIE/Hygro]载体是信号通路报告载体;含SIS-inducible element(SIE)元件;含萤火虫荧光素酶报告基因luc2P。
产品目录号: E4041
稳定性: 稳表达
组成型/诱导型: 组成型
病毒/非病毒: 非病毒

pGL4.47载体质粒图谱和多克隆位点信息

pGL4.47载体图谱



pGL4.47 载体特征

pGL4.47载体简介

The pGL4.47[luc2P/SIE/Hygro] Vector contains five copies of the SIS-inducible element (SIE) that drives transcription of the luciferase reporter gene luc2P (Photinus pyralis). luc2P is a synthetically-derived luciferase sequence with humanized codon optimization that is designed for high expression and reduced anomalous transcription. The luc2P gene contains hPEST, a protein destabilization sequence, which allows luc2P protein levels to respond more quickly than those of luc2 to induction of transcription. The vector backbone contains an ampicillin resistance gene to allow selection in E. coli and a gene for hygromycin resistance to allow selection of stably transfected mammalian cell lines.

Example Protocol
In this example protocol, the pGL4.47[luc2P/SIE/Hygro] vector is used to measure
activation of the SIE in HEK293 cells upon treatment with interleukin 6. In designing
such experiments, it is important that the chosen cell type can be transfected efficiently
and that it expresses the proper components of the signaling pathway of interest in order
to generate the biological response. Protocol optimization may be required for your
particular cell type and assay conditions.

实验材料
 DMEM (Life Technologies Cat.# 11995)
 FBS (HyClone Cat.# SH30070.03)
 Dulbecco’s PBS (DPBS; Life Technologies Cat. # 14190)
 0.05% Tryspin-EDTA (Life Technologies Cat.# 25300)
 Opti-MEM I (Life Technologies Cat.# 31985)
 FuGENE HD Transfection Reagent (Cat.# E2311)
 Human recombinant interleukin 6 (IL-6, Life Technologies Cat.# PHC0061)
 DMSO (Sigma Cat.# D2650)
 ONE-Glo Luciferase Assay System (Cat.# E6120)
 HEK293 cells

实验流程
Day 1: Plate Cells
1. Plate 10ml of HEK293 cells at 2 × 105 cells/ml in a 10cm dish in complete medium
(DMEM + 10% FBS).
2 Incubate for 24 hours in a 37°C, 5% CO2 incubator.

Day 2: Transfection
1. Dilute 10μg pGL4.47[luc2P/SIE/Hygro] Vector DNA in 500μl Opti-MEM I.
2. Add 30μl FuGENE HD to a 3:1 lipid:DNA ratio and mix. Incubate at room
temperature for 15 minutes.
3. Add DNA-lipid complex to cells and mix gently to ensure even distribution.
4 Incubate for 18 hours in a 37°C, 5% CO2 incubator.

Day 3: Medium Replacement and Cell Treatment
1. Wash cells with DPBS and treat with one volume of 0.05% trypsin-EDTA.
Resuspend cells in four volumes of complete medium.
2. Quantify the cells and dilute to 2 × 105 cells/ml in complete medium.
3. Plate 50μl per well into a solid, white 96-well plate (Corning Cat.# 3917).
4. Serially dilute human recombinant interleukin 6 into complete medium to give
2X stock solutions.
5. Add 50μl of the 2X dilutions of IL-6 to each well.
6. Incubate for 24 hours in a 37°C, 5% CO2 incubator.

.Day 4: Luminescence Measurement
1. Remove plates from the 37°C, 5% CO2 incubator and allow to cool to room
temperature for approximately 15 minutes.
2. Add 100μl ONE-Glo detection reagent to each well and measure luminescence
following the recommended protocol. (Refer to the ONE-Glo Luciferase Assay
System Technical Manual, #TM292 for details).

pGL4.47载体序列

LOCUS       JQ858512                6129 bp    DNA     circular SYN 02-JUL-2012
DEFINITION  Reporter vector PGL4.47[luc2P/SIE/Hygro], complete sequence.
ACCESSION   JQ858512
VERSION     JQ858512.1  GI:392934078
KEYWORDS    .
SOURCE      Reporter vector PGL4.47[luc2P/SIE/Hygro]
  ORGANISM  Reporter vector PGL4.47[luc2P/SIE/Hygro]
            other sequences; artificial sequences; vectors.
REFERENCE   1  (bases 1 to 6129)
  AUTHORS   McNamara,B.
  TITLE     Direct Submission
  JOURNAL   Submitted (28-MAR-2012) Scientific Communications, Promega
            Corporation, 2800 Woods Hollow Rd, Madison, WI 53711, USA
FEATURES             Location/Qualifiers
     source          1..6129
                     /organism="Reporter vector PGL4.47[luc2P/SIE/Hygro]"
                     /mol_type="other DNA"
                     /db_xref="taxon:1203006"
     regulatory      105..258
                     /regulatory_class="terminator"
                     /note="transcriptional pause site"
     regulatory      105..153
                     /regulatory_class="polyA_signal_sequence"
                     /note="synthetic poly(A) signal sequence"
     primer_bind     207..226
                     /note="RVprimer3 binding site"
     regulatory      285..409
                     /regulatory_class="enhancer"
                     /note="SIE response element"
     regulatory      455..485
                     /regulatory_class="promoter"
                     /note="minP"
     gene            518..2293
                     /gene="luc2P"
     CDS             518..2293
                     /gene="luc2P"
                     /codon_start=1
                     /transl_table=11
                     /product="luciferase"
                     /protein_id="AFM92223.1"
                     /db_xref="GI:392934079"
                     /translation="MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTD
                     AHIEVDITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAV
                     APANDIYNERELLNSMGISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQG
                     FQSMYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVR
                     FSHARDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRS
                     LQDYKIQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLP
                     GIRQGYGLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCV
                     RGPMIMSGYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVA
                     PAELESILLQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTT
                     AKKLRGGVVFVDEVPKGLTGKLDARKIREILIKAKKGGKIAVNSHGFPPEVEEQAAGT
                     LPMSCAQESGMDRHPAACASARINV"
     regulatory      2333..2554
                     /regulatory_class="polyA_signal_sequence"
                     /note="SV40 late poly(A) signal"
     regulatory      2602..3020
                     /regulatory_class="promoter"
                     /note="SV40 enhancer and early promoter"
     CDS             3045..4082
                     /note="hygromycin phosphotransferase"
                     /codon_start=1
                     /transl_table=11
                     /product="hygromycin resistance"
                     /protein_id="AFM92224.1"
                     /db_xref="GI:392934080"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQD
                     LPETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVY
                     HWQTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWS
                     EAMFGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQ
                     SLVDGNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPS
                     TRPRAKEVGRV"
     regulatory      4106..4154
                     /regulatory_class="polyA_signal_sequence"
                     /note="synthetic poly(A) signal sequence"
     primer_bind     complement(4221..4240)
                     /note="RVprimer4 binding site"
     rep_origin      4478..4514
     gene            complement(5269..6129)
                     /gene="bla"
     CDS             complement(5269..6129)
                     /gene="bla"
                     /note="AmpR"
                     /codon_start=1
                     /transl_table=11
                     /product="beta-lactamase"
                     /protein_id="AFM92225.1"
                     /db_xref="GI:392934081"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGY
                     IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVE
                     YSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRL
                     DRWEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPL
                     LRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIA
                     EIGASLIKHW"
ORIGIN      
        1 actcgtcctt tttcaatatt attgaagcat ttatcagggt tactagtacg tctctcaagg
       61 ataagtaagt aatattaagg tacgggaggt attggacagg ccgcaataaa atatctttat
      121 tttcattaca tctgtgtgtt ggttttttgt gtgaatcgat agtactaaca tacgctctcc
      181 atcaaaacaa aacgaaacaa aacaaactag caaaataggc tgtccccagt gcaagtgcag
      241 gtgccagaac atttctctgg cctaactggc cggtacctga gctcagcttc atttcccgta
      301 aatcgtcgaa gcttcatttc ccgtaaatcg tcgaagcttc atttcccgta aatcgtcgaa
      361 gcttcatttc ccgtaaatcg tcgaagcttc atttcccgta aatcgtcgac tcgaggatat
      421 caagatctgg cctcggcggc caagcttaga cactagaggg tatataatgg aagctcgact
      481 tccagcttgg caatccggta ctgttggtaa agccaccatg gaagatgcca aaaacattaa
      541 gaagggccca gcgccattct acccactcga agacgggacc gccggcgagc agctgcacaa
      601 agccatgaag cgctacgccc tggtgcccgg caccatcgcc tttaccgacg cacatatcga
      661 ggtggacatt acctacgccg agtacttcga gatgagcgtt cggctggcag aagctatgaa
      721 gcgctatggg ctgaatacaa accatcggat cgtggtgtgc agcgagaata gcttgcagtt
      781 cttcatgccc gtgttgggtg ccctgttcat cggtgtggct gtggccccag ctaacgacat
      841 ctacaacgag cgcgagctgc tgaacagcat gggcatcagc cagcccaccg tcgtattcgt
      901 gagcaagaaa gggctgcaaa agatcctcaa cgtgcaaaag aagctaccga tcatacaaaa
      961 gatcatcatc atggatagca agaccgacta ccagggcttc caaagcatgt acaccttcgt
     1021 gacttcccat ttgccacccg gcttcaacga gtacgacttc gtgcccgaga gcttcgaccg
     1081 ggacaaaacc atcgccctga tcatgaacag tagtggcagt accggattgc ccaagggcgt
     1141 agccctaccg caccgcaccg cttgtgtccg attcagtcat gcccgcgacc ccatcttcgg
     1201 caaccagatc atccccgaca ccgctatcct cagcgtggtg ccatttcacc acggcttcgg
     1261 catgttcacc acgctgggct acttgatctg cggctttcgg gtcgtgctca tgtaccgctt
     1321 cgaggaggag ctattcttgc gcagcttgca agactataag attcaatctg ccctgctggt
     1381 gcccacacta tttagcttct tcgctaagag cactctcatc gacaagtacg acctaagcaa
     1441 cttgcacgag atcgccagcg gcggggcgcc gctcagcaag gaggtaggtg aggccgtggc
     1501 caaacgcttc cacctaccag gcatccgcca gggctacggc ctgacagaaa caaccagcgc
     1561 cattctgatc acccccgaag gggacgacaa gcctggcgca gtaggcaagg tggtgccctt
     1621 cttcgaggct aaggtggtgg acttggacac cggtaagaca ctgggtgtga accagcgcgg
     1681 cgagctgtgc gtccgtggcc ccatgatcat gagcggctac gttaacaacc ccgaggctac
     1741 aaacgctctc atcgacaagg acggctggct gcacagcggc gacatcgcct actgggacga
     1801 ggacgagcac ttcttcatcg tggaccggct gaagagcctg atcaaataca agggctacca
     1861 ggtagcccca gccgaactgg agagcatcct gctgcaacac cccaacatct tcgacgccgg
     1921 ggtcgccggc ctgcccgacg acgatgccgg cgagctgccc gccgcagtcg tcgtgctgga
     1981 acacggtaaa accatgaccg agaaggagat cgtggactat gtggccagcc aggttacaac
     2041 cgccaagaag ctgcgcggtg gtgttgtgtt cgtggacgag gtgcctaaag gactgaccgg
     2101 caagttggac gcccgcaaga tccgcgagat tctcattaag gccaagaagg gcggcaagat
     2161 cgccgtgaat tctcacggct tccctcccga ggtggaggag caggccgccg gcaccctgcc
     2221 catgagctgc gcccaggaga gcggcatgga tagacaccct gctgcttgcg ccagcgccag
     2281 gatcaacgtc taaggccgcg actctagagt cggggcggcc ggccgcttcg agcagacatg
     2341 ataagataca ttgatgagtt tggacaaacc acaactagaa tgcagtgaaa aaaatgcttt
     2401 atttgtgaaa tttgtgatgc tattgcttta tttgtaacca ttataagctg caataaacaa
     2461 gttaacaaca acaattgcat tcattttatg tttcaggttc agggggaggt gtgggaggtt
     2521 ttttaaagca agtaaaacct ctacaaatgt ggtaaaatcg ataaggatcc gtttgcgtat
     2581 tgggcgctct tccgctgatc tgcgcagcac catggcctga aataacctct gaaagaggaa
     2641 cttggttagc taccttctga ggcggaaaga accagctgtg gaatgtgtgt cagttagggt
     2701 gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt
     2761 cagcaaccag gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc
     2821 atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc
     2881 cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg
     2941 ccgaggccgc ctctgcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc
     3001 taggcttttg caaaaagctc gattcttctg acactagcgc caccatgaag aagcccgaac
     3061 tcaccgctac cagcgttgaa aaatttctca tcgagaagtt cgacagtgtg agcgacctga
     3121 tgcagttgtc ggagggcgaa gagagccgag ccttcagctt cgatgtcggc ggacgcggct
     3181 atgtactgcg ggtgaatagc tgcgctgatg gcttctacaa agaccgctac gtgtaccgcc
     3241 acttcgccag cgctgcacta cccatccccg aagtgttgga catcggcgag ttcagcgaga
     3301 gcctgacata ctgcatcagt agacgcgccc aaggcgttac tctccaagac ctccccgaaa
     3361 cagagctgcc tgctgtgtta cagcctgtcg ccgaagctat ggatgctatt gccgccgccg
     3421 acctcagtca aaccagcggc ttcggcccat tcgggcccca aggcatcggc cagtacacaa
     3481 cctggcggga tttcatttgc gccattgctg atccccatgt ctaccactgg cagaccgtga
     3541 tggacgacac cgtgtccgcc agcgtagctc aagccctgga cgaactgatg ctgtgggccg
     3601 aagactgtcc cgaggtgcgc cacctcgtcc atgccgactt cggcagcaac aacgtcctga
     3661 ccgacaacgg ccgcatcacc gccgtaatcg actggtccga agctatgttc ggggacagtc
     3721 agtacgaggt ggccaacatc ttcttctggc ggccctggct ggcttgcatg gagcagcaga
     3781 ctcgctactt cgagcgccgg catcccgagc tggccggcag ccctcgtctg cgagcctaca
     3841 tgctgcgcat cggcctggat cagctctacc agagcctcgt ggacggcaac ttcgacgatg
     3901 ctgcctgggc tcaaggccgc tgcgatgcca tcgtccgcag cggggccggc accgtcggtc
     3961 gcacacaaat cgctcgccgg agcgcagccg tatggaccga cggctgcgtc gaggtgctgg
     4021 ccgacagcgg caaccgccgg cccagtacac gaccgcgcgc taaggaggta ggtcgagttt
     4081 aaactctaga accggtcatg gccgcaataa aatatcttta ttttcattac atctgtgtgt
     4141 tggttttttg tgtgttcgaa ctagatgctg tcgaccgatg cccttgagag ccttcaaccc
     4201 agtcagctcc ttccggtggg cgcggggcat gactatcgtc gccgcactta tgactgtctt
     4261 ctttatcatg caactcgtag gacaggtgcc ggcagcgctc ttccgcttcc tcgctcactg
     4321 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     4381 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     4441 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     4501 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     4561 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     4621 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     4681 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     4741 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     4801 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     4861 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     4921 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     4981 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     5041 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     5101 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     5161 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     5221 agtaaacttg gtctgacagc ggccgcaaat gctaaaccac tgcagtggtt accagtgctt
     5281 gatcagtgag gcaccgatct cagcgatctg cctatttcgt tcgtccatag tggcctgact
     5341 ccccgtcgtg tagatcacta cgattcgtga gggcttacca tcaggcccca gcgcagcaat
     5401 gatgccgcga gagccgcgtt caccggcccc cgatttgtca gcaatgaacc agccagcagg
     5461 gagggccgag cgaagaagtg gtcctgctac tttgtccgcc tccatccagt ctatgagctg
     5521 ctgtcgtgat gctagagtaa gaagttcgcc agtgagtagt ttccgaagag ttgtggccat
     5581 tgctactggc atcgtggtat cacgctcgtc gttcggtatg gcttcgttca actctggttc
     5641 ccagcggtca agccgggtca catgatcacc catattatga agaaatgcag tcagctcctt
     5701 agggcctccg atcgttgtca gaagtaagtt ggccgcggtg ttgtcgctca tggtaatggc
     5761 agcactacac aattctctta ccgtcatgcc atccgtaaga tgcttttccg tgaccggcga
     5821 gtactcaacc aagtcgtttt gtgagtagtg tatacggcga ccaagctgct cttgcccggc
     5881 gtctatacgg gacaacaccg cgccacatag cagtactttg aaagtgctca tcatcgggaa
     5941 tcgttcttcg gggcggaaag actcaaggat cttgccgcta ttgagatcca gttcgatata
     6001 gcccactctt gcacccagtt gatcttcagc atcttttact ttcaccagcg tttcggggtg
     6061 tgcaaaaaca ggcaagcaaa atgccgcaaa gaagggaatg agtgcgacac gaaaatgttg
     6121 gatgctcat
//

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