pGL4.48[luc2P/SBE/Hygro]

价格:1500元

联系方式:I47-825O-882O

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pGL4.48载体基本信息

出品公司: Promega
载体名称: pGL4.48 ; pGL4.48[luc2P/SBE/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: 6052 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: pGL4.48[luc2P/SBE/Hygro]载体是信号通路报告载体;含Smad-binding element (SBE)元件;含萤火 虫荧光素酶报告基因luc2P。
产品目录号: E3671
稳定性: 稳表达
组成型/诱导型: 组成型
病毒/非病毒: 非病毒

pGL4.48载体质粒图谱和多克隆位点信息

pGL4.48载体图谱



pGL4.48 载体特征

pGL4.48载体简介

pGL4.48载体简介
The pGL4.48[luc2P/SBE/Hygro] Vector contains three copies of a Smad-binding element (SBE) 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 pGL4.48[luc2P/SBE/Hygro] vector is used to measure activation
of the SBE in HEK293 cells upon treatment with TGF-β1. 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
 Dulbecco’s PBS (DPBS; Life Technologies Cat.# 14190)
 0.05% Trypsin-EDTA (Life Technologies Cat.# 25300)
 DMEM (Life Technologies Cat.# 11995)
 Complete medium (DMEM supplemented with 10% fetal bovine serum
[DMEM/FBS;Life Technologies Cat.# 16000] and
1X NEAA [Life Technologies Cat.# 11140])
 Opti-MEM I (Life Technologies Cat.# 31985)
 FuGENE HD Transfection Reagent (Cat.# E2311)
 Human TGF-β1 (Sigma Cat.# T7039-2UG)
 BSA (Sigma Cat.# B4287)
 ONE-Glo Luciferase Assay System (Cat.# E6120)
 HEK293 cells

实验流程
Day 1: Reverse Transfection
Preparation of Cells
1. Grow HEK293 cells in complete medium [DMEM + 10% FBS + 1X NEAA]. Wash
with DPBS and treat with one volume of 0.05% trypsin-EDTA. Resuspend cells in
four volumes of complete medium.
2. Pellet the cells by centrifugation at 233 x g for 5 minutes in a swinging-bucket rotor.
Resuspend in complete medium at a concentration of 2 × 105 cells/ml.
Preparation of Lipid:DNA Mixture
1. Dilute pGL4.48[luc2P/SBE/Hygro] DNA to 10ng DNA/μl in Opti-MEM I.
2. Add FuGENE HD to a 3:1 lipid:DNA ratio. Mix by pipetting. Incubate at room temperature
for 30 minutes.
3. Dilute lipid:DNA mixture 20-fold with 2 × 105 cells/ml cell suspension. Mix by inversion.
4. Plate 100μl per well into a solid, white 96-well plate (Corning Cat.# 3917).
5. Incubate for 18 hours in a 37°C, 5% CO2 incubator.

Day 2: Cell Treatment and Luminescence Measurement
1. Resuspend human TGF-β1 (hTGF-β1) to 50μg/ml in water. Dilute 50X into DPBS +
2 mg/ml BSA to give a 1μg/ml solution, and then serially dilute into the same buffer
to give 10X stocks.
2. Add 10μl of the 10X dilutions of hTGF-β1 to each well and incubate for 3 hours in a
in a 37°C, 5% CO2 incubator.
3. Remove plates from the 37°C, 5% CO2 incubator and allow them to cool to room
temperature for approximately 15 minutes.
4. Add ONE-Glo Luciferase Assay System 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).

pGL4.48载体序列

LOCUS       JQ858517                6052 bp    DNA     circular SYN 02-JUL-2012
DEFINITION  Reporter vector PGL4.48[luc2P/SBE/Hygro], complete sequence.
ACCESSION   JQ858517
VERSION     JQ858517.1  GI:392934098
KEYWORDS    .
SOURCE      Reporter vector PGL4.48[luc2P/SBE/Hygro]
  ORGANISM  Reporter vector PGL4.48[luc2P/SBE/Hygro]
            other sequences; artificial sequences; vectors.
REFERENCE   1  (bases 1 to 6052)
  AUTHORS   McNamara,B.
  TITLE     Direct Submission
  JOURNAL   Submitted (29-MAR-2012) Scientific Communications, Promega
            Corporation, 2800 Woods Hollow Rd, Madison, WI 53711, USA
FEATURES             Location/Qualifiers
     source          1..6052
                     /organism="Reporter vector PGL4.48[luc2P/SBE/Hygro]"
                     /mol_type="other DNA"
                     /db_xref="taxon:1203011"
     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"
     misc_feature    285..332
                     /note="SBE response element"
     regulatory      378..408
                     /regulatory_class="promoter"
                     /note="minP promoter"
     gene            441..2216
                     /gene="luc2P"
                     /note="synthetic luciferase"
     CDS             441..2216
                     /gene="luc2P"
                     /note="synthetic luciferase"
                     /codon_start=1
                     /transl_table=11
                     /product="luciferase"
                     /protein_id="AFM92239.1"
                     /db_xref="GI:392934100"
                     /translation="MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTD
                     AHIEVDITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAV
                     APANDIYNERELLNSMGISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQG
                     FQSMYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVR
                     FSHARDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRS
                     LQDYKIQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLP
                     GIRQGYGLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCV
                     RGPMIMSGYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVA
                     PAELESILLQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTT
                     AKKLRGGVVFVDEVPKGLTGKLDARKIREILIKAKKGGKIAVNSHGFPPEVEEQAAGT
                     LPMSCAQESGMDRHPAACASARINV"
     regulatory      2256..2477
                     /regulatory_class="polyA_signal_sequence"
                     /note="SV40 late poly(A) signal"
     regulatory      2525..2943
                     /regulatory_class="enhancer"
                     /note="SV40 enhancer and early promoter"
     CDS             2968..4005
                     /note="hygromycin resistance"
                     /codon_start=1
                     /transl_table=11
                     /product="hygromycin phosphotransferase"
                     /protein_id="AFM92240.1"
                     /db_xref="GI:392934101"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQD
                     LPETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVY
                     HWQTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWS
                     EAMFGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQ
                     SLVDGNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPS
                     TRPRAKEVGRV"
     regulatory      4029..4077
                     /regulatory_class="polyA_signal_sequence"
                     /note="synthetic poly(A) signal sequence"
     primer_bind     complement(4144..4163)
                     /note="RVprimer4 binding site"
     rep_origin      4401..4437
     gene            complement(5192..6052)
                     /gene="bla"
                     /note="beta-lactamase"
     CDS             complement(5192..6052)
                     /gene="bla"
                     /note="AmpR"
                     /codon_start=1
                     /transl_table=11
                     /product="beta-lactamase"
                     /protein_id="AFM92238.1"
                     /db_xref="GI:392934099"
                     /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 gctcagtatg tctagactga
      301 agtatgtcta gactgaagta tgtctagact gactcgagga tatcaagatc tggcctcggc
      361 ggccaagctt agacactaga gggtatataa tggaagctcg acttccagct tggcaatccg
      421 gtactgttgg taaagccacc atggaagatg ccaaaaacat taagaagggc ccagcgccat
      481 tctacccact cgaagacggg accgccggcg agcagctgca caaagccatg aagcgctacg
      541 ccctggtgcc cggcaccatc gcctttaccg acgcacatat cgaggtggac attacctacg
      601 ccgagtactt cgagatgagc gttcggctgg cagaagctat gaagcgctat gggctgaata
      661 caaaccatcg gatcgtggtg tgcagcgaga atagcttgca gttcttcatg cccgtgttgg
      721 gtgccctgtt catcggtgtg gctgtggccc cagctaacga catctacaac gagcgcgagc
      781 tgctgaacag catgggcatc agccagccca ccgtcgtatt cgtgagcaag aaagggctgc
      841 aaaagatcct caacgtgcaa aagaagctac cgatcataca aaagatcatc atcatggata
      901 gcaagaccga ctaccagggc ttccaaagca tgtacacctt cgtgacttcc catttgccac
      961 ccggcttcaa cgagtacgac ttcgtgcccg agagcttcga ccgggacaaa accatcgccc
     1021 tgatcatgaa cagtagtggc agtaccggat tgcccaaggg cgtagcccta ccgcaccgca
     1081 ccgcttgtgt ccgattcagt catgcccgcg accccatctt cggcaaccag atcatccccg
     1141 acaccgctat cctcagcgtg gtgccatttc accacggctt cggcatgttc accacgctgg
     1201 gctacttgat ctgcggcttt cgggtcgtgc tcatgtaccg cttcgaggag gagctattct
     1261 tgcgcagctt gcaagactat aagattcaat ctgccctgct ggtgcccaca ctatttagct
     1321 tcttcgctaa gagcactctc atcgacaagt acgacctaag caacttgcac gagatcgcca
     1381 gcggcggggc gccgctcagc aaggaggtag gtgaggccgt ggccaaacgc ttccacctac
     1441 caggcatccg ccagggctac ggcctgacag aaacaaccag cgccattctg atcacccccg
     1501 aaggggacga caagcctggc gcagtaggca aggtggtgcc cttcttcgag gctaaggtgg
     1561 tggacttgga caccggtaag acactgggtg tgaaccagcg cggcgagctg tgcgtccgtg
     1621 gccccatgat catgagcggc tacgttaaca accccgaggc tacaaacgct ctcatcgaca
     1681 aggacggctg gctgcacagc ggcgacatcg cctactggga cgaggacgag cacttcttca
     1741 tcgtggaccg gctgaagagc ctgatcaaat acaagggcta ccaggtagcc ccagccgaac
     1801 tggagagcat cctgctgcaa caccccaaca tcttcgacgc cggggtcgcc ggcctgcccg
     1861 acgacgatgc cggcgagctg cccgccgcag tcgtcgtgct ggaacacggt aaaaccatga
     1921 ccgagaagga gatcgtggac tatgtggcca gccaggttac aaccgccaag aagctgcgcg
     1981 gtggtgttgt gttcgtggac gaggtgccta aaggactgac cggcaagttg gacgcccgca
     2041 agatccgcga gattctcatt aaggccaaga agggcggcaa gatcgccgtg aattctcacg
     2101 gcttccctcc cgaggtggag gagcaggccg ccggcaccct gcccatgagc tgcgcccagg
     2161 agagcggcat ggatagacac cctgctgctt gcgccagcgc caggatcaac gtctaaggcc
     2221 gcgactctag agtcggggcg gccggccgct tcgagcagac atgataagat acattgatga
     2281 gtttggacaa accacaacta gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga
     2341 tgctattgct ttatttgtaa ccattataag ctgcaataaa caagttaaca acaacaattg
     2401 cattcatttt atgtttcagg ttcaggggga ggtgtgggag gttttttaaa gcaagtaaaa
     2461 cctctacaaa tgtggtaaaa tcgataagga tccgtttgcg tattgggcgc tcttccgctg
     2521 atctgcgcag caccatggcc tgaaataacc tctgaaagag gaacttggtt agctaccttc
     2581 tgaggcggaa agaaccagct gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc
     2641 tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac caggtgtgga
     2701 aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca
     2761 accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat
     2821 tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc cgcctctgcc
     2881 tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag
     2941 ctcgattctt ctgacactag cgccaccatg aagaagcccg aactcaccgc taccagcgtt
     3001 gaaaaatttc tcatcgagaa gttcgacagt gtgagcgacc tgatgcagtt gtcggagggc
     3061 gaagagagcc gagccttcag cttcgatgtc ggcggacgcg gctatgtact gcgggtgaat
     3121 agctgcgctg atggcttcta caaagaccgc tacgtgtacc gccacttcgc cagcgctgca
     3181 ctacccatcc ccgaagtgtt ggacatcggc gagttcagcg agagcctgac atactgcatc
     3241 agtagacgcg cccaaggcgt tactctccaa gacctccccg aaacagagct gcctgctgtg
     3301 ttacagcctg tcgccgaagc tatggatgct attgccgccg ccgacctcag tcaaaccagc
     3361 ggcttcggcc cattcgggcc ccaaggcatc ggccagtaca caacctggcg ggatttcatt
     3421 tgcgccattg ctgatcccca tgtctaccac tggcagaccg tgatggacga caccgtgtcc
     3481 gccagcgtag ctcaagccct ggacgaactg atgctgtggg ccgaagactg tcccgaggtg
     3541 cgccacctcg tccatgccga cttcggcagc aacaacgtcc tgaccgacaa cggccgcatc
     3601 accgccgtaa tcgactggtc cgaagctatg ttcggggaca gtcagtacga ggtggccaac
     3661 atcttcttct ggcggccctg gctggcttgc atggagcagc agactcgcta cttcgagcgc
     3721 cggcatcccg agctggccgg cagccctcgt ctgcgagcct acatgctgcg catcggcctg
     3781 gatcagctct accagagcct cgtggacggc aacttcgacg atgctgcctg ggctcaaggc
     3841 cgctgcgatg ccatcgtccg cagcggggcc ggcaccgtcg gtcgcacaca aatcgctcgc
     3901 cggagcgcag ccgtatggac cgacggctgc gtcgaggtgc tggccgacag cggcaaccgc
     3961 cggcccagta cacgaccgcg cgctaaggag gtaggtcgag tttaaactct agaaccggtc
     4021 atggccgcaa taaaatatct ttattttcat tacatctgtg tgttggtttt ttgtgtgttc
     4081 gaactagatg ctgtcgaccg atgcccttga gagccttcaa cccagtcagc tccttccggt
     4141 gggcgcgggg catgactatc gtcgccgcac ttatgactgt cttctttatc atgcaactcg
     4201 taggacaggt gccggcagcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
     4261 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
     4321 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     4381 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     4441 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     4501 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     4561 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     4621 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     4681 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     4741 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     4801 acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc
     4861 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     4921 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     4981 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     5041 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
     5101 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
     5161 agcggccgca aatgctaaac cactgcagtg gttaccagtg cttgatcagt gaggcaccga
     5221 tctcagcgat ctgcctattt cgttcgtcca tagtggcctg actccccgtc gtgtagatca
     5281 ctacgattcg tgagggctta ccatcaggcc ccagcgcagc aatgatgccg cgagagccgc
     5341 gttcaccggc ccccgatttg tcagcaatga accagccagc agggagggcc gagcgaagaa
     5401 gtggtcctgc tactttgtcc gcctccatcc agtctatgag ctgctgtcgt gatgctagag
     5461 taagaagttc gccagtgagt agtttccgaa gagttgtggc cattgctact ggcatcgtgg
     5521 tatcacgctc gtcgttcggt atggcttcgt tcaactctgg ttcccagcgg tcaagccggg
     5581 tcacatgatc acccatatta tgaagaaatg cagtcagctc cttagggcct ccgatcgttg
     5641 tcagaagtaa gttggccgcg gtgttgtcgc tcatggtaat ggcagcacta cacaattctc
     5701 ttaccgtcat gccatccgta agatgctttt ccgtgaccgg cgagtactca accaagtcgt
     5761 tttgtgagta gtgtatacgg cgaccaagct gctcttgccc ggcgtctata cgggacaaca
     5821 ccgcgccaca tagcagtact ttgaaagtgc tcatcatcgg gaatcgttct tcggggcgga
     5881 aagactcaag gatcttgccg ctattgagat ccagttcgat atagcccact cttgcaccca
     5941 gttgatcttc agcatctttt actttcacca gcgtttcggg gtgtgcaaaa acaggcaagc
     6001 aaaatgccgc aaagaaggga atgagtgcga cacgaaaatg ttggatgctc at
//

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