pGL4.39[luc2P/ATF6 RE/Hygro]

价格:1500元

联系方式:I47-825O-882O

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pGL4.39载体基本信息

出品公司: Promega
载体名称: pGL4.39
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: 6139 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: pGL4.39[luc2P/ATF6 RE/Hygro]载体是信号通路报告载体;含ATF6应答元件;含萤火虫荧光素酶报告基因luc2P。
产品目录号: E3661
稳定性: 稳表达
组成型/诱导型: 组成型
病毒/非病毒: 非病毒

pGL4.39载体质粒图谱和多克隆位点信息

pGL4.39载体图谱



pGL4.39 载体特征

pGL4.39载体简介

The pGL4.39[luc2P/ATF6 RE/Hygro] Vector contains five copies of an ATF6 response element (ATF6 RE) 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 pGL4.39[luc2P/ATF6 RE/Hygro] Vector is used to measure
activation of the ATF6 RE in HeLa cells upon treatment with tunicamycin.. The pGL4.75
Vector (encoding Renilla luciferase) is used as a normalization control. 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)
 Complete medium [DMEM supplemented with 10% fetal bovine serum (DMEM/FBS;
Life Technologies Cat.# 16000] and 1X NEAA [Life Technologies Cat.# 11140])
 Dulbecco’s PBS (DPBS; Life Technologies Cat. # 14190)
 0.05% Tryspin-EDTA (Life Technologies Cat.# 25300)
 Charcoal-stripped FBS (Life Technologies Cat.# 126776-011)
 Opti-MEM I (Life Technologies Cat.# 31985)
 FuGENE HD Transfection Reagent (Cat.# E2311)
 Tunicamycin (Calbiochem Cat.# 654380)
 DMSO (Sigma Cat.# D2650)
 Dual-Glo Luciferase Assay System (Cat.# E2940)
 HeLa cells
 pGL4.75[hRluc/CMV] Vector (Cat.# E6931)

实验流程
Day 1: Reverse Transfection
Preparation of Cells
1. Grow HeLa 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 200 × g for 5 minutes in a swinging-bucket rotor.
Resuspend in complete medium at a concentration of 1 × 105 cells/ml.
Preparation of Lipid:DNA Mixture
1. Dilute pGL4.39[luc2P/ATF6 RE/Hygro] and pGL4.75 [hRluc/CMV] Renilla luciferase
vector constructs in a 10:1 mass ratio, respectively, to 10ng total 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 1 × 105 cells/ml cell suspension and 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: Medium Replacement and Cell Treatment
1. Resuspend tunicamycin to 10mM in DMSO. Serially dilute into DMSO to give
1,000X stocks. Dilute 100-fold into DMEM to give 10X stocks.
2. Remove existing medium from cells and replace with 72μl of DMEM + 0.5% charcoal-
stripped FBS per well.
3. Add 8μl of the 10X dilutions of tunicamycin and incubate for 18 hours in a 37°C,
5% CO2 incubator.

Day 3: Luminescence Measurement
1. Remove plates from the 37°C, 5% CO2 incubator and allow to cool to room temperature
for approximately 15 minutes.
4. Add 80μl of the Dual-Glo Luciferase Assay System detection reagents and measure
luminescence following the recommended protocol (Refer to the Dual-Glo
Luciferase Assay System Technical Manual, #TM058 for details).

pGL4.39载体序列

LOCUS       JQ858519                6139 bp    DNA     circular SYN 02-JUL-2012
DEFINITION  Reporter vector pGL4.39[luc2P/ATF6 RE/Hygro], complete sequence.
ACCESSION   JQ858519
VERSION     JQ858519.1  GI:392934106
KEYWORDS    .
SOURCE      Reporter vector pGL4.39[luc2P/ATF6 RE/Hygro]
  ORGANISM  Reporter vector pGL4.39[luc2P/ATF6 RE/Hygro]
            other sequences; artificial sequences; vectors.
REFERENCE   1  (bases 1 to 6139)
  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..6139
                     /organism="Reporter vector pGL4.39[luc2P/ATF6 RE/Hygro]"
                     /mol_type="other DNA"
                     /db_xref="taxon:1203013"
     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..419
                     /regulatory_class="enhancer"
                     /note="ATF6 response element"
     regulatory      465..495
                     /regulatory_class="promoter"
                     /note="minP promoter"
     gene            528..2303
                     /gene="luc2P"
                     /note="synthetic luciferase"
     CDS             528..2303
                     /gene="luc2P"
                     /note="synthetic luciferase"
                     /codon_start=1
                     /transl_table=11
                     /product="luciferase"
                     /protein_id="AFM92245.1"
                     /db_xref="GI:392934108"
                     /translation="MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTD
                     AHIEVDITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAV
                     APANDIYNERELLNSMGISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQG
                     FQSMYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVR
                     FSHARDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRS
                     LQDYKIQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLP
                     GIRQGYGLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCV
                     RGPMIMSGYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVA
                     PAELESILLQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTT
                     AKKLRGGVVFVDEVPKGLTGKLDARKIREILIKAKKGGKIAVNSHGFPPEVEEQAAGT
                     LPMSCAQESGMDRHPAACASARINV"
     regulatory      2343..2564
                     /regulatory_class="polyA_signal_sequence"
                     /note="SV40 late poly(A) signal"
     regulatory      2612..3030
                     /regulatory_class="enhancer"
                     /note="SV40 early enhancer/promoter"
     CDS             3055..4092
                     /note="hygromycin resistance"
                     /codon_start=1
                     /transl_table=11
                     /product="hygromycin phosphotransferase"
                     /protein_id="AFM92246.1"
                     /db_xref="GI:392934109"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQD
                     LPETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVY
                     HWQTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWS
                     EAMFGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQ
                     SLVDGNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPS
                     TRPRAKEVGRV"
     regulatory      4116..4164
                     /regulatory_class="polyA_signal_sequence"
                     /note="synthetic poly(A) signal sequence"
     primer_bind     complement(4231..4250)
                     /note="RVprimer4 binding site"
     rep_origin      4488..4524
     gene            complement(5279..6139)
                     /gene="bla"
                     /note="AmpR"
     CDS             complement(5279..6139)
                     /gene="bla"
                     /note="AmpR"
                     /codon_start=1
                     /transl_table=11
                     /product="beta-lactamase"
                     /protein_id="AFM92244.1"
                     /db_xref="GI:392934107"
                     /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 gctcatcgag acaggtgctg
      301 acgtggcatt catcgagaca ggtgctgacg tggcattcat cgagacaggt gctgacgtgg
      361 cattcatcga gacaggtgct gacgtggcat tcatcgagac aggtgctgac gtggcattcc
      421 tcgaggatat caagatctgg cctcggcggc caagcttaga cactagaggg tatataatgg
      481 aagctcgact tccagcttgg caatccggta ctgttggtaa agccaccatg gaagatgcca
      541 aaaacattaa gaagggccca gcgccattct acccactcga agacgggacc gccggcgagc
      601 agctgcacaa agccatgaag cgctacgccc tggtgcccgg caccatcgcc tttaccgacg
      661 cacatatcga ggtggacatt acctacgccg agtacttcga gatgagcgtt cggctggcag
      721 aagctatgaa gcgctatggg ctgaatacaa accatcggat cgtggtgtgc agcgagaata
      781 gcttgcagtt cttcatgccc gtgttgggtg ccctgttcat cggtgtggct gtggccccag
      841 ctaacgacat ctacaacgag cgcgagctgc tgaacagcat gggcatcagc cagcccaccg
      901 tcgtattcgt gagcaagaaa gggctgcaaa agatcctcaa cgtgcaaaag aagctaccga
      961 tcatacaaaa gatcatcatc atggatagca agaccgacta ccagggcttc caaagcatgt
     1021 acaccttcgt gacttcccat ttgccacccg gcttcaacga gtacgacttc gtgcccgaga
     1081 gcttcgaccg ggacaaaacc atcgccctga tcatgaacag tagtggcagt accggattgc
     1141 ccaagggcgt agccctaccg caccgcaccg cttgtgtccg attcagtcat gcccgcgacc
     1201 ccatcttcgg caaccagatc atccccgaca ccgctatcct cagcgtggtg ccatttcacc
     1261 acggcttcgg catgttcacc acgctgggct acttgatctg cggctttcgg gtcgtgctca
     1321 tgtaccgctt cgaggaggag ctattcttgc gcagcttgca agactataag attcaatctg
     1381 ccctgctggt gcccacacta tttagcttct tcgctaagag cactctcatc gacaagtacg
     1441 acctaagcaa cttgcacgag atcgccagcg gcggggcgcc gctcagcaag gaggtaggtg
     1501 aggccgtggc caaacgcttc cacctaccag gcatccgcca gggctacggc ctgacagaaa
     1561 caaccagcgc cattctgatc acccccgaag gggacgacaa gcctggcgca gtaggcaagg
     1621 tggtgccctt cttcgaggct aaggtggtgg acttggacac cggtaagaca ctgggtgtga
     1681 accagcgcgg cgagctgtgc gtccgtggcc ccatgatcat gagcggctac gttaacaacc
     1741 ccgaggctac aaacgctctc atcgacaagg acggctggct gcacagcggc gacatcgcct
     1801 actgggacga ggacgagcac ttcttcatcg tggaccggct gaagagcctg atcaaataca
     1861 agggctacca ggtagcccca gccgaactgg agagcatcct gctgcaacac cccaacatct
     1921 tcgacgccgg ggtcgccggc ctgcccgacg acgatgccgg cgagctgccc gccgcagtcg
     1981 tcgtgctgga acacggtaaa accatgaccg agaaggagat cgtggactat gtggccagcc
     2041 aggttacaac cgccaagaag ctgcgcggtg gtgttgtgtt cgtggacgag gtgcctaaag
     2101 gactgaccgg caagttggac gcccgcaaga tccgcgagat tctcattaag gccaagaagg
     2161 gcggcaagat cgccgtgaat tctcacggct tccctcccga ggtggaggag caggccgccg
     2221 gcaccctgcc catgagctgc gcccaggaga gcggcatgga tagacaccct gctgcttgcg
     2281 ccagcgccag gatcaacgtc taaggccgcg actctagagt cggggcggcc ggccgcttcg
     2341 agcagacatg ataagataca ttgatgagtt tggacaaacc acaactagaa tgcagtgaaa
     2401 aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta tttgtaacca ttataagctg
     2461 caataaacaa gttaacaaca acaattgcat tcattttatg tttcaggttc agggggaggt
     2521 gtgggaggtt ttttaaagca agtaaaacct ctacaaatgt ggtaaaatcg ataaggatcc
     2581 gtttgcgtat tgggcgctct tccgctgatc tgcgcagcac catggcctga aataacctct
     2641 gaaagaggaa cttggttagc taccttctga ggcggaaaga accagctgtg gaatgtgtgt
     2701 cagttagggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat
     2761 ctcaattagt cagcaaccag gtgtggaaag tccccaggct ccccagcagg cagaagtatg
     2821 caaagcatgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg
     2881 cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt
     2941 tatgcagagg ccgaggccgc ctctgcctct gagctattcc agaagtagtg aggaggcttt
     3001 tttggaggcc taggcttttg caaaaagctc gattcttctg acactagcgc caccatgaag
     3061 aagcccgaac tcaccgctac cagcgttgaa aaatttctca tcgagaagtt cgacagtgtg
     3121 agcgacctga tgcagttgtc ggagggcgaa gagagccgag ccttcagctt cgatgtcggc
     3181 ggacgcggct atgtactgcg ggtgaatagc tgcgctgatg gcttctacaa agaccgctac
     3241 gtgtaccgcc acttcgccag cgctgcacta cccatccccg aagtgttgga catcggcgag
     3301 ttcagcgaga gcctgacata ctgcatcagt agacgcgccc aaggcgttac tctccaagac
     3361 ctccccgaaa cagagctgcc tgctgtgtta cagcctgtcg ccgaagctat ggatgctatt
     3421 gccgccgccg acctcagtca aaccagcggc ttcggcccat tcgggcccca aggcatcggc
     3481 cagtacacaa cctggcggga tttcatttgc gccattgctg atccccatgt ctaccactgg
     3541 cagaccgtga tggacgacac cgtgtccgcc agcgtagctc aagccctgga cgaactgatg
     3601 ctgtgggccg aagactgtcc cgaggtgcgc cacctcgtcc atgccgactt cggcagcaac
     3661 aacgtcctga ccgacaacgg ccgcatcacc gccgtaatcg actggtccga agctatgttc
     3721 ggggacagtc agtacgaggt ggccaacatc ttcttctggc ggccctggct ggcttgcatg
     3781 gagcagcaga ctcgctactt cgagcgccgg catcccgagc tggccggcag ccctcgtctg
     3841 cgagcctaca tgctgcgcat cggcctggat cagctctacc agagcctcgt ggacggcaac
     3901 ttcgacgatg ctgcctgggc tcaaggccgc tgcgatgcca tcgtccgcag cggggccggc
     3961 accgtcggtc gcacacaaat cgctcgccgg agcgcagccg tatggaccga cggctgcgtc
     4021 gaggtgctgg ccgacagcgg caaccgccgg cccagtacac gaccgcgcgc taaggaggta
     4081 ggtcgagttt aaactctaga accggtcatg gccgcaataa aatatcttta ttttcattac
     4141 atctgtgtgt tggttttttg tgtgttcgaa ctagatgctg tcgaccgatg cccttgagag
     4201 ccttcaaccc agtcagctcc ttccggtggg cgcggggcat gactatcgtc gccgcactta
     4261 tgactgtctt ctttatcatg caactcgtag gacaggtgcc ggcagcgctc ttccgcttcc
     4321 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
     4381 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
     4441 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     4501 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     4561 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     4621 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     4681 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     4741 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     4801 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     4861 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     4921 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     4981 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     5041 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     5101 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
     5161 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     5221 agtatatatg agtaaacttg gtctgacagc ggccgcaaat gctaaaccac tgcagtggtt
     5281 accagtgctt gatcagtgag gcaccgatct cagcgatctg cctatttcgt tcgtccatag
     5341 tggcctgact ccccgtcgtg tagatcacta cgattcgtga gggcttacca tcaggcccca
     5401 gcgcagcaat gatgccgcga gagccgcgtt caccggcccc cgatttgtca gcaatgaacc
     5461 agccagcagg gagggccgag cgaagaagtg gtcctgctac tttgtccgcc tccatccagt
     5521 ctatgagctg ctgtcgtgat gctagagtaa gaagttcgcc agtgagtagt ttccgaagag
     5581 ttgtggccat tgctactggc atcgtggtat cacgctcgtc gttcggtatg gcttcgttca
     5641 actctggttc ccagcggtca agccgggtca catgatcacc catattatga agaaatgcag
     5701 tcagctcctt agggcctccg atcgttgtca gaagtaagtt ggccgcggtg ttgtcgctca
     5761 tggtaatggc agcactacac aattctctta ccgtcatgcc atccgtaaga tgcttttccg
     5821 tgaccggcga gtactcaacc aagtcgtttt gtgagtagtg tatacggcga ccaagctgct
     5881 cttgcccggc gtctatacgg gacaacaccg cgccacatag cagtactttg aaagtgctca
     5941 tcatcgggaa tcgttcttcg gggcggaaag actcaaggat cttgccgcta ttgagatcca
     6001 gttcgatata gcccactctt gcacccagtt gatcttcagc atcttttact ttcaccagcg
     6061 tttcggggtg tgcaaaaaca ggcaagcaaa atgccgcaaa gaagggaatg agtgcgacac
     6121 gaaaatgttg gatgctcat
//

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