| 出品公司: | Promega |
|---|---|
| 载体名称: | pGL4.43 ; pGL4.43[luc2P/XRE/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: | 6067 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: | pGL4.43[luc2P/XRE/Hygro]载体是信号通路报告载体；含异己物应答元件XRE；含萤火虫荧光素酶报告基因luc2P。 |
| 产品目录号: | E3751 |
| 稳定性: | 稳表达 |
| 组成型/诱导型: | 组成型 |
| 病毒/非病毒: | 非病毒 |
买家导航
pGL4.43载体基本信息
pGL4.43载体图谱质粒图谱和多克隆位点信息
pGL4.43载体简介
pGL4.43载体简介 The pGL4.43[luc2P/XRE/Hygro] Vector contains three copies of a xenobiotic response element (XRE) 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 pGL4.43[luc2P/XRE/Hygro] Vector is used to measure activation of the XRE in HepG2 cells upon treatment with TCDD. 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) TCDD (2,3,7,8-Tetrachlorodibenzo-p-dioxin; AccuStandard Cat.# D-404N) DMSO (Sigma Cat.# D2650) Dual-Glo Luciferase Assay System (Cat.# E2940) HepG2 cells pGL4.75[hRluc/CMV] Vector (Cat.# E6931) 实验流程 Day 1: Plate Cells 1. Grow HepG2 cells in complete medium (DMEM + 10% FBS + 1X NEAA). Wash twice with DPBS and treat with one volume of 0.05% trypsin-EDTA, followed by four volumes of complete medium. 2. Vigorously resuspend the cells by pipetting and allow cell clumps to settle. Remove the cell suspension from any cell clumps, quantify the cells and dilute in complete medium to 1 × 105 cells/ml. 3. Plate 100μl per well to a solid, white 96-well plate (Corning Cat.# 3917). 4. Incubate for 24 hours in a 37°C, 5% CO2 incubator. .Day 2: Transfection 1. Dilute pGL4.43[luc2P/XRE/hygro] and pGL4.75 [hRluc/CMV] Renilla luciferase vector constructs in a 10:1 mass ratio, respectively, to 12.5ng total DNA/μl in Opti-MEM I. 2. Add FuGENE HD to a 4.5:1 lipid:DNA ratio. Mix by pipetting. Incubate at room temperature for 20 minutes. 3. Add 8μl transfection complex per well (100ng DNA/well) and incubate for 18 hours in a 37°C, 5% CO2 incubator. Day 3: Medium Replacement and Cell Treatment 1. Resuspend TCDD to 31.6μM in DMSO. Serially dilute by half logs into DMSO to give concentrated stock solutions (316X). Dilute these 31.6-fold into Opti-MEM I 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 TCDD dilutions and 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 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).
pGL4.43载体序列
LOCUS JQ858513 6067 bp DNA circular SYN 02-JUL-2012
DEFINITION Reporter vector PGL4.43[luc2P/XRE/Hygro], complete sequence.
ACCESSION JQ858513
VERSION JQ858513.1 GI:392934082
KEYWORDS .
SOURCE Reporter vector PGL4.43[luc2P/XRE/Hygro]
ORGANISM Reporter vector PGL4.43[luc2P/XRE/Hygro]
other sequences; artificial sequences; vectors.
REFERENCE 1 (bases 1 to 6067)
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..6067
/organism="Reporter vector PGL4.43[luc2P/XRE/Hygro]"
/mol_type="other DNA"
/db_xref="taxon:1203007"
regulatory 105..258
/regulatory_class="terminator"
/note="transcriptional pause site"
regulatory 105..153
/regulatory_class="polyA_signal_sequence"
/note="synthetic poly(A) sequence"
primer_bind 207..226
/note="RVprimer3 binding site"
misc_feature 285..347
/note="XRE response element"
regulatory 393..423
/regulatory_class="promoter"
/note="minP promoter"
gene 456..2231
/gene="luc2P"
/note="luciferase"
CDS 456..2231
/gene="luc2P"
/codon_start=1
/transl_table=11
/product="luciferase"
/protein_id="AFM92227.1"
/db_xref="GI:392934084"
/translation="MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTD
AHIEVDITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAV
APANDIYNERELLNSMGISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQG
FQSMYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVR
FSHARDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRS
LQDYKIQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLP
GIRQGYGLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCV
RGPMIMSGYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVA
PAELESILLQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTT
AKKLRGGVVFVDEVPKGLTGKLDARKIREILIKAKKGGKIAVNSHGFPPEVEEQAAGT
LPMSCAQESGMDRHPAACASARINV"
regulatory 2271..2492
/regulatory_class="polyA_signal_sequence"
/note="SV40 late poly(A) signal"
regulatory 2540..2958
/regulatory_class="enhancer"
/note="SV40 enhancer and early promoter"
CDS 2983..4020
/note="hygromycin resistance"
/codon_start=1
/transl_table=11
/product="hygromycin phosphotransferase"
/protein_id="AFM92228.1"
/db_xref="GI:392934085"
/translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQD
LPETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVY
HWQTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWS
EAMFGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQ
SLVDGNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPS
TRPRAKEVGRV"
regulatory 4044..4092
/regulatory_class="polyA_signal_sequence"
/note="synthetic poly(A) sequence"
primer_bind complement(4159..4178)
/note="RVprimer4 binding site"
rep_origin 4416..4452
gene complement(5207..6067)
/gene="bla"
CDS complement(5207..6067)
/gene="bla"
/note="AmpR"
/codon_start=1
/transl_table=11
/product="beta-lactamase"
/protein_id="AFM92226.1"
/db_xref="GI:392934083"
/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 gctctgagtt ctcacgctag
301 cagattgagt tctcacgcta gcagattgag ttctcacgct agcagatctc gaggatatca
361 agatctggcc tcggcggcca agcttagaca ctagagggta tataatggaa gctcgacttc
421 cagcttggca atccggtact gttggtaaag ccaccatgga agatgccaaa aacattaaga
481 agggcccagc gccattctac ccactcgaag acgggaccgc cggcgagcag ctgcacaaag
541 ccatgaagcg ctacgccctg gtgcccggca ccatcgcctt taccgacgca catatcgagg
601 tggacattac ctacgccgag tacttcgaga tgagcgttcg gctggcagaa gctatgaagc
661 gctatgggct gaatacaaac catcggatcg tggtgtgcag cgagaatagc ttgcagttct
721 tcatgcccgt gttgggtgcc ctgttcatcg gtgtggctgt ggccccagct aacgacatct
781 acaacgagcg cgagctgctg aacagcatgg gcatcagcca gcccaccgtc gtattcgtga
841 gcaagaaagg gctgcaaaag atcctcaacg tgcaaaagaa gctaccgatc atacaaaaga
901 tcatcatcat ggatagcaag accgactacc agggcttcca aagcatgtac accttcgtga
961 cttcccattt gccacccggc ttcaacgagt acgacttcgt gcccgagagc ttcgaccggg
1021 acaaaaccat cgccctgatc atgaacagta gtggcagtac cggattgccc aagggcgtag
1081 ccctaccgca ccgcaccgct tgtgtccgat tcagtcatgc ccgcgacccc atcttcggca
1141 accagatcat ccccgacacc gctatcctca gcgtggtgcc atttcaccac ggcttcggca
1201 tgttcaccac gctgggctac ttgatctgcg gctttcgggt cgtgctcatg taccgcttcg
1261 aggaggagct attcttgcgc agcttgcaag actataagat tcaatctgcc ctgctggtgc
1321 ccacactatt tagcttcttc gctaagagca ctctcatcga caagtacgac ctaagcaact
1381 tgcacgagat cgccagcggc ggggcgccgc tcagcaagga ggtaggtgag gccgtggcca
1441 aacgcttcca cctaccaggc atccgccagg gctacggcct gacagaaaca accagcgcca
1501 ttctgatcac ccccgaaggg gacgacaagc ctggcgcagt aggcaaggtg gtgcccttct
1561 tcgaggctaa ggtggtggac ttggacaccg gtaagacact gggtgtgaac cagcgcggcg
1621 agctgtgcgt ccgtggcccc atgatcatga gcggctacgt taacaacccc gaggctacaa
1681 acgctctcat cgacaaggac ggctggctgc acagcggcga catcgcctac tgggacgagg
1741 acgagcactt cttcatcgtg gaccggctga agagcctgat caaatacaag ggctaccagg
1801 tagccccagc cgaactggag agcatcctgc tgcaacaccc caacatcttc gacgccgggg
1861 tcgccggcct gcccgacgac gatgccggcg agctgcccgc cgcagtcgtc gtgctggaac
1921 acggtaaaac catgaccgag aaggagatcg tggactatgt ggccagccag gttacaaccg
1981 ccaagaagct gcgcggtggt gttgtgttcg tggacgaggt gcctaaagga ctgaccggca
2041 agttggacgc ccgcaagatc cgcgagattc tcattaaggc caagaagggc ggcaagatcg
2101 ccgtgaattc tcacggcttc cctcccgagg tggaggagca ggccgccggc accctgccca
2161 tgagctgcgc ccaggagagc ggcatggata gacaccctgc tgcttgcgcc agcgccagga
2221 tcaacgtcta aggccgcgac tctagagtcg gggcggccgg ccgcttcgag cagacatgat
2281 aagatacatt gatgagtttg gacaaaccac aactagaatg cagtgaaaaa aatgctttat
2341 ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt ataagctgca ataaacaagt
2401 taacaacaac aattgcattc attttatgtt tcaggttcag ggggaggtgt gggaggtttt
2461 ttaaagcaag taaaacctct acaaatgtgg taaaatcgat aaggatccgt ttgcgtattg
2521 ggcgctcttc cgctgatctg cgcagcacca tggcctgaaa taacctctga aagaggaact
2581 tggttagcta ccttctgagg cggaaagaac cagctgtgga atgtgtgtca gttagggtgt
2641 ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca
2701 gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat
2761 ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg
2821 cccagttccg cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc
2881 gaggccgcct ctgcctctga gctattccag aagtagtgag gaggcttttt tggaggccta
2941 ggcttttgca aaaagctcga ttcttctgac actagcgcca ccatgaagaa gcccgaactc
3001 accgctacca gcgttgaaaa atttctcatc gagaagttcg acagtgtgag cgacctgatg
3061 cagttgtcgg agggcgaaga gagccgagcc ttcagcttcg atgtcggcgg acgcggctat
3121 gtactgcggg tgaatagctg cgctgatggc ttctacaaag accgctacgt gtaccgccac
3181 ttcgccagcg ctgcactacc catccccgaa gtgttggaca tcggcgagtt cagcgagagc
3241 ctgacatact gcatcagtag acgcgcccaa ggcgttactc tccaagacct ccccgaaaca
3301 gagctgcctg ctgtgttaca gcctgtcgcc gaagctatgg atgctattgc cgccgccgac
3361 ctcagtcaaa ccagcggctt cggcccattc gggccccaag gcatcggcca gtacacaacc
3421 tggcgggatt tcatttgcgc cattgctgat ccccatgtct accactggca gaccgtgatg
3481 gacgacaccg tgtccgccag cgtagctcaa gccctggacg aactgatgct gtgggccgaa
3541 gactgtcccg aggtgcgcca cctcgtccat gccgacttcg gcagcaacaa cgtcctgacc
3601 gacaacggcc gcatcaccgc cgtaatcgac tggtccgaag ctatgttcgg ggacagtcag
3661 tacgaggtgg ccaacatctt cttctggcgg ccctggctgg cttgcatgga gcagcagact
3721 cgctacttcg agcgccggca tcccgagctg gccggcagcc ctcgtctgcg agcctacatg
3781 ctgcgcatcg gcctggatca gctctaccag agcctcgtgg acggcaactt cgacgatgct
3841 gcctgggctc aaggccgctg cgatgccatc gtccgcagcg gggccggcac cgtcggtcgc
3901 acacaaatcg ctcgccggag cgcagccgta tggaccgacg gctgcgtcga ggtgctggcc
3961 gacagcggca accgccggcc cagtacacga ccgcgcgcta aggaggtagg tcgagtttaa
4021 actctagaac cggtcatggc cgcaataaaa tatctttatt ttcattacat ctgtgtgttg
4081 gttttttgtg tgttcgaact agatgctgtc gaccgatgcc cttgagagcc ttcaacccag
4141 tcagctcctt ccggtgggcg cggggcatga ctatcgtcgc cgcacttatg actgtcttct
4201 ttatcatgca actcgtagga caggtgccgg cagcgctctt ccgcttcctc gctcactgac
4261 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
4321 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
4381 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
4441 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
4501 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
4561 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
4621 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
4681 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
4741 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
4801 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga
4861 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
4921 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
4981 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
5041 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
5101 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
5161 taaacttggt ctgacagcgg ccgcaaatgc taaaccactg cagtggttac cagtgcttga
5221 tcagtgaggc accgatctca gcgatctgcc tatttcgttc gtccatagtg gcctgactcc
5281 ccgtcgtgta gatcactacg attcgtgagg gcttaccatc aggccccagc gcagcaatga
5341 tgccgcgaga gccgcgttca ccggcccccg atttgtcagc aatgaaccag ccagcaggga
5401 gggccgagcg aagaagtggt cctgctactt tgtccgcctc catccagtct atgagctgct
5461 gtcgtgatgc tagagtaaga agttcgccag tgagtagttt ccgaagagtt gtggccattg
5521 ctactggcat cgtggtatca cgctcgtcgt tcggtatggc ttcgttcaac tctggttccc
5581 agcggtcaag ccgggtcaca tgatcaccca tattatgaag aaatgcagtc agctccttag
5641 ggcctccgat cgttgtcaga agtaagttgg ccgcggtgtt gtcgctcatg gtaatggcag
5701 cactacacaa ttctcttacc gtcatgccat ccgtaagatg cttttccgtg accggcgagt
5761 actcaaccaa gtcgttttgt gagtagtgta tacggcgacc aagctgctct tgcccggcgt
5821 ctatacggga caacaccgcg ccacatagca gtactttgaa agtgctcatc atcgggaatc
5881 gttcttcggg gcggaaagac tcaaggatct tgccgctatt gagatccagt tcgatatagc
5941 ccactcttgc acccagttga tcttcagcat cttttacttt caccagcgtt tcggggtgtg
6001 caaaaacagg caagcaaaat gccgcaaaga agggaatgag tgcgacacga aaatgttgga
6061 tgctcat
//
