| 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 |
买家导航
pGL4.39[luc2P/ATF6 RE/Hygro]载体pGL4.39质粒基本信息
pGL4.39[luc2P/ATF6 RE/Hygro]质粒图谱pGL4.39载体图谱和pGL4.39[luc2P/ATF6 RE/Hygro]载体序列pGL4.39质粒序列多克隆位点信息
pGL4.39[luc2P/ATF6 RE/Hygro]质粒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[luc2P/ATF6 RE/Hygro]质粒序列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
//
