| 出品公司: | ZYbscience |
|---|---|
| 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: | ZY3661 |
| 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[luc2P/ATF6 RE/Hygro]载体](https://res.dxycdn.com/trademd/upload/pic/2016/10/13/B14763476825327qripd5ucf.png!small.t.1)
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保存条件 ：-20℃低温保存
保质期 ：三年
英文名 ：pGL4.39[luc2P/ATF6 RE/Hygro]
库存 ：20
供应商 ：泽叶生物
pGL4.39载体基本信息
pGL4.39载体质粒图谱和多克隆位点信息
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 ProtocolIn this example protocol, the pGL4.39[luc2P/ATF6 RE/Hygro] Vector is used to measureactivation of the ATF6 RE in HeLa cells upon treatment with tunicamycin.. The pGL4.75Vector (encoding Renilla luciferase) is used as a normalization control. In designing suchexperiments, it is important that the chosen cell type can be transfected efficiently andthat it expresses the proper components of the signaling pathway of interest in order togenerate the biological response. Protocol optimization may be required for your particularcell 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 TransfectionPreparation of Cells1. Grow HeLa cells in complete medium (DMEM + 10% FBS + 1X NEAA). Wash withDPBS and treat with one volume of 0.05% trypsin-EDTA. Resuspend cells in fourvolumes 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 Mixture1. Dilute pGL4.39[luc2P/ATF6 RE/Hygro] and pGL4.75 [hRluc/CMV] Renilla luciferasevector 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 temperaturefor 30 minutes.3. Dilute lipid:DNA mixture 20-fold with 1 × 105 cells/ml cell suspension and mix byinversion.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 Treatment1. Resuspend tunicamycin to 10mM in DMSO. Serially dilute into DMSO to give1,000X 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 Measurement1. Remove plates from the 37°C, 5% CO2 incubator and allow to cool to room temperaturefor approximately 15 minutes.4. Add 80μl of the Dual-Glo Luciferase Assay System detection reagents and measureluminescence following the recommended protocol (Refer to the Dual-GloLuciferase Assay System Technical Manual, #TM058 for details).
pGL4.39载体序列
LOCUS JQ858519 6139 bp DNA circular SYN 02-JUL-2012DEFINITION Reporter vector pGL4.39[luc2P/ATF6 RE/Hygro], complete sequence.ACCESSION JQ858519VERSION JQ858519.1 GI:392934106KEYWORDS .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, USAFEATURES 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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