常用pGEX载体图谱.docx

1、常用pGEX载体图谱Rosetta系列的表达菌株可以提供T7 RNA聚合酶，它能表达PET系列载体上的外源基因.pGEX系列载体上的外源基因不需要T7 RNA聚合酶，普通的大肠杆菌经IPTG诱导便可表达之五兆芳芳创作Tac启动子是一组由Lac和trp启动子人工构建的杂合启动子，受Lac阻遏蛋白的负调节，它的启动能力比Lac和trp都强.其中Tac 1是由Trp启动子的35区加上一个分解的46 bp DNA片段(包含Pribnow盒)和Lac把持基因组成，Tac 12是由Trp的启动子-35区和Lac启动子的-10区，加上Lac把持子中的把持基因部分和SD序列融合而成蛋白标签：A myc tag

2、 is a polypeptideprotein tagderived from thec-mycgene product that can be added to a protein usingrecombinant DNAtechnology. It can be used foraffinity chromatography, then used to separate recombinant, overexpressed protein from wild type protein expressed by the host organism. It can also be used

3、in the isolation of protein complexes with multiple subunits.A myc tag can be used in many different assays that require recognition by anantibody. If there is no antibody against the studied protein, adding a myc-tag allows one to follow the protein with an antibody against the Myc epitope. Example

4、s are cellular localization studies by immunofluorescence or detection byWestern blotting.The peptide sequence of the myc-tag is (in 1- and 3-letter codes, respectively): N-EQKLISEEDL-C, N-Glu-Gln-Lys-Leu-Ile-Ser-Glu-Glu-Asp-Leu-C, where N stands forAmino-terminusand C stands forCarboxy terminus. Th

5、e tag is approximately 1202Daltonsin atomic mass and has 10 amino acids.It can be fused to theC-terminusand theN-terminusof a protein. It is advisable not to fuse the tag directly behind the signal peptide of a secretory protein, since it can interfere with translocation into thesecretory pathway.A

6、monoclonal antibody against the mycepitope, named 9E10, is available from the non-commercialDevelopmental Studies Hybridoma BankpGEX4T1载体根本信息出品公司:GE别号:pGEX-4T-1, pGEX4T1, pGEX 4T 1质粒类型:大肠杆菌蛋白表达载体表达水平:高拷贝启动子:Tac克隆办法:多克隆位点，限制性内切酶载体大小:4969 bp5 测序引物及序列:pGEX5: GGGCTGGCAAGCCACGTTTGGTG3 测序引物及序列:pGEX3: CCGG

7、GAGCTGCATGTGTCAGAGG载体标签:N-GST载体抗性:Ampicillin 氨苄备注:复制子是pMB1产品目录号:27-4580-01稳定性:瞬时表达 Transient组成型:诱导表达病毒/非病毒:非病毒pGEX4T1载体质粒图谱和多克隆位点信息原核生物DNA复制起始点，是DNA链上独特的具有起始DNA复制功效的碱基序列.大肠杆菌的复制起点包含OriC和OriHThrombin 凝血酶pGEX4T1载体简介pGEX4T1载体序列LOCUS pGEX-4T-1 4969 bp DNA circular SYNDEFINITION pGEX-4T-1ACCESSION KEYWOR

8、DS SOURCE ORGANISM other sequences; artificial sequences; vectors.COMMENT This file is created by Vector NTICOMMENT VNTAUTHORNAME|FEATURES Location/Qualifierssource 1.4969 /organism=pGEX-4T-1 /mol_type=other DNApromoter 184.212 /label=tac_promotermisc_feature 224.246 /label=M13_pUC_rev_primergene 25

9、8.977 /label=GST (variant) /gene=GST (variant) CDS 258.977 /label=ORF frame 3misc_feature complement(1019.1041) /label=pGEX_3_primerpromoter 1307.1335 /label=AmpR_promotergene 1377.2237 /label=Ampicillin /gene=Ampicillin CDS 1377.2237 /label=ORF frame 3rep_origin 2392.3011 /label=pBR322_originmisc_f

10、eature 3309.4400 /label=lacI CDS 3441.4400 /label=ORF frame 3promoter 4449.4478 /label=lac_promotermisc_feature 4492.4514 /label=M13_pUC_rev_primerpromoter 4513.4531 /label=M13_reverse_primer CDS 4523.81 /label=ORF frame 2misc_feature 4540.4695 /label=lacZ_apromoter complement(4543.4559) /label=M13_

11、forward20_primermisc_feature complement(4552.4574) /label=M13_pUC_fwd_primerpGEX4T2载体根本信息出品公司:GE别号:pGEX-4T-2, pGEX4T2, pGEX 4T 2质粒类型:大肠杆菌蛋白表达载体表达水平:高拷贝启动子:Tac克隆办法:多克隆位点，限制性内切酶载体大小:4970 bp5 测序引物及序列:pGEX5: GGGCTGGCAAGCCACGTTTGGTG3 测序引物及序列:pGEX3: CCGGGAGCTGCATGTGTCAGAGG载体标签:N-GST载体抗性:Ampicillin 氨苄备注:复制

12、子是pMB1产品目录号:27-4581-01稳定性:瞬时表达 Transient组成型:诱导表达病毒/非病毒:非病毒pGEX4T2载体质粒图谱和多克隆位点信息pGEX4T2载体简介pGEX4T2载体序列LOCUS pGEX-4T-2 4970 bp DNA circular SYNDEFINITION pGEX-4T-2ACCESSION KEYWORDS SOURCE ORGANISM other sequences; artificial sequences; vectors.COMMENT This file is created by Vector NTICOMMENT VNTAUTH

13、ORNAME|FEATURES Location/Qualifierssource 1.4970 /organism=pGEX-4T-2 /mol_type=other DNApromoter 184.212 /label=tac_promotermisc_feature 224.246 /label=M13_pUC_rev_primergene 258.978 /label=GST (variant) /gene=GST (variant) CDS 258.974 /label=ORF frame 3misc_feature complement(1020.1042) /label=pGEX

14、_3_primerpromoter 1308.1336 /label=AmpR_promotergene 1378.2238 /label=Ampicillin /gene=Ampicillin CDS 1378.2238 /label=ORF frame 1rep_origin 2393.3012 /label=pBR322_originmisc_feature 3310.4401 /label=lacI CDS 3442.4401 /label=ORF frame 1promoter 4450.4479 /label=lac_promotermisc_feature 4493.4515 /

15、label=M13_pUC_rev_primerpromoter 4514.4532 /label=M13_reverse_primer CDS 4524.81 /label=ORF frame 3misc_feature 4541.4696 /label=lacZ_apromoter complement(4544.4560) /label=M13_forward20_primermisc_feature complement(4553.4575) /label=M13_pUC_fwd_primerpGEX4T3载体质粒图谱和多克隆位点信息pGEX4T3载体简介pGEX4T3载体序列LOCU

16、S pGEX-4T-3 4968 bp DNA circular SYNDEFINITION pGEX-4T-3ACCESSION KEYWORDS SOURCE ORGANISM other sequences; artificial sequences; vectors.COMMENT This file is created by Vector NTICOMMENT VNTAUTHORNAME|FEATURES Location/Qualifierssource 1.4968 /organism=pGEX-4T-3 /mol_type=other DNApromoter 184.212

17、/label=tac_promotermisc_feature 224.246 /label=M13_pUC_rev_primergene 258.976 /label=GST (variant) /gene=GST (variant) CDS 258.980 /label=ORF frame 3misc_feature complement(1018.1040) /label=pGEX_3_primerpromoter 1306.1334 /label=AmpR_promotergene 1376.2236 /label=Ampicillin /gene=Ampicillin CDS 137

18、6.2236 /label=ORF frame 2rep_origin 2391.3010 /label=pBR322_originmisc_feature 3308.4399 /label=lacI CDS 3440.4399 /label=ORF frame 2promoter 4448.4477 /label=lac_promotermisc_feature 4491.4513 /label=M13_pUC_rev_primerpromoter 4512.4530 /label=M13_reverse_primer CDS 4522.81 /label=ORF frame 1misc_f

19、eature 4539.4694 /label=lacZ_apromoter complement(4542.4558) /label=M13_forward20_primermisc_feature complement(4551.4573) /label=M13_pUC_fwd_primerpGEX6P1载体根本信息出品公司:GE别号:pGEX-6P-1, pGEX6P1, pGEX 6P 1质粒类型:大肠杆菌蛋白表达载体表达水平:高拷贝启动子:Tac克隆办法:多克隆位点，限制性内切酶载体大小:4984 bp5 测序引物及序列:pGEX5: GGGCTGGCAAGCCACGTTTGGTG3

20、 测序引物及序列:pGEX3: CCGGGAGCTGCATGTGTCAGAGG载体标签:N-GST载体抗性:Ampicillin 氨苄备注:复制子是pMB1产品目录号:27-4597-01稳定性:瞬时表达 Transient组成型:诱导表达病毒/非病毒:非病毒pGEX6P1载体质粒图谱和多克隆位点信息pGEX6P1载体简介pGEX6P1载体序列LOCUS pGEX-6P-1 4984 bp DNA circular SYNDEFINITION pGEX-6P-1ACCESSION KEYWORDS SOURCE ORGANISM other sequences; artificial sequ

21、ences; vectors.COMMENT This file is created by Vector NTICOMMENT VNTAUTHORNAME|FEATURES Location/Qualifierssource 1.4984 /organism=pGEX-6P-1 /mol_type=other DNApromoter 184.212 /label=tac_promotermisc_feature 224.246 /label=M13_pUC_rev_primergene 258.992 /label=GST /gene=GST CDS 258.992 /label=ORF f

22、rame 3misc_feature 918.938 /label=precisionmisc_feature complement(1034.1056) /label=pGEX_3_primerpromoter 1322.1350 /label=AmpR_promotergene 1392.2252 /label=Ampicillin /gene=Ampicillin CDS 1392.2252 /label=ORF frame 3rep_origin 2407.3026 /label=pBR322_originmisc_feature 3324.4415 /label=lacI CDS 3

23、456.4415 /label=ORF frame 3promoter 4464.4493 /label=lac_promotermisc_feature 4507.4529 /label=M13_pUC_rev_primerpromoter 4528.4546 /label=M13_reverse_primer CDS 4538.81 /label=ORF frame 2misc_feature 4555.4710 /label=lacZ_apromoter complement(4558.4574) /label=M13_forward20_primermisc_feature compl

24、ement(4567.4589) /label=M13_pUC_fwd_primerpET28a载体根本信息出品公司:EMD Biosciences (Novagen)别号:pET28a, pet 28a, pET-28a(+)质粒类型:大肠杆菌蛋白表达表达水平:高克隆办法:多克隆位点，限制性内切酶载体大小:5369 bp5 测序引物及序列:T7: 5-TAATACGACTCACTATAGGG-33 测序引物序列:T7t: 5-GCTAGTTATTGCTCAGCGG-3载体标签:N-His, N-Thrombin, N-T7, C-His载体抗性:卡那备注:N端含有Thrombin蛋白酶切位点

25、; pET28a, b, c的差别仅仅存在于多克隆位点处.产品目录号:69864-3稳定性:瞬时表达 Transient组成型:组成型 Constitutive病毒/非病毒:非病毒pET28a载体质粒图谱和多克隆位点信息pET28a载体简介pET-28a-c(+)载体带有一个N端的His/Thrombin/T7蛋白标签，同时含有一个可以选择的C端His标签.pET28a载体的单一的多克隆位点见上面的环状质粒图谱.注意：载体序列是以pBR322质粒的编码端方进行编码的，所以T7蛋白表达区在质粒图谱上面是反向的.T7 RNA聚合酶启动的克隆和表达区域在质粒图谱中也被标注了出来.质粒的F1复制子是被

26、定向的，所以在T7噬菌体聚合酶的作用下，包含有蛋白编码序列的病毒粒子能够产生，并启动蛋白表达，同时蛋白表达将被T7终止子序列(Cat. No. 69337-3)的作用下终止蛋白翻译.pET30a载体根本信息出品公司:EMD Biosciences (Novagen)别号:pET30a, pet 30a质粒类型:大肠杆菌蛋白表达表达水平:高克隆办法:多克隆位点，限制性内切酶载体大小:5422 bp5 测序引物及序列:T7: 5-TAATACGACTCACTATAGGG-33 测序引物序列:T7t: 5-GCTAGTTATTGCTCAGCGG-3载体标签:N-His, N-S, N-Thrombi

27、n, N-EK, C-His载体抗性:卡那备注:pET30a, b, c的差别只存在于多克隆位点，其他位点不存在.产品目录号:69909-3稳定性:瞬时表达组成型:组成型病毒/非病毒:非病毒pET30a载体质粒图谱和多克隆位点信息pET30a载体简介pET-30a(+)载体携带有一个N端的His标签/Thrombin酶切位点/ S标签/ EK蛋白酶切标签以及一个可选择的C端His标签.载体的多克隆位点见上面的图谱.需要注意的是图谱中的核苷酸编码顺序是以pBR322载体的复制标的目的为正标的目的，所以T7表达区域在图谱中是处于反向的位置.T7 RNA聚合酶启动的克隆和表达区域在质粒图谱中也被标注

28、了出来.质粒的F1复制子是被定向的，所以在T7噬菌体聚合酶的作用下，包含有蛋白编码序列的病毒粒子能够产生，并启动蛋白表达，同时蛋白表达将被T7终止子序列(Cat. No. 69337-3)的作用下终止蛋白翻译.pet32a载体根本信息出品公司:EMD Biosciences(Novagen)别号:pET32a, pet 32a质粒类型:大肠杆菌蛋白表达表达水平:高克隆办法:多克隆位点，限制性内切酶载体大小:5900bp5 测序引物:T7： 5-TAATACGACTCACTATAGGG-33 测序引物:T7t: 5-GCTAGTTATTGCTCAGCGG-3载体标签:thioredoxin (N

29、端); His (中间和C端)载体抗性:氨苄备注:pET32a载体用于表达可溶性，具有活性功效的蛋白;pET-32a(+)载体具有N端Thrombin（凝血酶）酶切位点，N端肠激酶酶切位点.pET32啊，pET32b，pET32c载体的差别主要是在于多克隆位点，其他地方完全一致.产品货号:69015-3稳定性:瞬时表达组成型:组成型病毒/非病毒:非病毒pet32a载体质粒图谱和多克隆位点信息pet32a载体简介pET32系列载体是设计用来克隆构建和高水平融合表达表达带有109个氨基酸Trx标签的蛋白质序列.将基因拔出到载体的多克隆位点，能够取得融合表达蛋白质.同时融合蛋白包含有His标签和S标签，这些可以用于蛋白表达检测和纯化进程.载体的多克