常用pGEX載體圖譜

1、Rosetta系列的表達(dá)菌株可以提供T7 RNA聚合酶，它能表達(dá)PET系列載體上的外源基因。pGEX系列載體上的外源基因不需要T7 RNA聚合酶，普通的大腸桿菌經(jīng)IPTG誘導(dǎo)即可表達(dá)Tac啟動(dòng)子是一組由Lac和trp啟動(dòng)子人工構(gòu)建的雜合啟動(dòng)子，受Lac阻遏蛋白的負(fù)調(diào)節(jié)，它的啟動(dòng)能力比Lac和trp都強(qiáng)。其中Tac 1是由Trp啟動(dòng)子的35區(qū)加上一個(gè)合成的46 bp DNA片段(包括Pribnow 盒)和Lac操縱基因構(gòu)成，Tac 12是由Trp的啟動(dòng)子-35區(qū)和Lac啟動(dòng)子的-10區(qū)，加上Lac操縱子中的操縱基因部分和SD序列融合而成蛋白標(biāo)簽：A myc tag is a polypeptid

2、e protein tag derived from the c-myc gene product that can be added to a protein using recombinant DNA technology. It can be used for affinity chromatography, then used to separate recombinant, overexpressed protein from wild type protein expressed by the host orga

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

4、against the Myc epitope. Examples are cellular localization studies by immunofluorescence or detection by Western 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 for Amino-

5、terminus and C stands for Carboxy terminus. The tag is approximately 1202 Daltons in atomic mass and has 10 amino acids.It can be fused to the C-terminus and the N-terminus of a protein. It is advisable not to fuse the tag directly behind the signal peptide of

6、 a secretory protein, since it can interfere with translocation into the secretory pathway.A monoclonal antibody against the myc epitope, named 9E10, is available from the non-commercial Developmental Studies Hybridoma BankpGEX4T1載體基本信息出品公司:GE別名:pGEX-4T-1, pGEX4T1, pGEX 4T 1質(zhì)粒類型:大腸桿菌蛋

7、白表達(dá)載體表達(dá)水平:高拷貝啟動(dòng)子:Tac克隆方法:多克隆位點(diǎn)，限制性內(nèi)切酶載體大小:4969 bp5' 測(cè)序引物及序列:pGEX5':  GGGCTGGCAAGCCACGTTTGGTG3' 測(cè)序引物及序列:pGEX3': CCGGGAGCTGCATGTGTCAGAGG載體標(biāo)簽:N-GST載體抗性:Ampicillin 氨芐備注:復(fù)制子是pMB1產(chǎn)品目錄號(hào):27-4580-01穩(wěn)定性:瞬時(shí)表達(dá) Transient組成型:誘導(dǎo)表達(dá)病毒/非病毒:非病毒pGEX4T1載體質(zhì)粒圖譜和多克隆位點(diǎn)信息原核生物DNA復(fù)制起始點(diǎn)，是DNA鏈上獨(dú)特的具有起始DNA復(fù)制功能

8、的堿基序列。大腸桿菌的復(fù)制起點(diǎn)包括OriC和OriHThrombin 凝血酶pGEX4T1載體簡(jiǎn)介pGEX4T1載體序列LOCUS pGEX-4T-14969 bp DNA circularSYNDEFINITION pGEX-4T-1ACCESSION KEYWORDS SOURCE ORGANISM other sequences; artificial sequences; vectors.COMMENT     This file is created by Vector NTI     

9、0;      COMMENT     VNTAUTHORNAME|FEATURES Location/Qualifiers source 1.4969 /organism="pGEX-4T-1" /mol_type="other DNA" promoter 184.212 /label="tac_promoter" misc_feature 224.246 /label="M13_pUC_rev_primer" gene 2

10、58.977 /label="GST (variant)" /gene="GST (variant)" CDS 258.977 /label="ORF frame 3" misc_feature complement(1019.1041) /label="pGEX_3_primer" promoter 1307.1335 /label="AmpR_promoter" gene 1377.2237 /label="Ampicillin" /gene="Ampicill

11、in" CDS 1377.2237 /label="ORF frame 3" rep_origin 2392.3011 /label="pBR322_origin" misc_feature 3309.4400 /label="lacI" CDS 3441.4400 /label="ORF frame 3" promoter 4449.4478 /label="lac_promoter" misc_feature 4492.4514 /label="M13_pUC_rev_p

12、rimer" promoter 4513.4531 /label="M13_reverse_primer" CDS 4523.81 /label="ORF frame 2" misc_feature 4540.4695 /label="lacZ_a" promoter complement(4543.4559) /label="M13_forward20_primer" misc_feature complement(4552.4574) /label="M13_pUC_fwd_primer&q

13、uot;pGEX4T2載體基本信息出品公司:GE別名:pGEX-4T-2, pGEX4T2, pGEX 4T 2質(zhì)粒類型:大腸桿菌蛋白表達(dá)載體表達(dá)水平:高拷貝啟動(dòng)子:Tac克隆方法:多克隆位點(diǎn)，限制性內(nèi)切酶載體大小:4970 bp5' 測(cè)序引物及序列:pGEX5':  GGGCTGGCAAGCCACGTTTGGTG3' 測(cè)序引物及序列:pGEX3': CCGGGAGCTGCATGTGTCAGAGG載體標(biāo)簽:N-GST載體抗性:Ampicillin 氨芐備注:復(fù)制子是pMB1產(chǎn)品目錄號(hào):27-4581-01穩(wěn)定性:瞬時(shí)表達(dá) Transient組成型:誘導(dǎo)

14、表達(dá)病毒/非病毒:非病毒pGEX4T2載體質(zhì)粒圖譜和多克隆位點(diǎn)信息pGEX4T2載體簡(jiǎn)介pGEX4T2載體序列LOCUS pGEX-4T-24970 bp DNA circular SYNDEFINITION pGEX-4T-2ACCESSION KEYWORDS SOURCE ORGANISM other sequences; artificial sequences; vectors.COMMENT     This file is created by Vector NTI      &#

15、160;     COMMENT     VNTAUTHORNAME|FEATURES Location/Qualifiers source 1.4970 /organism="pGEX-4T-2" /mol_type="other DNA" promoter 184.212 /label="tac_promoter" misc_feature 224.246 /label="M13_pUC_rev_primer" gene 258.9

16、78 /label="GST (variant)" /gene="GST (variant)" CDS 258.974 /label="ORF frame 3" misc_feature complement(1020.1042) /label="pGEX_3_primer" promoter 1308.1336 /label="AmpR_promoter" gene 1378.2238 /label="Ampicillin" /gene="Ampicillin&q

17、uot; CDS 1378.2238 /label="ORF frame 1" rep_origin 2393.3012 /label="pBR322_origin" misc_feature 3310.4401 /label="lacI" CDS 3442.4401 /label="ORF frame 1" promoter 4450.4479 /label="lac_promoter" misc_feature 4493.4515 /label="M13_pUC_rev_prime

18、r" promoter 4514.4532 /label="M13_reverse_primer" CDS 4524.81 /label="ORF frame 3" misc_feature 4541.4696 /label="lacZ_a" promoter complement(4544.4560) /label="M13_forward20_primer" misc_feature complement(4553.4575) /label="M13_pUC_fwd_primer"

19、pGEX4T3載體質(zhì)粒圖譜和多克隆位點(diǎn)信息pGEX4T3載體簡(jiǎn)介pGEX4T3載體序列LOCUS pGEX-4T-34968 bp DNA circularSYNDEFINITION pGEX-4T-3ACCESSION KEYWORDS SOURCE ORGANISM other sequences; artificial sequences; vectors.COMMENT This file is created by Vector NTI COMMENT VNTAUTHORNAME|FEATURES Location/Qualifiers source 1.4968 /organism

20、="pGEX-4T-3" /mol_type="other DNA" promoter 184.212 /label="tac_promoter" misc_feature 224.246 /label="M13_pUC_rev_primer" gene 258.976 /label="GST (variant)" /gene="GST (variant)" CDS 258.980 /label="ORF frame 3" misc_feature com

21、plement(1018.1040) /label="pGEX_3_primer" promoter 1306.1334 /label="AmpR_promoter" gene 1376.2236 /label="Ampicillin" /gene="Ampicillin" CDS 1376.2236 /label="ORF frame 2" rep_origin 2391.3010 /label="pBR322_origin" misc_feature 3308.4399

22、/label="lacI" CDS 3440.4399 /label="ORF frame 2" promoter 4448.4477 /label="lac_promoter" misc_feature 4491.4513 /label="M13_pUC_rev_primer" promoter 4512.4530 /label="M13_reverse_primer" CDS 4522.81 /label="ORF frame 1" misc_feature 4539.4

23、694 /label="lacZ_a" promoter complement(4542.4558) /label="M13_forward20_primer" misc_feature complement(4551.4573) /label="M13_pUC_fwd_primer"pGEX6P1載體基本信息出品公司:GE別名:pGEX-6P-1, pGEX6P1, pGEX 6P 1質(zhì)粒類型:大腸桿菌蛋白表達(dá)載體表達(dá)水平:高拷貝啟動(dòng)子:Tac克隆方法:多克隆位點(diǎn)，限制性內(nèi)切酶載體大小:4984 bp5' 測(cè)序引物及序列:p

24、GEX5':  GGGCTGGCAAGCCACGTTTGGTG3' 測(cè)序引物及序列:pGEX3': CCGGGAGCTGCATGTGTCAGAGG載體標(biāo)簽:N-GST載體抗性:Ampicillin 氨芐備注:復(fù)制子是pMB1產(chǎn)品目錄號(hào):27-4597-01穩(wěn)定性:瞬時(shí)表達(dá) Transient組成型:誘導(dǎo)表達(dá)病毒/非病毒:非病毒pGEX6P1載體質(zhì)粒圖譜和多克隆位點(diǎn)信息pGEX6P1載體簡(jiǎn)介pGEX6P1載體序列LOCUS pGEX-6P-14984 bp DNA circular SYNDEFINITION pGEX-6P-1ACCESSION KEYWORD

25、S SOURCE ORGANISM other sequences; artificial sequences; vectors.COMMENT     This file is created by Vector NTI            COMMENT     VNTAUTHORNAME|FEATURES Location/Qualifiers source 1.4984 /organism=&qu

26、ot;pGEX-6P-1" /mol_type="other DNA" promoter 184.212 /label="tac_promoter" misc_feature 224.246 /label="M13_pUC_rev_primer" gene 258.992 /label="GST" /gene="GST" CDS 258.992 /label="ORF frame 3" misc_feature 918.938 /label="precis

27、ion" misc_feature complement(1034.1056) /label="pGEX_3_primer" promoter 1322.1350 /label="AmpR_promoter" gene 1392.2252 /label="Ampicillin" /gene="Ampicillin" CDS 1392.2252 /label="ORF frame 3" rep_origin 2407.3026 /label="pBR322_origin&quo

28、t; misc_feature 3324.4415 /label="lacI" CDS 3456.4415 /label="ORF frame 3" promoter 4464.4493 /label="lac_promoter" misc_feature 4507.4529 /label="M13_pUC_rev_primer" promoter 4528.4546 /label="M13_reverse_primer" CDS 4538.81 /label="ORF frame 2

29、" misc_feature 4555.4710 /label="lacZ_a" promoter complement(4558.4574) /label="M13_forward20_primer" misc_feature complement(4567.4589) /label="M13_pUC_fwd_primer"pET28a載體基本信息出品公司:EMD Biosciences (Novagen)別名:pET28a, pet 28a, pET-28a(+)質(zhì)粒類型:大腸桿菌蛋白表達(dá)表達(dá)水平:高克隆方法:多克隆位點(diǎn)

30、，限制性內(nèi)切酶載體大小:5369 bp5' 測(cè)序引物及序列:T7: 5'-TAATACGACTCACTATAGGG-3'3' 測(cè)序引物序列:  T7t: 5'-GCTAGTTATTGCTCAGCGG-3'載體標(biāo)簽:N-His, N-Thrombin, N-T7, C-His載體抗性:卡那備注:N端含有Thrombin蛋白酶切位點(diǎn); pET28a, b, c的差異僅僅存在于多克隆位點(diǎn)處。產(chǎn)品目錄號(hào):69864-3穩(wěn)定性:瞬時(shí)表達(dá) Transient組成型:組成型 Constitutive病毒/非病毒:非病毒pET28a載體質(zhì)粒圖

31、譜和多克隆位點(diǎn)信息pET28a載體簡(jiǎn)介pET-28a-c(+)載體帶有一個(gè)N端的His/Thrombin/T7蛋白標(biāo)簽，同時(shí)含有一個(gè)可以選擇的C端His標(biāo)簽。pET28a載體的單一的多克隆位點(diǎn)見上面的環(huán)狀質(zhì)粒圖譜。注意：載體序列是以pBR322質(zhì)粒的編碼規(guī)矩進(jìn)行編碼的，所以T7蛋白表達(dá)區(qū)在質(zhì)粒圖譜上面是反向的。T7 RNA聚合酶啟動(dòng)的克隆和表達(dá)區(qū)域在質(zhì)粒圖譜中也被標(biāo)注了出來(lái)。質(zhì)粒的F1復(fù)制子是被定向的，所以在T7噬菌體聚合酶的作用下，包含有蛋白編碼序列的病毒粒子能夠產(chǎn)生，并啟動(dòng)蛋白表達(dá)，同時(shí)蛋白表達(dá)將被T7終止子序列(Cat. No. 69337-3)的作用下終止蛋白翻譯。pET30a載體基本

32、信息出品公司:EMD Biosciences (Novagen)別名:pET30a, pet 30a質(zhì)粒類型:大腸桿菌蛋白表達(dá)表達(dá)水平:高克隆方法:多克隆位點(diǎn)，限制性內(nèi)切酶載體大小:5422 bp5' 測(cè)序引物及序列:T7: 5'-TAATACGACTCACTATAGGG-3'3' 測(cè)序引物序列:  T7t: 5'-GCTAGTTATTGCTCAGCGG-3'載體標(biāo)簽:N-His, N-S, N-Thrombin, N-EK, C-His載體抗性:卡那備注:pET30a, b, c的差異只存在于多克隆位點(diǎn)，其他位點(diǎn)不存在。產(chǎn)品

33、目錄號(hào):69909-3穩(wěn)定性:瞬時(shí)表達(dá)組成型:組成型病毒/非病毒:非病毒pET30a載體質(zhì)粒圖譜和多克隆位點(diǎn)信息pET30a載體簡(jiǎn)介pET-30a(+)載體攜帶有一個(gè)N端的His標(biāo)簽/Thrombin酶切位點(diǎn)/ S標(biāo)簽/ EK蛋白酶切標(biāo)簽以及一個(gè)可選擇的C端His標(biāo)簽。載體的多克隆位點(diǎn)見上面的圖譜。需要注意的是圖譜中的核苷酸編碼順序是以pBR322載體的復(fù)制方向?yàn)檎较颍訲7表達(dá)區(qū)域在圖譜中是處于反向的位 置。T7 RNA聚合酶啟動(dòng)的克隆和表達(dá)區(qū)域在質(zhì)粒圖譜中也被標(biāo)注了出來(lái)。質(zhì)粒的F1復(fù)制子是被定向的，所以在T7噬菌體聚合酶的作用下，包含有蛋白編碼序列的病毒 粒子能夠產(chǎn)生，并啟動(dòng)蛋白表達(dá)，

34、同時(shí)蛋白表達(dá)將被T7終止子序列(Cat. No. 69337-3)的作用下終止蛋白翻譯。pet32a載體基本信息出品公司:EMD Biosciences(Novagen)別名:pET32a, pet 32a質(zhì)粒類型:大腸桿菌蛋白表達(dá)表達(dá)水平:高克隆方法:多克隆位點(diǎn)，限制性內(nèi)切酶載體大小:5900bp5' 測(cè)序引物:T7： 5'-TAATACGACTCACTATAGGG-3'3' 測(cè)序引物:  T7t: 5'-GCTAGTTATTGCTCAGCGG-3'載體標(biāo)簽:thioredoxin (N端); His (中間和C端)載體抗性

35、:氨芐備注:pET32a載體用于表達(dá)可溶性，具有活性功能的蛋白;pET-32a(+)載體具有N端Thrombin（凝血酶）酶切位點(diǎn)，N端腸激酶酶切位點(diǎn)。pET32啊，pET32b，pET32c載體的差異主要是在于多克隆位點(diǎn)，其他地方完全一致。 產(chǎn)品貨號(hào):69015-3穩(wěn)定性:瞬時(shí)表達(dá)組成型:組成型病毒/非病毒:非病毒pet32a載體質(zhì)粒圖譜和多克隆位點(diǎn)信息pet32a載體簡(jiǎn)介pET32系列載體是設(shè)計(jì)用來(lái)克隆構(gòu)建和高水平融合表達(dá)表達(dá)帶有109個(gè)氨基酸Trx標(biāo)簽的蛋白質(zhì)序列。將基因插入到載體的多克隆位點(diǎn)，能夠獲得融合表達(dá)蛋白質(zhì)。同時(shí)融合蛋白包含有His標(biāo)簽和S標(biāo)簽，這些可以用于蛋白表達(dá)檢

36、測(cè)和純化過(guò)程。載體的多克隆位點(diǎn)見上面的圖譜。需要注意的是圖譜中的核苷酸編碼順序是以pBR322載體的復(fù)制方向?yàn)檎较?，所以T7表達(dá)區(qū)域在圖譜中是處于反向的位置。T7 RNA聚合酶啟動(dòng)的克隆和表達(dá)區(qū)域在質(zhì)粒圖譜中也被標(biāo)注了出來(lái)。質(zhì)粒的F1復(fù)制子是被定向的，所以在T7噬菌體聚合酶的作用下，包含有蛋白編碼序列的病毒 粒子能夠產(chǎn)生，并啟動(dòng)蛋白表達(dá)，同時(shí)蛋白表達(dá)將被T7終止子序列(Cat. No. 69337-3)的作用下終止蛋白翻譯。pET23a載體基本信息出品公司:EMD Biosciences(Novagen)別名:pET23a, pet 23a質(zhì)粒類型:大腸桿菌蛋白表達(dá)表達(dá)水平:高克隆方法:多克隆位點(diǎn)，限制性內(nèi)切酶載體大小:3666bp5' 測(cè)序引物:T75' 測(cè)序引物序列:  5'-TAATACGACTCACTATAGGG-3'載體標(biāo)簽:N-T7, C-His載體抗性:Ampicillin克隆菌株:DH5a表達(dá)菌株:BL21系列備注:Same as pET21abcd(+) but no lac; a,b,c,d vary by MCS.產(chǎn)品目錄號(hào): 69745-3穩(wěn)定性:穩(wěn)表達(dá)組成型:組成型病毒/非病毒:非病毒pET23a載體