pET-48b(+)大肠杆菌表达载体说明

pET-48b(+)

编号 载体名称

北京华越洋生物VECT4670 pET-­‐48b(+)

pET48b载体基本信息

别名: pET48b, p ET 48b

质粒类型: 大肠杆菌蛋白表达

表达水平: 高

克隆方法: 多克隆位点，限制性内切酶

载体大小: 5605 b p

5' 测序引物序列: T7: 5'-­‐TAATACGACTCACTATAGGG-­‐3';

Trx-­‐F: 5'-­‐TTCCTCGACGCTAACCTG-­‐3'

3' 测序引物序列: T7t: 5'-­‐TGCTAGTTATTGCTCAGCGG-­‐3'

载体标签: N-­‐Trx, N-­‐His,N-­‐HRV 3C, C-­‐S, C-­‐Thrombin

载体抗性: Kanamycin (卡那霉素)

备注: Same as pET47 but also has Nterm Trx Tag; contains HRV 3C Protease

cleavage site for fusion tag removal at low temperatures; Cterm

thrombin c leavage s ite.

稳定性: 瞬时表达

组成型: 组成型

病毒/非病毒: 非病毒

pET48b载体质粒图谱和多克隆位点信息

pET48b载体简介

pET-­‐48b载体含有N端Trx和His标签，在标签后面紧跟着的是HRV 3C蛋白酶切位点。HRV 3C蛋白酶能够高特异性的识别LEVLFQ↓GP蛋白序列，能够在低温下高效切割掉融合标签序列。pET-­‐48b载体还含有一个可选择的C端Thrombin蛋白酶切位点，紧接着位点后是S标签。

pET48b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质粒的编码规矩进行编码的，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. N o. 69337-­‐3)的作用下终止蛋白翻译。载体的单链测序应该使用S 18引物(Cat. N o. 71262-­‐3)。

The pET-­‐48b(+) vector carries N-­‐terminal Trx•Tag™ and His•Tag? coding sequences followed b y a r ecognition s ite f or t he h uman r hinovirus (HRV) 3C p rotease. T his p rotease is highly specifi c for cleavage of the sequence LEVLFQ↓GP (1), and is active at low temperatures (2). pET-­‐48b(+) also contains an optional C-­‐terminal thrombin recognition site followed by an S•Tag™ coding sequence. Unique restriction sites are shown on the circle map. Note that the sequence is numbered by the pBR322 convention, so the T7 expression region is reversed on the circle map. The cloning/expression region of the coding strand transcribed by T7 RNA polymerase is shown below. The f1 origin is oriented so that infection with the helper phage will produce virions containing single-­‐stranded DNA that corresponds to the coding strand. Therefore, single-­‐stranded sequencing s hould b e p erformed u sing t he A S S•Tag 18mer P rimer (Cat. N o. 71262-­‐3).

pET48b载体序列

ORIGIN

1 ATCCGGATAT AGTTCCTCCT TTCAGCAAAA AACCCCTCAA GACCCGTTTA GAGGCCCCAA

61 GGGGTTATGC TAGTTATTGC TCAGCGGTGG CAGCAGCCAA CTCAGCTTCC TTTCGGGCTT

121 TGTTTAGCAG CCTAGGTTAA TTAAGCCTCG AGAGCAGCAG AAGTAGAGCT GTCCATGTGC

181 TGGCGTTCGA ATTTAGCAGC AGCGGTTTCT TTACTACCGC GTGGCACCAG AGCGAGCTCT

241 GCGGCCGCAA GCTTGTCGAC GGACGTCGGG CGCGCCAAGG CCTGTACAGA ATTCGGATCC

301 TGGTACCCGG GTCCCTGAAA GAGGACTTCA AGAGCCGCGG AGTGATGGTG ATGGTGATGA

361 CCAGAACCAC TAGATGGAGT AGGAGTAGGA GGATTGTTAT TAGAACCGCC ACCACCACTA

421 GTATGGCCAG AACCAGAACC GGCCAGGTTA GCGTCGAGGA ACTCTTTCAA CTGACCTTTA

481 GACAGTGCAC CCACTTTGGT TGCCGCCACT TCACCGTTTT TGAACAGCAG CAGAGTCGGG

541 ATACCACGGA TGCCATATTT CGGCGCAGTG CCAGGGTTTT GATCGATGTT CAGTTTTGCA

601 ACGGTCAGTT TGCCCTGATA TTCGTCAGCG ATTTCATCCA GAATCGGGGC GATCATTTTG

661 CACGGACCGC ACCACTCTGC CCAGAAATCG ACGAGGATCG CCCCGTCCGC TTTGAGTACA

721 TCCGTGTCAA AACTGTCGTC AGTCAGGTGA ATAATTTTAT CGCTCATATG TATATCTCCT

781 TCTTAAAGTT AAACAAAATT ATTTCTAGAG GGGAATTGTT ATCCGCTCAC AATTCCCCTA

841 TAGTGAGTCG TATTAATTTC GCGGGATCGA GATCGATCTC GATCCTCTAC GCCGGACGCA

901 TCGTGGCCGG CATCACCGGC GCCACAGGTG CGGTTGCTGG CGCCTATATC GCCGACATCA

961 CCGATGGGGA AGATCGGGCT CGCCACTTCG GGCTCATGAG CGCTTGTTTC GGCGTGGGTA

1021 TGGTGGCAGG CCCCGTGGCC GGGGGACTGT TGGGCGCCAT CTCCTTGCAT GCACCATTCC 1081 TTGCGGCGGC GGTGCTCAAC GGCCTCAACC TACTACTGGG CTGCTTCCTA ATGCAGGAGT 1141 CGCATAAGGG AGAGCGTCGA GATCCCGGAC ACCATCGAAT GGCGCAAAAC CTTTCGCGGT 1201 ATGGCATGAT AGCGCCCGGA AGAGAGTCAA TTCAGGGTGG TGAATGTGAA ACCAGTAACG 1261 TTATACGATG TCGCAGAGTA TGCCGGTGTC TCTTATCAGA CCGTTTCCCG CGTGGTGAAC 1321 CAGGCCAGCC ACGTTTCTGC GAAAACGCGG GAAAAAGTGG AAGCGGCGAT GGCGGAGCTG 1381 AATTACATTC CCAACCGCGT GGCACAACAA CTGGCGGGCA AACAGTCGTT GCTGATTGGC 1441 GTTGCCACCT CCAGTCTGGC CCTGCACGCG CCGTCGCAAA TTGTCGCGGC GATTAAATCT 1501 CGCGCCGATC AACTGGGTGC CAGCGTGGTG GTGTCGATGG TAGAACGAAG CGGCGTCGAA 1561 GCCTGTAAAG CGGCGGTGCA CAATCTTCTC GCGCAACGCG TCAGTGGGCT GATCATTAAC