pIRES哺乳动物表达载体说明

pIRES

编号 载体名称

北京华越洋生物VECT6163 pIRES

pIRES载体基本信息

载体名称: pIRES

质粒类型: 哺乳动物细胞表达载体；双顺反子载体；双表达载体启动子: CMV

表达水平: 高

克隆方法: 多克隆位点，限制性内切酶

载体大小: 6092 bp

5' 测序引物: CMV-F: 5'-CGCAAATGGGCGGTAGGCGTG-3'

3' 测序引物: pIRES-R:5'-GCCCTAGATGCATGCTCG-3'

载体标签: 无

载体抗性: 氨苄青霉素（Ampicillin）

筛选标记: 新霉素（Neomycin）

备注: pIRES载体含有IRES元件，可以同时表达两个基因。稳定性: 稳表达或瞬表达

组成型: 组成型

病毒/非病毒: 非病毒

pIRES载体质粒图谱和多克隆位点信息

pIRES载体简介

pIRES i s a m ammalian e xpression v ector t hat a llows h igh l evel e xpression o f t wo g enes o f interest from the same bicistronic mRNA transcript. The vector contains the encephalomyocarditis virus (ECMV) internal ribosome entry site (IRES) flanked by two multiple cloning sites (MCS A and B), an arrangement that allows cap-­‐independent translation of the gene cloned into MCS B (1–3). pIRES utilizes a partially disabled IRES sequence (1) that reduces the rate at which the gene cloned into MCS B is translated relative t o t hat o f M CS A.

Expression of the bicistronic transcript is driven by the constitutively active cytomegalovirus immediate early promoter (PCMV IE), located upstream of MCS A. An intervening sequence (IVS) known to enhance the stability of mRNA (4) is located between PCMV IE and MCS A, and is efficiently spliced out following transcription. SV40 polyadenylation signals downstream of MCS B direct proper processing of the 3' end of the mRNA. Bacteriophage T7 and T3 promoters are located upstream of MCS A and downstream o f M CS B, r espectively. p IRES i ncludes a n eomycin r esistance g ene (Neor) t o aid in the selection of transfected cells. Neor is expressed from the SV40 enhancer/promoter, a nd a s ynthetic p olyadenyla¬tion s ignal d irects p roper p rocessing o f the 3' e nd o f t he N eor m RNA. T he S V40 o rigin a l¬lows f or r eplication i n m ammalian c ells expressing the SV40 T antigen. The vector also contains an ampicillin resistance gene (Ampr), a nd a C olE1 o rigin o f r eplication f or s e¬lection a nd p ropagation i n E. c oli, a nd a n f1 o rigin f or s ingle-­‐stranded D NA p roduction.

pIRES载体应用

Genes cloned into either MCS must contain ATG start codons. pIRES and derivatives can be introduced into mammalian cells by any standard transfection method. Transformed cells can be selected by growth in medium containing the antibiotic G418. Sense or antisense R NA c an b e t ranscribed f rom t he T7 a nd T3 p romoters, r espectively.

pIRES载体序列

ORIGIN

1 TCAATATTGG CCATTAGCCA TATTATTCAT TGGTTATATA GCATAAATCA ATATTGGCTA 61 TTGGCCATTG CATACGTTGT ATCTATATCA TAATATGTAC ATTTATATTG GCTCATGTCC 121 AATATGACCG CCATGTTGGC ATTGATTATT GACTAGTTAT TAATAGTAAT CAATTACGGG 181 GTCATTAGTT CATAGCCCAT ATATGGAGTT CCGCGTTACA TAACTTACGG TAAATGGCCC 241 GCCTGGCTGA CCGCCCAACG ACCCCCGCCC ATTGACGTCA ATAATGACGT ATGTTCCCAT 301 AGTAACGCCA ATAGGGACTT TCCATTGACG TCAATGGGTG GAGTATTTAC GGTAAACTGC 361 CCACTTGGCA GTACATCAAG TGTATCATAT GCCAAGTCCG CCCCCTATTG ACGTCAATGA 421 CGGTAAATGG CCCGCCTGGC ATTATGCCCA GTACATGACC TTACGGGACT TTCCTACTTG 481 GCAGTACATC TACGTATTAG TCATCGCTAT TACCATGGTG ATGCGGTTTT GGCAGTACAC 541 CAATGGGCGT GGATAGCGGT TTGACTCACG GGGATTTCCA AGTCTCCACC CCATTGACGT 601 CAATGGGAGT TTGTTTTGGC ACCAAAATCA ACGGGACTTT CCAAAATGTC GTAACAACTG 661 CGATCGCCCG CCCCGTTGAC GCAAATGGGC GGTAGGCGTG TACGGTGGGA GGTCTATATA