pIRES2-ZsGreen1哺乳动物表达载体说明

pIRES2-ZsGreen1

编号

北京华越洋生物VECT6062

pIRES2-­‐ZsGreen1载体基本信息

载体名称: 质粒类型: 高拷贝/低拷贝: 克隆方法: 启动子: 载体大小: 5' 测序引物及序列: 3' 测序引物及序列:

载体标签: 载体抗性: 筛选标记: 克隆菌株: 宿主细胞（系）:

备注: 稳定性: 组成型/诱导型: 病毒/非病毒:

pIRES2-ZsGreen1

载体名称

pIRES2-­‐ZsGreen1

哺乳动物细胞载体；荧光报告载体；双顺反子载体 高拷贝

限制性内切酶，多克隆位点 CMV 5283 bp

CMV-F: 5'-CGCAAATGGGCGGTAGGCGTG-3' --

绿色荧光蛋白ZsGreen1(C-端） 卡那霉素

新霉素（Neomycin ） DH5α, HB101

常规细胞系，293、CV-1、CHO 等

pIRES2-ZsGreen1载体含有IRES 元件，目的基因与报告基因ZsGreen1共转录为一个mRNA ，但分别翻译；ZsGreen1是亮度最高的绿色荧光蛋白

, 适合流式细胞仪筛选。 瞬表达 或 稳表达 组成型 非病毒

pIRES2-­‐ZsGreen1载体质粒图谱和多克隆位点信息

pIRES2-ZsGreen1载体描述

pIRES2-ZsGreen1 contains the internal ribosome entry site (IRES; 1, 2) of the encephalomyocarditis virus (ECMV) between an MCS and the Zoanthus sp. green fluorescent protein (ZsGreen; 3, 4)coding region. This design permits both the gene of interest (cloned into the MCS) and the ZsGreen1 gene to be translated from a single bicistronic mRNA. pIRES2-ZsGreen1 is designed for the efficient selection (by flow cytometry or other methods) of transiently transfected mammalian cells expressing ZsGreen1 and the protein of interest. This vector can be used to obtain stably transfected cell lines without time-consuming drug and clonal selection.

ZsGreen1 is a human codon-optimized (5) ZsGreen variant that encodes the brightest commercially available green fluorescent protein. The MCS in pIRES2-ZsGreen1 is located between the immediate early promoter of cytomegalovirus (PCMV IE) and the IRES sequence. SV40 polyadenylation signals downstream of the ZsGreen1

gene

direct proper processing of the 3' end of the bicistronic mRNA. The vector backbone also contains an SV40 origin for replication in mammalian cells expressing the SV40 T antigen. A neomycin-resistance cassette (Neor), consisting of the SV40 early promoter, the neomycin/kanamycin resistance gene of Tn5, and polyadenylation signals from the herpes simplex virus thymidine kinase (HSV TK) gene, allows stably transfected eukaryotic cells to be selected using G418. A bacterial promoter upstream of this cassette expresses kanamycin resistance in E.coli. The pIRES2-ZsGreen1 backbone also provides a pUC origin of replication for propagation in E. coli and an f1 origin for single-stranded DNA production.

pIRES2-ZsGreen1 can be used to quickly identify cells expressing a gene of interest by screening for ZsGreen1 fluorescence. Genes inserted into the MCS should include an initiating ATG codon. Selection of ZsGreen1-positive cells is possible 24–36 hours after transfection by flow cytometry or fluorescence microscopy. pIRES2-ZsGreen1 and its derivatives can be introduced into mammalian cells using any standard transfection method. If required, stable transformants can be selected using G418 (6).

Selection of Stable Transfectants

Selectable marker: plasmid confers resistance to G418 (500 μg/ml)

Propagation in E. coli

Suitable host strains: DH5α, HB101, and other general purpose strains. Single-stranded DNA production requires

a host containing an F plasmid such as JM101 or XL1-Blue.

Selectable marker: plasmid confers resistance to kanamycin (50 μg/ml) in E. coli hosts.

E. coli replication origin: pUC Copy number: high

pIRES2-­‐ZsGreen1载体序列

ORIGIN

1 TAGTTATTAA TAGTAATCAA TTACGGGGTC ATTAGTTCAT AGCCCATATA TGGAGTTCCG 61 CGTTACATAA CTTACGGTAA ATGGCCCGCC TGGCTGACCG CCCAACGACC CCCGCCCATT 121 GACGTCAATA ATGACGTATG TTCCCATAGT AACGCCAATA GGGACTTTCC ATTGACGTCA 181 ATGGGTGGAG TATTTACGGT AAACTGCCCA CTTGGCAGTA CATCAAGTGT ATCATATGCC 241 AAGTACGCCC CCTATTGACG TCAATGACGG TAAATGGCCC GCCTGGCATT ATGCCCAGTA 301 CATGACCTTA TGGGACTTTC CTACTTGGCA GTACATCTAC GTATTAGTCA TCGCTATTAC 361 CATGGTGATG CGGTTTTGGC AGTACATCAA TGGGCGTGGA TAGCGGTTTG ACTCACGGGG 421 ATTTCCAAGT CTCCACCCCA TTGACGTCAA TGGGAGTTTG TTTTGGCACC AAAATCAACG 481 GGACTTTCCA AAATGTCGTA ACAACTCCGC CCCATTGACG CAAATGGGCG GTAGGCGTGT 541 ACGGTGGGAG GTCTATATAA GCAGAGCTGG TTTAGTGAAC CGTCAGATCC GCTAGCGCTA 601 CCGGACTCAG ATCTCGAGCT CAAGCTTCGA ATTCTGCAGT CGACGGTACC GCGGGCCCGG 661 GATCCGCCCC TCTCCCTCCC CCCCCCCTAA CGTTACTGGC CGAAGCCGCT TGGAATAAGG 721 CCGGTGTGCG TTTGTCTATA TGTTATTTTC CACCATATTG CCGTCTTTTG GCAATGTGAG

781 GGCCCGGAAA CCTGGCCCTG TCTTCTTGAC GAGCATTCCT AGGGGTCTTT CCCCTCTCGC 841 CAAAGGAATG CAAGGTCTGT TGAATGTCGT GAAGGAAGCA GTTCCTCTGG AAGCTTCTTG 901 AAGACAAACA ACGTCTGTAG CGACCCTTTG CAGGCAGCGG AACCCCCCAC CTGGCGACAG 961 GTGCCTCTGC GGCCAAAAGC CACGTGTATA AGATACACCT GCAAAGGCGG CACAACCCCA 1021 GTGCCACGTT GTGAGTTGGA TAGTTGTGGA AAGAGTCAAA TGGCTCTCCT CAAGCGTATT 1081 CAACAAGGGG CTGAAGGATG CCCAGAAGGT ACCCCATTGT ATGGGATCTG ATCTGGGGCC 1141 TCGGTACACA TGCTTTACAT GTGTTTAGTC GAGGTTAAAA AAACGTCTAG GCCCCCCGAA 1201 CCACGGGGAC GTGGTTTTCC TTTGAAAAAC ACGATGATAA TATGGCCACA ACCATGGCCC 1261 AGTCCAAGCA CGGCCTGACC AAGGAGATGA CCATGAAGTA CCGCATGGAG GGCTGCGTGG 1321 ACGGCCACAA GTTCGTGATC ACCGGCGAGG GCATCGGCTA CCCCTTCAAG GGCAAGCAGG 1381 CCATCAACCT GTGCGTGGTG GAGGGCGGCC CCTTGCCCTT CGCCGAGGAC ATCTTGTCCG 1441 CCGCCTTCAT GTACGGCAAC CGCGTGTTCA CCGAGTACCC CCAGGACATC GTCGACTACT 1501 TCAAGAACTC CTGCCCCGCC GGCTACACCT GGGACCGCTC CTTCCTGTTC GAGGACGGCG 1561 CCGTGTGCAT CTGCAACGCC GACATCACCG TGAGCGTGGA GGAGAACTGC ATGTACCACG 1621 AGTCCAAGTT CTACGGCGTG AACTTCCCCG CCGACGGCCC CGTGATGAAG AAGATGACCG 1681 ACAACTGGGA GCCCTCCTGC GAGAAGATCA TCCCCGTGCC CAAGCAGGGC ATCTTGAAGG 1741 GCGACGTGAG CATGTACCTG CTGCTGAAGG ACGGTGGCCG CTTGCGCTGC CAGTTCGACA 1801 CCGTGTACAA GGCCAAGTCC GTGCCCCGCA AGATGCCCGA CTGGCACTTC ATCCAGCACA 1861 AGCTGACCCG CGAGGACCGC AGCGACGCCA AGAACCAGAA GTGGCACCTG ACCGAGCACG 1921 CCATCGCCTC CGGCTCCGCC TTGCCCTGAG CGGCCGCGAC TCTAGATCAT AATCAGCCAT 1981 ACCACATTTG TAGAGGTTTT ACTTGCTTTA AAAAACCTCC CACACCTCCC CCTGAACCTG 2041 AAACATAAAA TGAATGCAAT TGTTGTTGTT AACTTGTTTA TTGCAGCTTA TAATGGTTAC 2101 AAATAAAGCA ATAGCATCAC AAATTTCACA AATAAAGCAT TTTTTTCACT GCATTCTAGT 2161 TGTGGTTTGT CCAAACTCAT CAATGTATCT TAAGGCGTAA ATTGTAAGCG TTAATATTTT 2221 GTTAAAATTC GCGTTAAATT TTTGTTAAAT CAGCTCATTT TTTAACCAAT AGGCCGAAAT 2281 CGGCAAAATC CCTTATAAAT CAAAAGAATA GACCGAGATA GGGTTGAGTG TTGTTCCAGT 2341 TTGGAACAAG AGTCCACTAT TAAAGAACGT GGACTCCAAC GTCAAAGGGC GAAAAACCGT 2401 CTATCAGGGC GATGGCCCAC TACGTGAACC ATCACCCTAA TCAAGTTTTT TGGGGTCGAG 2461 GTGCCGTAAA GCACTAAATC GGAACCCTAA AGGGAGCCCC CGATTTAGAG CTTGACGGGG 2521 AAAGCCGGCG AACGTGGCGA GAAAGGAAGG GAAGAAAGCG AAAGGAGCGG GCGCTAGGGC 2581 GCTGGCAAGT GTAGCGGTCA CGCTGCGCGT AACCACCACA CCCGCCGCGC TTAATGCGCC 2641 GCTACAGGGC GCGTCAGGTG GCACTTTTCG GGGAAATGTG CGCGGAACCC CTATTTGTTT 2701 ATTTTTCTAA ATACATTCAA ATATGTATCC GCTCATGAGA CAATAACCCT GATAAATGCT 2761 TCAATAATAT TGAAAAAGGA AGAGTCCTGA GGCGGAAAGA ACCAGCTGTG GAATGTGTGT 2821 CAGTTAGGGT GTGGAAAGTC CCCAGGCTCC CCAGCAGGCA GAAGTATGCA AAGCATGCAT 2881 CTCAATTAGT CAGCAACCAG GTGTGGAAAG TCCCCAGGCT CCCCAGCAGG CAGAAGTATG 2941 CAAAGCATGC ATCTCAATTA GTCAGCAACC ATAGTCCCGC CCCTAACTCC GCCCATCCCG 3001 CCCCTAACTC CGCCCAGTTC CGCCCATTCT CCGCCCCATG GCTGACTAAT TTTTTTTATT 3061 TATGCAGAGG CCGAGGCCGC CTCGGCCTCT GAGCTATTCC AGAAGTAGTG AGGAGGCTTT 3121 TTTGGAGGCC TAGGCTTTTG CAAAGATCGA TCAAGAGACA GGATGAGGAT CGTTTCGCAT 3181 GATTGAACAA GATGGATTGC ACGCAGGTTC TCCGGCCGCT TGGGTGGAGA GGCTATTCGG 3241 CTATGACTGG GCACAACAGA CAATCGGCTG CTCTGATGCC GCCGTGTTCC GGCTGTCAGC 3301 GCAGGGGCGC CCGGTTCTTT TTGTCAAGAC CGACCTGTCC GGTGCCCTGA ATGAACTGCA 3361 AGACGAGGCA GCGCGGCTAT CGTGGCTGGC CACGACGGGC GTTCCTTGCG CAGCTGTGCT

3421 CGACGTTGTC ACTGAAGCGG GAAGGGACTG GCTGCTATTG GGCGAAGTGC CGGGGCAGGA 3481 TCTCCTGTCA TCTCACCTTG CTCCTGCCGA GAAAGTATCC ATCATGGCTG ATGCAATGCG 3541 GCGGCTGCAT ACGCTTGATC CGGCTACCTG CCCATTCGAC CACCAAGCGA AACATCGCAT 3601 CGAGCGAGCA CGTACTCGGA TGGAAGCCGG TCTTGTCGAT CAGGATGATC TGGACGAAGA 3661 GCATCAGGGG CTCGCGCCAG CCGAACTGTT CGCCAGGCTC AAGGCGAGCA TGCCCGACGG 3721 CGAGGATCTC GTCGTGACCC ATGGCGATGC CTGCTTGCCG AATATCATGG TGGAAAATGG 3781 CCGCTTTTCT GGATTCATCG ACTGTGGCCG GCTGGGTGTG GCGGACCGCT ATCAGGACAT 3841 AGCGTTGGCT ACCCGTGATA TTGCTGAAGA GCTTGGCGGC GAATGGGCTG ACCGCTTCCT 3901 CGTGCTTTAC GGTATCGCCG CTCCCGATTC GCAGCGCATC GCCTTCTATC GCCTTCTTGA 3961 CGAGTTCTTC TGAGCGGGAC TCTGGGGTTC GAAATGACCG ACCAAGCGAC GCCCAACCTG 4021 CCATCACGAG ATTTCGATTC CACCGCCGCC TTCTATGAAA GGTTGGGCTT CGGAATCGTT 4081 TTCCGGGACG CCGGCTGGAT GATCCTCCAG CGCGGGGATC TCATGCTGGA GTTCTTCGCC 4141 CACCCTAGGG GGAGGCTAAC TGAAACACGG AAGGAGACAA TACCGGAAGG AACCCGCGCT 4201 ATGACGGCAA TAAAAAGACA GAATAAAACG CACGGTGTTG GGTCGTTTGT TCATAAACGC 4261 GGGGTTCGGT CCCAGGGCTG GCACTCTGTC GATACCCCAC CGAGACCCCA TTGGGGCCAA 4321 TACGCCCGCG TTTCTTCCTT TTCCCCACCC CACCCCCCAA GTTCGGGTGA AGGCCCAGGG 4381 CTCGCAGCCA ACGTCGGGGC GGCAGGCCCT GCCATAGCCT CAGGTTACTC ATATATACTT 4441 TAGATTGATT TAAAACTTCA TTTTTAATTT AAAAGGATCT AGGTGAAGAT CCTTTTTGAT 4501 AATCTCATGA CCAAAATCCC TTAACGTGAG TTTTCGTTCC ACTGAGCGTC AGACCCCGTA 4561 GAAAAGATCA AAGGATCTTC TTGAGATCCT TTTTTTCTGC GCGTAATCTG CTGCTTGCAA 4621 ACAAAAAAAC CACCGCTACC AGCGGTGGTT TGTTTGCCGG ATCAAGAGCT ACCAACTCTT 4681 TTTCCGAAGG TAACTGGCTT CAGCAGAGCG CAGATACCAA ATACTGTTCT TCTAGTGTAG 4741 CCGTAGTTAG GCCACCACTT CAAGAACTCT GTAGCACCGC CTACATACCT CGCTCTGCTA 4801 ATCCTGTTAC CAGTGGCTGC TGCCAGTGGC GATAAGTCGT GTCTTACCGG GTTGGACTCA 4861 AGACGATAGT TACCGGATAA GGCGCAGCGG TCGGGCTGAA CGGGGGGTTC GTGCACACAG 4921 CCCAGCTTGG AGCGAACGAC CTACACCGAA CTGAGATACC TACAGCGTGA GCTATGAGAA 4981 AGCGCCACGC TTCCCGAAGG GAGAAAGGCG GACAGGTATC CGGTAAGCGG CAGGGTCGGA 5041 ACAGGAGAGC GCACGAGGGA GCTTCCAGGG GGAAACGCCT GGTATCTTTA TAGTCCTGTC 5101 GGGTTTCGCC ACCTCTGACT TGAGCGTCGA TTTTTGTGAT GCTCGTCAGG GGGGCGGAGC 5161 CTATGGAAAA ACGCCAGCAA CGCGGCCTTT TTACGGTTCC TGGCCTTTTG CTGGCCTTTT 5221 GCTCACATGT TCTTTCCTGC GTTATCCCCT GATTCTGTGG ATAACCGTAT TACCGCCATG 5281 CAT //

其他哺乳动物表达载体：

pCHO1.0 pcDNA3.1/His C pcDNA3.1/His B pcDNA3.1/His A pIRESpuro3 pIRES2-EGFP pTT5

pNFkB-DD-tdTomato pBI-CMV5

pBApo-CMV-Pur

pcDNA5/FRT/V5-His-TOPO pcDNA5/FRT/TO-TOPO pcDNA5/TO pcDNA5/FRT/TO pcDNA5/FRT pFLAG-CMV2

pcDNA3.1/CT-GFP-TOPO pcDNA3.1/NT-GFP-TOPO

pOPRSVI pREP4 pDual-GC pBK-RSV pBK-CMV pBI-CMV4

pcDNA4/TO/Myc-His/LacZ pOPI3CAT pGene/V5-His B

pSEAP2-Basic pSEAP2-Control pBI-CMV3 pBI-CMV2 pBI-CMV1

pNF κB-MetLuc2-Reporter pCRE-MetLuc2-Reporter pAcGFP1-Actin

pAcGFP1-N In-Fusion Ready pAcGFP1-C3 pAcGFP1-C pAcGFP1-p53 pAcGFP1-Mito pAcGFP1-Mem pAcGFP1-Lam pAcGFP1-Golgi pAcGFP1-F pAcGFP1-Hyg-C1 pAsRed2-N1 ptdTomato-N1 pCMV-tdTomato pCRE-DD-tdTomato pCMV-DsRed-Express2 pEF1α-tdTomato pCRE-hrGFP ptdTomato-C1 pAsRed2-C1 pGL3-Promoter pGL3 basic pAcGFP1-C2 pAcGFP1-C1 pAcGFP1-N3 pAcGFP1-N2 pAcGFP1-N1

pAcGFP1-C In-Fusion Ready pCRE-DD-AmCyan1 pNFkB-DD-AmCyan1 pDsRed2-Bid pDsRED2-Mito

pDD-AmCyan1 Reporter pAmCyan1-N1 pAmCyan1-C1

pEF1α-IRES-DsRed-Express2 pEF1α-DsRed-Monomer-N1

pOptiVEC-TOPO pCMV-MEKK1 pCMV-MEK1 pCMV-PKA

pcDNA6.2/nTC-Tag-DEST pcDNA6.2/cTC-Tag-DEST pcDNA3.2/V5/GW/D-TOPO pcDNA6.2/V5/GW/D-TOPO

pcDNA6.2/nGeneBLAzer-GW/D-TOPO pcDNA6.2/C-YFP-DEST pcDNA6.2/cGeneBLAzer-DEST pcDNA6/V5-His A pcDNA6/V5-His B pcDNA6/V5-His C pcDNA6/myc-His C pcDNA6/myc-His A pcDNA6/myc-His B

pcDNA6.2/nGeneBLAzer-DEST pcDNA4/HisMax-TOPO pcDNA6.2/nLumio-DEST pcDNA6.2/cLumio-DEST pcDNA4/myc-His C pcDNA4/HisMax C pcDNA4/HisMax A c-Flag pcDNA3 pcDNA4/HisMax B pcDNA4/myc-His B pcDNA4/myc-His A pcDNA4/His C pcDNA4/His B pcDNA4/His A pcDNA6/TR

pcDNA4/TO/Myc-His A pcDNA4/TO

pcDNA4/TO/Myc-His B pcDNA4/TO/Myc-His C pcDNA3.3-TOPO pBudCE4.1 pFLAG-CMV-4 pFLAG-CMV-3 pFLAG-CMV-2 pFLAG-CMV-5a p3XFLAG-CMV-9 p3xFLAG-CMV-10

pSwitch pCMVLacI pVgRxR pIND pTRE3G-Luc pTRE3G pTRE2-hygro pTRE-Tight pTK-hyg pTet-On pTet-Off

pTet on advanced pRevTRE pRevTet-On pRevTet-Off pCMV-Tet3G pTRE2 pBD-NF-κB pCMV-AD pCMV-BD pBIND-Id Control pBIND pG5 luciferase pACT-MyoD pACT

pCMV-SPORT6 pGL4.13 pGL4.19 pGL4.26 pGL4.20 pGL4.29 pGL4.30 pGL4.27 pGL4.75 pGL4.10 pGRN145 pSecTag2 A pEBVHis B pEBVHis A pCMV-Tag 3C pCMV-Tag 3A pCMV-Tag 3B pCMV-Tag 5C pCMV-Tag 5A

pDsRED-Monomer-N1 pDsRed-Express-N1 p3XFLAG-CMV-7 pDsRed-Express-C1 pIRES2-ZsGreen1 pDsRed-Express2-C1 pDsRed-Express2-N1 pEF1α-DsRed-Express2 pIRES2-DsRed-Express pIRES2-DsRed-Express2 pIRES-hrGFP-1a pIRESneo2 pIRESneo pIREShyg3

p3XFLAG-CMV-8 p3XFLAG-CMV-7.1

pDsRed-Monomer-N In-Fusion Ready p3XFLAG-CMV-13 p3XFLAG-CMV-14 plRES2-ZsGreen1 pBApo-EF1α-pur pBApo-EF1α-neo pBApo-CMV pBApo-CMV-neo pIRES-EGFP pIRESneo3 pDsRed-Monomer pIRES

pCMV-Tag 4A pCMV-Tag 5B pCMV-Tag 4B pCMV-Tag 2C pCMV-Tag 2B pCMV-Tag 2A pCMV-LacZ pCMV-Myc

pEF1α-IRES-AcGFP1 pEF1α-IRES-ZsGreen1 pEF1α-AcGFP1-N1 pIRES2-DsRed2 pIRES2-AcGFP1