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哺乳动物过表达质粒

pIRES2-zsGreen1 哺乳动物表达载体;双顺反子载体;荧光报告载体
发布时间:2018-05-27 20:53:02 | 浏览次数:


测序及更多信息:链接:https://pan.baidu.com/s/1hh_7AXyFvUjzIbMarABtIQ 
提取码:c37w

采购地址:淘宝店铺地址

pIRES2-ZsGreen1

载体基本信息

载体名称:

pIRES2-ZsGreen1

质粒类型:

哺乳动物细胞载体;荧光报告载体;双顺反子载体

高拷贝/低拷贝:

高拷贝

克隆方法:

限制性内切酶,多克隆位点

启动子:

CMV

载体大小:

5283 bp

5' 测序引物及序列:

CMV-F: 5'-CGCAAATGGGCGGTAGGCGTG-3'

3' 测序引物及序列:

--

载体标签:

绿色荧光蛋白ZsGreen1(C-端)

载体抗性:

卡那霉素

筛选标记:

新霉素(Neomycin

克隆菌株:

DH5α, HB101

宿主细胞(系):

常规细胞系,293CV-1CHO

备注:

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。

稳定性:

瞬表达 稳表达

组成型/诱导型:

组成型

病毒/非病毒:

非病毒



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


全序列

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



 
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