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pIRES2 EGFP载体基本信息
| Clontech |
| pIRES2-EGFP, pIRES2 EGFP |
| 哺乳动物细胞表达载体 |
| CMV |
| 高 |
| 多克隆位点，限制性内切酶 |
| 5308 bp |
| CMV-F: 5'-CGCAAATGGGCGGTAGGCGTG-3' |
| EGFP-N: 5'-CGTCGCCGTCCAGCTCGACCAG-3' |
| / |
| 卡那 |
| 新霉素(G418) |
通过一个转录mRNA，能够同时表达EGFP蛋白和一个目的基因。 |
| 6029-1 |
| 稳表达 |
| 组成型 |
| 非病毒 |
pIRES2 EGFP载体质粒图谱和多克隆位点信息
pIRES2 EGFP载体简介
Description:
pIRES2-EGFP contains the internal ribosome entry site (IRES; 1, 2) of the encephalomyocarditis virus (ECMV) between the MCS and the enhanced green fluorescent protein (EGFP) coding region. This permits both the gene of interest (cloned into the MCS) and the EGFP gene to be translated from a single bicistronic mRNA. pIRES2-EGFP is designed for the efficient selection (by flow cytometry or other methods) of transiently transfected mammalian cells expressing EGFP and the protein of interest. This vector can also be used to express EGFP alone or to obtain stably transfected cell lines without time-consuming drug and clonal selection. EGFP is a red-shifted variant of wild-type GFP (3–5) which has been optimized for brighter fluorescence and higher expression in mammalian cells. (Excitation maximum = 488 nm; emission maximum = 507 nm.) EGFP encodes the GFPmut1 variant (6) which contains the double-amino-acid substitution of Phe-64 to Leu and Ser-65 to Thr. The coding sequence of the EGFP gene contains more than 190 silent base changes which correspond to human codon-usage preferences (7). Sequences flanking EGFP have been converted to a Kozak consensus translation initiation site (8) to further increase the translation efficiency in eukaryotic cells. The MCS in pIRES2-EGFP is between the immediate early promoter of cytomegalovirus (PCMV IE) and the IRES sequence. SV40 polyadenylation signals downstream of the EGFP 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-EGFP backbone also provides a pUC origin of replication for propagation in E. coli and an f1 origin for single-stranded DNA production. pIRES2-EGFP replaces (but is not derived from) the pIRES-EGFP Vector previously sold by BD Biosciences Clontech. pIRES2-EGFP is functionally similarly to pIRES-EGFP; however, pIRES2- EGFP gives brighter EGFP fluorescence than the older vector. Note that the Xba I site at position 1987 is methylated in the DNA provided by BD Biosciences Clontech. If you wish to digest the vector with this enzyme, you will need to transform the vector into a dam– host and make fresh DNA.
Use:
Genes inserted into the MCS should include the initiating ATG codon. pIRES2-EGFP and its derivatives can be introduced into mammalian cells using any standard transfection method. If required, stable transformants can be selected using G418 (9).
pIRES2 EGFP载体序列
DEFINITION pIRES2-EGFP
ACCESSION
KEYWORDS
SOURCE
ORGANISM other sequences; artificial sequences; vectors.
COMMENT This file is created by Vector NTI
http://www.biofeng.com/
COMMENT ORIGDB|GenBank
COMMENT VNTAUTHORNAME|biofeng.com|
FEATURES Location/Qualifiers
source 1..5308
/organism="pIRES2-EGFP"
/mol_type="other DNA"
promoter 10..562
/label="CMV_immearly_promoter"
misc_feature 65..352
/label="CAG_enhancer"
misc_feature 519..539
/label="CMV_fwd_primer"
promoter 520..589
/label="CMV_promoter"
misc_feature 666..1250
/label="IRES"
CDS 1242..1973
/label="ORF frame 3"
gene 1257..1970
/label="EGFP"
/gene="EGFP"
misc_feature complement(1299..1320)
/label="EGFP_N_primer"
misc_feature 1437..1466
/label="Y66 (EGFP)"
misc_feature 1907..1928
/label="EGFP_C_primer"
misc_feature 2187..2206
/label="EBV_rev_primer"
rep_origin complement(2356..2662)
/label="f1_origin"
promoter 2741..2769
/label="AmpR_promoter"
misc_feature complement(2835..2855)
/label="pBABE_3_primer"
misc_feature complement(2841..3056)
/label="SV40_enhancer"
promoter 2853..3121
/label="SV40_promoter"
rep_origin 3020..3097
/label="SV40_origin"
misc_feature 3082..3101
/label="SV40pro_F_primer"
CDS 3204..3998
/label="ORF frame 3"
gene 3207..3995
/label="NeoR/KanR"
/gene="NeoR/KanR"
CDS complement(3513..4049)
/label="ORF frame 3"
terminator 4173..4442
/label="TK_PA_terminator"
rep_origin 4590..5209
/label="pBR322_origin"
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 TCGGTGCACA TGCTTTACAT GTGTTTAGTC GAGGTTAAAA AAACGTCTAG GCCCCCCGAA
1201 CCACGGGGAC GTGGTTTTCC TTTGAAAAAC ACGATGATAA TATGGCCACA ACCATGGTGA
1261 GCAAGGGCGA GGAGCTGTTC ACCGGGGTGG TGCCCATCCT GGTCGAGCTG GACGGCGACG
1321 TAAACGGCCA CAAGTTCAGC GTGTCCGGCG AGGGCGAGGG CGATGCCACC TACGGCAAGC
1381 TGACCCTGAA GTTCATCTGC ACCACCGGCA AGCTGCCCGT GCCCTGGCCC ACCCTCGTGA
1441 CCACCCTGAC CTACGGCGTG CAGTGCTTCA GCCGCTACCC CGACCACATG AAGCAGCACG
1501 ACTTCTTCAA GTCCGCCATG CCCGAAGGCT ACGTCCAGGA GCGCACCATC TTCTTCAAGG
1561 ACGACGGCAA CTACAAGACC CGCGCCGAGG TGAAGTTCGA GGGCGACACC CTGGTGAACC
1621 GCATCGAGCT GAAGGGCATC GACTTCAAGG AGGACGGCAA CATCCTGGGG CACAAGCTGG
1681 AGTACAACTA CAACAGCCAC AACGTCTATA TCATGGCCGA CAAGCAGAAG AACGGCATCA
1741 AGGTGAACTT CAAGATCCGC CACAACATCG AGGACGGCAG CGTGCAGCTC GCCGACCACT
1801 ACCAGCAGAA CACCCCCATC GGCGACGGCC CCGTGCTGCT GCCCGACAAC CACTACCTGA
1861 GCACCCAGTC CGCCCTGAGC AAAGACCCCA ACGAGAAGCG CGATCACATG GTCCTGCTGG
1921 AGTTCGTGAC CGCCGCCGGG ATCACTCTCG GCATGGACGA GCTGTACAAG TAAAGCGGCC
1981 GCGACTCTAG ATCATAATCA GCCATACCAC ATTTGTAGAG GTTTTACTTG CTTTAAAAAA
2041 CCTCCCACAC CTCCCCCTGA ACCTGAAACA TAAAATGAAT GCAATTGTTG TTGTTAACTT
2101 GTTTATTGCA GCTTATAATG GTTACAAATA AAGCAATAGC ATCACAAATT TCACAAATAA
2161 AGCATTTTTT TCACTGCATT CTAGTTGTGG TTTGTCCAAA CTCATCAATG TATCTTAAGG
2221 CGTAAATTGT AAGCGTTAAT ATTTTGTTAA AATTCGCGTT AAATTTTTGT TAAATCAGCT
2281 CATTTTTTAA CCAATAGGCC GAAATCGGCA AAATCCCTTA TAAATCAAAA GAATAGACCG
2341 AGATAGGGTT GAGTGTTGTT CCAGTTTGGA ACAAGAGTCC ACTATTAAAG AACGTGGACT
2401 CCAACGTCAA AGGGCGAAAA ACCGTCTATC AGGGCGATGG CCCACTACGT GAACCATCAC
2461 CCTAATCAAG TTTTTTGGGG TCGAGGTGCC GTAAAGCACT AAATCGGAAC CCTAAAGGGA
2521 GCCCCCGATT TAGAGCTTGA CGGGGAAAGC CGGCGAACGT GGCGAGAAAG GAAGGGAAGA
2581 AAGCGAAAGG AGCGGGCGCT AGGGCGCTGG CAAGTGTAGC GGTCACGCTG CGCGTAACCA
2641 CCACACCCGC CGCGCTTAAT GCGCCGCTAC AGGGCGCGTC AGGTGGCACT TTTCGGGGAA
2701 ATGTGCGCGG AACCCCTATT TGTTTATTTT TCTAAATACA TTCAAATATG TATCCGCTCA
2761 TGAGACAATA ACCCTGATAA ATGCTTCAAT AATATTGAAA AAGGAAGAGT CCTGAGGCGG
2821 AAAGAACCAG CTGTGGAATG TGTGTCAGTT AGGGTGTGGA AAGTCCCCAG GCTCCCCAGC
2881 AGGCAGAAGT ATGCAAAGCA TGCATCTCAA TTAGTCAGCA ACCAGGTGTG GAAAGTCCCC
2941 AGGCTCCCCA GCAGGCAGAA GTATGCAAAG CATGCATCTC AATTAGTCAG CAACCATAGT
3001 CCCGCCCCTA ACTCCGCCCA TCCCGCCCCT AACTCCGCCC AGTTCCGCCC ATTCTCCGCC
3061 CCATGGCTGA CTAATTTTTT TTATTTATGC AGAGGCCGAG GCCGCCTCGG CCTCTGAGCT
3121 ATTCCAGAAG TAGTGAGGAG GCTTTTTTGG AGGCCTAGGC TTTTGCAAAG ATCGATCAAG
3181 AGACAGGATG AGGATCGTTT CGCATGATTG AACAAGATGG ATTGCACGCA GGTTCTCCGG
3241 CCGCTTGGGT GGAGAGGCTA TTCGGCTATG ACTGGGCACA ACAGACAATC GGCTGCTCTG
3301 ATGCCGCCGT GTTCCGGCTG TCAGCGCAGG GGCGCCCGGT TCTTTTTGTC AAGACCGACC
3361 TGTCCGGTGC CCTGAATGAA CTGCAAGACG AGGCAGCGCG GCTATCGTGG CTGGCCACGA
3421 CGGGCGTTCC TTGCGCAGCT GTGCTCGACG TTGTCACTGA AGCGGGAAGG GACTGGCTGC
3481 TATTGGGCGA AGTGCCGGGG CAGGATCTCC TGTCATCTCA CCTTGCTCCT GCCGAGAAAG
3541 TATCCATCAT GGCTGATGCA ATGCGGCGGC TGCATACGCT TGATCCGGCT ACCTGCCCAT
3601 TCGACCACCA AGCGAAACAT CGCATCGAGC GAGCACGTAC TCGGATGGAA GCCGGTCTTG
3661 TCGATCAGGA TGATCTGGAC GAAGAGCATC AGGGGCTCGC GCCAGCCGAA CTGTTCGCCA
3721 GGCTCAAGGC GAGCATGCCC GACGGCGAGG ATCTCGTCGT GACCCATGGC GATGCCTGCT
3781 TGCCGAATAT CATGGTGGAA AATGGCCGCT TTTCTGGATT CATCGACTGT GGCCGGCTGG
3841 GTGTGGCGGA CCGCTATCAG GACATAGCGT TGGCTACCCG TGATATTGCT GAAGAGCTTG
3901 GCGGCGAATG GGCTGACCGC TTCCTCGTGC TTTACGGTAT CGCCGCTCCC GATTCGCAGC
3961 GCATCGCCTT CTATCGCCTT CTTGACGAGT TCTTCTGAGC GGGACTCTGG GGTTCGAAAT
4021 GACCGACCAA GCGACGCCCA ACCTGCCATC ACGAGATTTC GATTCCACCG CCGCCTTCTA
4081 TGAAAGGTTG GGCTTCGGAA TCGTTTTCCG GGACGCCGGC TGGATGATCC TCCAGCGCGG
4141 GGATCTCATG CTGGAGTTCT TCGCCCACCC TAGGGGGAGG CTAACTGAAA CACGGAAGGA
4201 GACAATACCG GAAGGAACCC GCGCTATGAC GGCAATAAAA AGACAGAATA AAACGCACGG
4261 TGTTGGGTCG TTTGTTCATA AACGCGGGGT TCGGTCCCAG GGCTGGCACT CTGTCGATAC
4321 CCCACCGAGA CCCCATTGGG GCCAATACGC CCGCGTTTCT TCCTTTTCCC CACCCCACCC
4381 CCCAAGTTCG GGTGAAGGCC CAGGGCTCGC AGCCAACGTC GGGGCGGCAG GCCCTGCCAT
4441 AGCCTCAGGT TACTCATATA TACTTTAGAT TGATTTAAAA CTTCATTTTT AATTTAAAAG
4501 GATCTAGGTG AAGATCCTTT TTGATAATCT CATGACCAAA ATCCCTTAAC GTGAGTTTTC
4561 GTTCCACTGA GCGTCAGACC CCGTAGAAAA GATCAAAGGA TCTTCTTGAG ATCCTTTTTT
4621 TCTGCGCGTA ATCTGCTGCT TGCAAACAAA AAAACCACCG CTACCAGCGG TGGTTTGTTT
4681 GCCGGATCAA GAGCTACCAA CTCTTTTTCC GAAGGTAACT GGCTTCAGCA GAGCGCAGAT
4741 ACCAAATACT GTCCTTCTAG TGTAGCCGTA GTTAGGCCAC CACTTCAAGA ACTCTGTAGC
4801 ACCGCCTACA TACCTCGCTC TGCTAATCCT GTTACCAGTG GCTGCTGCCA GTGGCGATAA
4861 GTCGTGTCTT ACCGGGTTGG ACTCAAGACG ATAGTTACCG GATAAGGCGC AGCGGTCGGG
4921 CTGAACGGGG GGTTCGTGCA CACAGCCCAG CTTGGAGCGA ACGACCTACA CCGAACTGAG
4981 ATACCTACAG CGTGAGCTAT GAGAAAGCGC CACGCTTCCC GAAGGGAGAA AGGCGGACAG
5041 GTATCCGGTA AGCGGCAGGG TCGGAACAGG AGAGCGCACG AGGGAGCTTC CAGGGGGAAA
5101 CGCCTGGTAT CTTTATAGTC CTGTCGGGTT TCGCCACCTC TGACTTGAGC GTCGATTTTT
5161 GTGATGCTCG TCAGGGGGGC GGAGCCTATG GAAAAACGCC AGCAACGCGG CCTTTTTACG
5221 GTTCCTGGCC TTTTGCTGGC CTTTTGCTCA CATGTTCTTT CCTGCGTTAT CCCCTGATTC
5281 TGTGGATAAC CGTATTACCG CCATGCAT
//
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