| 出品公司: | Clontech |
|---|---|
| 别名: | pEGFP-N3, pEGFP N3 |
| 质粒类型: | 哺乳动物细胞表达载体 |
| 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: | 4729bp |
| 5' 测序引物: | CMV-F |
| 5' 测序引物序列: | 5'-CGCAAATGGGCGGTAGGCGTG-3' |
| 3' 测序引物: | EGFP-N |
| 3' 测序引物序列: | 5'-CGTCGCCGTCCAGCTCGACCAG-3' |
| 载体标签: | C-EGFP |
| 载体抗性: | Kanamycin |
| 筛选标记: | Neomycin |
| 备注: |
This plasmid has been discontinued by Clontech. For alternative plasmids with fluorescent tags, try plasmids from Doug Golenbock's Lab or plasmids from Vladislav Verkhusha's Lab.
|
| 产品目录号: | 6080-1 |
| 稳定性: | 稳表达 Stable |
| 组成型: | 组成型 Constitutive |
| 病毒/非病毒: | 非病毒 |
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pEGFP N3载体基本信息
pEGFP N3载体图谱质粒图谱和多克隆位点信息
pEGFP N3载体简介
Description:
pEGFP-N3 encodes a red-shifted variant of wild-type GFP (1–3) which has been optimized for brighter fluorescence and higher expression in mammalian cells. (Excitation maximum = 488 nm;emission maximum = 507 nm.) pEGFP-N3 encodes the GFPmut1 variant (4) 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 (5). Sequences flanking EGFP have been converted to a Kozak consensus translation initiation site (6) to further increase the translation efficiency in eukaryotic cells. The MCS in pEGFP-N3 is between the immediate early promoter of CMV (PCMV IE) and the EGFP coding sequences. Genes cloned into the MCS will be expressed as fusions to the N terminus of EGFP if they are in the same reading frame as EGFP and there are no intervening stop codons. SV40polyadenylation signals downstream of the EGFP gene direct proper processing of the 3' end of the EGFP 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 tobe selected using G418. A bacterial promoter upstream of this cassette expresses kanamycin resistance in E. coli. The pEGFP-N3 backbone also provides a pUC origin of replication for propagation in E. coli and an f1 origin for single-stranded DNA production.
Use:
Fusions to the N terminus of EGFP retain the fluorescent properties of the native protein allowing the localization of the fusion protein in vivo . The target gene should be cloned into pEGFP-N3 so that it is in frame with the EGFP coding sequences, with no intervening in-frame stop codons. The inserted gene should include the initiating ATG codon. The recombinant EGFP vector can be transfected into mammalian cells using any standard transfection method. If required, stable transformants can be selected using G418 (7). pEGFP-N3 can also be used simply to express EGFP in a cell line of interest (e.g., as a transfection marker).
pEGFP N3载体序列
LOCUS pEGFP-N3 4729 bp DNA circular SYN DEFINITION pEGFP-N3 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..4729 /organism="pEGFP-N3" /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" CDS 675..1394 /label="ORF frame 3" gene 678..1391 /label="EGFP" /gene="EGFP" misc_feature complement(720..741) /label="EGFP_N_primer" misc_feature 858..887 /label="Y66 (EGFP)" misc_feature 1328..1349 /label="EGFP_C_primer" misc_feature 1608..1627 /label="EBV_rev_primer" rep_origin complement(1777..2083) /label="f1_origin" promoter 2162..2190 /label="AmpR_promoter" misc_feature complement(2256..2276) /label="pBABE_3_primer" misc_feature complement(2262..2477) /label="SV40_enhancer" promoter 2274..2542 /label="SV40_promoter" rep_origin 2441..2518 /label="SV40_origin" misc_feature 2503..2522 /label="SV40pro_F_primer" CDS 2625..3419 /label="ORF frame 3" gene 2628..3416 /label="NeoR/KanR" /gene="NeoR/KanR" CDS complement(2934..3470) /label="ORF frame 3" terminator 3594..3863 /label="TK_PA_terminator" rep_origin 4011..4630 /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 GATCCATCGC CACCATGGTG AGCAAGGGCG AGGAGCTGTT CACCGGGGTG GTGCCCATCC 721 TGGTCGAGCT GGACGGCGAC GTAAACGGCC ACAAGTTCAG CGTGTCCGGC GAGGGCGAGG 781 GCGATGCCAC CTACGGCAAG CTGACCCTGA AGTTCATCTG CACCACCGGC AAGCTGCCCG 841 TGCCCTGGCC CACCCTCGTG ACCACCCTGA CCTACGGCGT GCAGTGCTTC AGCCGCTACC 901 CCGACCACAT GAAGCAGCAC GACTTCTTCA AGTCCGCCAT GCCCGAAGGC TACGTCCAGG 961 AGCGCACCAT CTTCTTCAAG GACGACGGCA ACTACAAGAC CCGCGCCGAG GTGAAGTTCG 1021 AGGGCGACAC CCTGGTGAAC CGCATCGAGC TGAAGGGCAT CGACTTCAAG GAGGACGGCA 1081 ACATCCTGGG GCACAAGCTG GAGTACAACT ACAACAGCCA CAACGTCTAT ATCATGGCCG 1141 ACAAGCAGAA GAACGGCATC AAGGTGAACT TCAAGATCCG CCACAACATC GAGGACGGCA 1201 GCGTGCAGCT CGCCGACCAC TACCAGCAGA ACACCCCCAT CGGCGACGGC CCCGTGCTGC 1261 TGCCCGACAA CCACTACCTG AGCACCCAGT CCGCCCTGAG CAAAGACCCC AACGAGAAGC 1321 GCGATCACAT GGTCCTGCTG GAGTTCGTGA CCGCCGCCGG GATCACTCTC GGCATGGACG 1381 AGCTGTACAA GTAAAGCGGC CGCGACTCTA GATCATAATC AGCCATACCA CATTTGTAGA 1441 GGTTTTACTT GCTTTAAAAA ACCTCCCACA CCTCCCCCTG AACCTGAAAC ATAAAATGAA 1501 TGCAATTGTT GTTGTTAACT TGTTTATTGC AGCTTATAAT GGTTACAAAT AAAGCAATAG 1561 CATCACAAAT TTCACAAATA AAGCATTTTT TTCACTGCAT TCTAGTTGTG GTTTGTCCAA 1621 ACTCATCAAT GTATCTTAAG GCGTAAATTG TAAGCGTTAA TATTTTGTTA AAATTCGCGT 1681 TAAATTTTTG TTAAATCAGC TCATTTTTTA ACCAATAGGC CGAAATCGGC AAAATCCCTT 1741 ATAAATCAAA AGAATAGACC GAGATAGGGT TGAGTGTTGT TCCAGTTTGG AACAAGAGTC 1801 CACTATTAAA GAACGTGGAC TCCAACGTCA AAGGGCGAAA AACCGTCTAT CAGGGCGATG 1861 GCCCACTACG TGAACCATCA CCCTAATCAA GTTTTTTGGG GTCGAGGTGC CGTAAAGCAC 1921 TAAATCGGAA CCCTAAAGGG AGCCCCCGAT TTAGAGCTTG ACGGGGAAAG CCGGCGAACG 1981 TGGCGAGAAA GGAAGGGAAG AAAGCGAAAG GAGCGGGCGC TAGGGCGCTG GCAAGTGTAG 2041 CGGTCACGCT GCGCGTAACC ACCACACCCG CCGCGCTTAA TGCGCCGCTA CAGGGCGCGT 2101 CAGGTGGCAC TTTTCGGGGA AATGTGCGCG GAACCCCTAT TTGTTTATTT TTCTAAATAC 2161 ATTCAAATAT GTATCCGCTC ATGAGACAAT AACCCTGATA AATGCTTCAA TAATATTGAA 2221 AAAGGAAGAG TCCTGAGGCG GAAAGAACCA GCTGTGGAAT GTGTGTCAGT TAGGGTGTGG 2281 AAAGTCCCCA GGCTCCCCAG CAGGCAGAAG TATGCAAAGC ATGCATCTCA ATTAGTCAGC 2341 AACCAGGTGT GGAAAGTCCC CAGGCTCCCC AGCAGGCAGA AGTATGCAAA GCATGCATCT 2401 CAATTAGTCA GCAACCATAG TCCCGCCCCT AACTCCGCCC ATCCCGCCCC TAACTCCGCC 2461 CAGTTCCGCC CATTCTCCGC CCCATGGCTG ACTAATTTTT TTTATTTATG CAGAGGCCGA 2521 GGCCGCCTCG GCCTCTGAGC TATTCCAGAA GTAGTGAGGA GGCTTTTTTG GAGGCCTAGG 2581 CTTTTGCAAA GATCGATCAA GAGACAGGAT GAGGATCGTT TCGCATGATT GAACAAGATG 2641 GATTGCACGC AGGTTCTCCG GCCGCTTGGG TGGAGAGGCT ATTCGGCTAT GACTGGGCAC 2701 AACAGACAAT CGGCTGCTCT GATGCCGCCG TGTTCCGGCT GTCAGCGCAG GGGCGCCCGG 2761 TTCTTTTTGT CAAGACCGAC CTGTCCGGTG CCCTGAATGA ACTGCAAGAC GAGGCAGCGC 2821 GGCTATCGTG GCTGGCCACG ACGGGCGTTC CTTGCGCAGC TGTGCTCGAC GTTGTCACTG 2881 AAGCGGGAAG GGACTGGCTG CTATTGGGCG AAGTGCCGGG GCAGGATCTC CTGTCATCTC 2941 ACCTTGCTCC TGCCGAGAAA GTATCCATCA TGGCTGATGC AATGCGGCGG CTGCATACGC 3001 TTGATCCGGC TACCTGCCCA TTCGACCACC AAGCGAAACA TCGCATCGAG CGAGCACGTA 3061 CTCGGATGGA AGCCGGTCTT GTCGATCAGG ATGATCTGGA CGAAGAGCAT CAGGGGCTCG 3121 CGCCAGCCGA ACTGTTCGCC AGGCTCAAGG CGAGCATGCC CGACGGCGAG GATCTCGTCG 3181 TGACCCATGG CGATGCCTGC TTGCCGAATA TCATGGTGGA AAATGGCCGC TTTTCTGGAT 3241 TCATCGACTG TGGCCGGCTG GGTGTGGCGG ACCGCTATCA GGACATAGCG TTGGCTACCC 3301 GTGATATTGC TGAAGAGCTT GGCGGCGAAT GGGCTGACCG CTTCCTCGTG CTTTACGGTA 3361 TCGCCGCTCC CGATTCGCAG CGCATCGCCT TCTATCGCCT TCTTGACGAG TTCTTCTGAG 3421 CGGGACTCTG GGGTTCGAAA TGACCGACCA AGCGACGCCC AACCTGCCAT CACGAGATTT 3481 CGATTCCACC GCCGCCTTCT ATGAAAGGTT GGGCTTCGGA ATCGTTTTCC GGGACGCCGG 3541 CTGGATGATC CTCCAGCGCG GGGATCTCAT GCTGGAGTTC TTCGCCCACC CTAGGGGGAG 3601 GCTAACTGAA ACACGGAAGG AGACAATACC GGAAGGAACC CGCGCTATGA CGGCAATAAA 3661 AAGACAGAAT AAAACGCACG GTGTTGGGTC GTTTGTTCAT AAACGCGGGG TTCGGTCCCA 3721 GGGCTGGCAC TCTGTCGATA CCCCACCGAG ACCCCATTGG GGCCAATACG CCCGCGTTTC 3781 TTCCTTTTCC CCACCCCACC CCCCAAGTTC GGGTGAAGGC CCAGGGCTCG CAGCCAACGT 3841 CGGGGCGGCA GGCCCTGCCA TAGCCTCAGG TTACTCATAT ATACTTTAGA TTGATTTAAA 3901 ACTTCATTTT TAATTTAAAA GGATCTAGGT GAAGATCCTT TTTGATAATC TCATGACCAA 3961 AATCCCTTAA CGTGAGTTTT CGTTCCACTG AGCGTCAGAC CCCGTAGAAA AGATCAAAGG 4021 ATCTTCTTGA GATCCTTTTT TTCTGCGCGT AATCTGCTGC TTGCAAACAA AAAAACCACC 4081 GCTACCAGCG GTGGTTTGTT TGCCGGATCA AGAGCTACCA ACTCTTTTTC CGAAGGTAAC 4141 TGGCTTCAGC AGAGCGCAGA TACCAAATAC TGTCCTTCTA GTGTAGCCGT AGTTAGGCCA 4201 CCACTTCAAG AACTCTGTAG CACCGCCTAC ATACCTCGCT CTGCTAATCC TGTTACCAGT 4261 GGCTGCTGCC AGTGGCGATA AGTCGTGTCT TACCGGGTTG GACTCAAGAC GATAGTTACC 4321 GGATAAGGCG CAGCGGTCGG GCTGAACGGG GGGTTCGTGC ACACAGCCCA GCTTGGAGCG 4381 AACGACCTAC ACCGAACTGA GATACCTACA GCGTGAGCTA TGAGAAAGCG CCACGCTTCC 4441 CGAAGGGAGA AAGGCGGACA GGTATCCGGT AAGCGGCAGG GTCGGAACAG GAGAGCGCAC 4501 GAGGGAGCTT CCAGGGGGAA ACGCCTGGTA TCTTTATAGT CCTGTCGGGT TTCGCCACCT 4561 CTGACTTGAG CGTCGATTTT TGTGATGCTC GTCAGGGGGG CGGAGCCTAT GGAAAAACGC 4621 CAGCAACGCG GCCTTTTTAC GGTTCCTGGC CTTTTGCTGG CCTTTTGCTC ACATGTTCTT 4681 TCCTGCGTTA TCCCCTGATT CTGTGGATAA CCGTATTACC GCCATGCAT //
