| 质粒名称: | pEGFP-N1-FLAG |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 60360 |
| 存储实验室: | Patrick Calsou |
|
载体骨架基本信息 |
|
| 载体名称: | pEGFP-N1 |
| 载体出品公司: | Clontech |
| 载体抗性: | 卡那霉素 |
| 空载体大小: | -- |
| 完整质粒大小: | 4733 bp |
| 载体修饰: | 在GFP前面插入一个标志标签以及一个ATG和一个强kozak序列。 |
| 载体类型: | 哺乳细胞表达 |
| 筛选标记: | 新霉素（G418） |
| 高拷贝/低拷贝: | 高拷贝 |
| 生长条件: | 37 ℃ |
|
插入基因信息 |
|
| 插入基因名称: | -- |
| 物种来源: | -- |
| 插入大小: | -- |
| 基因突变: | -- |
| GenBank编号 | -- |
| 启动子 | CMV |
| 标签/融合蛋白: |
FLAG（主干上的N个终端）
EGFP（主干上的C端）
|
| 克隆方法: | 限制性内切酶 |
| 5' 克隆位点: | -- |
| 3' 克隆位点: | -- |
| 5' 测序引物 | CMV Forward |
| 3' 测序引物 | EGFP-N |
|
文献引用方法 |
|
| 材料和方法部分: | pEGFP-N1-FLAG was a gift from Patrick Calsou (Addgene plasmid # 60360 ; http://n2t.net/addgene:60360 ; RRID:Addgene_60360) |
| 参考文献部分: | DNA damage triggers SAF-A and RNA biogenesis factors exclusion from chromatin coupled to R-loops removal. Britton S, Dernoncourt E, Delteil C, Froment C, Schiltz O, Salles B, Frit P, Calsou P. Nucleic Acids Res. 2014 Oct 1;42(14):9047-62. doi: 10.1093/nar/gku601. Epub 2014 Jul 16. 10.1093/nar/gku601 PubMed 25030905 |
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载体基本信息
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载体简介
载体序列
LOCUS pEGFP-N1-FLAG 4777 bp ds-DNA circular SYN 06-AUG-2018 DEFINITION Plasmid for cloning and expression of FLAG-GFP tagged proteins (C-terminal tag). Confers resistance to G418.. ACCESSION . VERSION . KEYWORDS pEGFP-N1-FLAG SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 4777) AUTHORS Britton S, Dernoncourt E, Delteil C, Froment C, Schiltz O, Salles B, Frit P, Calsou P TITLE DNA damage triggers SAF-A and RNA biogenesis factors exclusion from chromatin coupled to R-loops removal. JOURNAL Nucleic Acids Res. 2014 Oct 1;42(14):9047-62. doi: 10.1093/nar/gku601. Epub 2014 Jul 16. PUBMED 25030905 REFERENCE 2 (bases 1 to 4777) AUTHORS . TITLE Direct Submission JOURNAL Exported Aug 6, 2018 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..4777 /organism="synthetic DNA construct" /mol_type="other DNA" enhancer 1..304 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 305..508 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 459..479 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" misc_feature 549..605 /label=MCS /note="multiple cloning site" CDS 624..647 /codon_start=1 /product="FLAG(R) epitope tag, followed by an enterokinase cleavage site" /label=FLAG /translation="DYKDDDDK" regulatory 657..666 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 663..1382 /codon_start=1 /product="enhanced GFP" /label=EGFP /note="mammalian codon-optimized" /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" primer_bind complement(708..729) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(969..988) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 1316..1337 /label=EGFP-C /note="EGFP, forward primer" polyA_signal 1505..1626 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(1542..1561) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 1596..1615 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" rep_origin complement(1633..2088) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(1770..1791) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 1982..2001 /label=F1ori-R /note="F1 origin, reverse primer" promoter 2115..2219 /gene="bla" /label=AmpR promoter promoter 2221..2578 /label=SV40 promoter /note="SV40 enhancer and early promoter" primer_bind complement(2244..2264) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" rep_origin 2429..2564 /label=SV40 ori /note="SV40 origin of replication" primer_bind 2491..2510 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" CDS 2613..3407 /codon_start=1 /gene="aph(3')-II (or nptII)" /product="aminoglycoside phosphotransferase from Tn5" /label=NeoR/KanR /note="confers resistance to neomycin, kanamycin, and G418 (Geneticin(R))" /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF" primer_bind complement(2667..2686) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 3277..3296 /label=Neo-F /note="Neomycin resistance gene, forward primer" primer_bind complement(3595..3614) /label=TK-pA-R /note="Thymidine kinase polyA, reverse primer" polyA_signal 3639..3686 /label=HSV TK poly(A) signal /note="herpes simplex virus thymidine kinase polyadenylation signal (Cole and Stacy, 1985)" rep_origin 4015..4603 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 4504..4523 /label=pBR322ori-F /note="pBR322 origin, forward primer" ORIGIN 1 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt 61 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca 121 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc 181 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta 241 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac 301 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg 361 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg 421 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt 481 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta 541 ccggactcag atctcgagct caagcttcga attctgcagt cgacggtacc gcgggcccgg 601 gatccgccgc caccatgacg cgtgattaca aggatgacga cgataagcca ccggtcgcca 661 ccatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg 721 acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct 781 acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca 841 ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc gaccacatga 901 agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct 961 tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc 1021 tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc 1081 acaagctgga gtacaactac aacagccaca acgtctatat catggccgac aagcagaaga 1141 acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc gtgcagctcg 1201 ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc 1261 actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc gatcacatgg 1321 tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaagt 1381 aaagcggccg cgactctaga tcataatcag ccataccaca tttgtagagg ttttacttgc 1441 tttaaaaaac ctcccacacc tccccctgaa cctgaaacat aaaatgaatg caattgttgt 1501 tgttaacttg tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt 1561 cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt 1621 atcttaaggc gtaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt 1681 aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag 1741 aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga 1801 acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg 1861 aaccatcacc ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc 1921 ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg 1981 aagggaagaa agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc 2041 gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca gggcgcgtca ggtggcactt 2101 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt 2161 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagtc 2221 ctgaggcgga aagaaccagc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg 2281 ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg 2341 aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc 2401 aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca 2461 ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg ccgcctcggc 2521 ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaga 2581 tcgatcaaga gacaggatga ggatcgtttc gcatgattga acaagatgga ttgcacgcag 2641 gttctccggc cgcttgggtg gagaggctat tcggctatga ctgggcacaa cagacaatcg 2701 gctgctctga tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca 2761 agaccgacct gtccggtgcc ctgaatgaac tgcaagacga ggcagcgcgg ctatcgtggc 2821 tggccacgac gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg 2881 actggctgct attgggcgaa gtgccggggc aggatctcct gtcatctcac cttgctcctg 2941 ccgagaaagt atccatcatg gctgatgcaa tgcggcggct gcatacgctt gatccggcta 3001 cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact cggatggaag 3061 ccggtcttgt cgatcaggat gatctggacg aagagcatca ggggctcgcg ccagccgaac 3121 tgttcgccag gctcaaggcg agcatgcccg acggcgagga tctcgtcgtg acccatggcg 3181 atgcctgctt gccgaatatc atggtggaaa atggccgctt ttctggattc atcgactgtg 3241 gccggctggg tgtggcggac cgctatcagg acatagcgtt ggctacccgt gatattgctg 3301 aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc gccgctcccg 3361 attcgcagcg catcgccttc tatcgccttc ttgacgagtt cttctgagcg ggactctggg 3421 gttcgaaatg accgaccaag cgacgcccaa cctgccatca cgagatttcg attccaccgc 3481 cgccttctat gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct 3541 ccagcgcggg gatctcatgc tggagttctt cgcccaccct agggggaggc taactgaaac 3601 acggaaggag acaataccgg aaggaacccg cgctatgacg gcaataaaaa gacagaataa 3661 aacgcacggt gttgggtcgt ttgttcataa acgcggggtt cggtcccagg gctggcactc 3721 tgtcgatacc ccaccgagac cccattgggg ccaatacgcc cgcgtttctt ccttttcccc 3781 accccacccc ccaagttcgg gtgaaggccc agggctcgca gccaacgtcg gggcggcagg 3841 ccctgccata gcctcaggtt actcatatat actttagatt gatttaaaac ttcattttta 3901 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg 3961 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga 4021 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt 4081 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag 4141 agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa 4201 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag 4261 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca 4321 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac 4381 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa 4441 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc 4501 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg 4561 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc 4621 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc 4681 ccctgattct gtggataacc gtattaccgc catgcattag ttattaatag taatcaatta 4741 cggggtcatt agttcatagc ccatatatgg agttccg //
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