| 质粒名称: | pcDNA3.1(+) eGFP |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 129020 |
| 存储实验室: | Jeremy Wilusz |
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载体骨架基本信息 |
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| 载体名称: | pcDNA3.1(+) |
| 载体出品公司: | Life Technologies |
| 空载体大小: | 6107bp |
| 完整质粒大小: | 6118bp |
| 载体类型: | 哺乳细胞表达 |
| 筛选标记: | 新霉素（G418） |
| 载体抗性: | 氨苄霉素 |
| 生长温度: | 37 ℃ |
| 宿主菌: | DH5a |
| 拷贝数: | 高拷贝 |
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插入基因信息 |
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| 插入基因名称: | eGFP |
| 启动子: | CMV |
| 克隆方法: | 限制性内切酶 |
| 5’克隆位点: | AgeI(未破坏) |
| 3’克隆位点: | XbaI (not destroyed) |
| 5’测序引物 | CGCAAATGGGCGGTAGGCGTG |
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文献引用方法 |
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| 材料和方法部分: | pcDNA3.1(+) eGFP was a gift from Jeremy Wilusz (Addgene plasmid # 129020 ; http://n2t.net/addgene:129020 ; RRID:Addgene_129020) |
| 参考文献部分: | An improved method for circular RNA purification using RNase R that efficiently removes linear RNAs containing G-quadruplexes or structured 3' ends. Xiao MS, Wilusz JE. Nucleic Acids Res. 2019 Jul 3. pii: 5527976. doi: 10.1093/nar/gkz576. 10.1093/nar/gkz576 PubMed 31269210 |
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载体基本信息
载体图谱质粒图谱和多克隆位点信息
 eGFP质粒图谱.png)
载体简介
载体序列
LOCUS pcDNA3.1(+) eGFP 6118 bp DNA circular SYN 25-AUG-2024 DEFINITION Expresses eGFP. ACCESSION . VERSION . KEYWORDS . SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6118) AUTHORS Xiao MS, Wilusz JE TITLE An improved method for circular RNA purification using RNase R that efficiently removes linear RNAs containing G-quadruplexes or structured 3' ends. JOURNAL Nucleic Acids Res. 2019 Jul 3. pii: 5527976. doi: 10.1093/nar/gkz576. PUBMED 31269210 REFERENCE 2 (bases 1 to 6118) AUTHORS . TITLE Direct Submission JOURNAL Exported Aug 25, 2024 from SnapGene Server 7.0.3 https://www.biofeng.com COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Nucleic Acids Res. 2019 Jul 3. pii: 5527976. doi: 10.1093/nar/gkz576." FEATURES Location/Qualifiers source 1..6118 /mol_type="other DNA" /organism="synthetic DNA construct" promoter complement(45..149) /gene="bla" /label=AmpR promoter primer_bind complement(224..243) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" enhancer 415..794 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 795..998 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 949..969 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" primer_bind 1043..1062 /label=T7 /note="T7 promoter, forward primer" promoter 1043..1061 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" regulatory 1123..1132 /label=Kozak sequence /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" /regulatory_class="other" CDS 1129..1848 /codon_start=1 /product="enhanced GFP with monomerizing A206K mutation (Zacharias et al., 2002)" /label=mEGFP /note="mammalian codon-optimized" /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" primer_bind complement(1174..1195) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(1435..1454) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 1782..1803 /label=EGFP-C /note="EGFP, forward primer" primer_bind complement(1889..1906) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 1895..2119 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" rep_origin 2165..2593 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(2252..2271) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 2462..2483 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(2602..2622) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 2607..2936 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 2787..2922 /label=SV40 ori /note="SV40 origin of replication" primer_bind 2849..2868 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" CDS 3003..3797 /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 GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF" primer_bind complement(3057..3076) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 3667..3686 /label=Neo-F /note="Neomycin resistance gene, forward primer" polyA_signal 3971..4092 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(4008..4027) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 4062..4081 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(4141..4157) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4141..4157) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(4154..4176) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 4165..4181 /label=lac operator /bound_moiety="lac repressor encoded by lacI" /note="The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-beta-D-thiogalactopyranoside (IPTG)." promoter complement(4189..4219) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 4234..4255 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(4372..4389) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(4543..5131) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(4623..4642) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(join(5302..6118,1..44)) /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind 5925..5944 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" ORIGIN 1 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa 61 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt 121 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc 181 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg 241 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 301 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 361 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 421 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 481 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 541 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 601 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt 661 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 721 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 781 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 841 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 901 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 961 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 1021 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc 1081 gtttaaactt aagcttggca atccggtact gttggtaaat aagccaccat ggtgagcaag 1141 ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac 1201 ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc 1261 ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc 1321 ctgacctacg gcgtgcagtg cttcagccgc taccccgacc acatgaagca gcacgacttc 1381 ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac 1441 ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc 1501 gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac 1561 aactacaaca gccacaacgt ctatatcatg gccgacaagc agaagaacgg catcaaggtg 1621 aacttcaaga tccgccacaa catcgaggac ggcagcgtgc agctcgccga ccactaccag 1681 cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagcacc 1741 cagtccaagc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc 1801 gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaagtaagg ccgcgactct 1861 agagggcccg tttaaacccg ctgatcagcc tcgactgtgc cttctagttg ccagccatct 1921 gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt 1981 tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg 2041 ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg 2101 gatgcggtgg gctctatggc ttctgaggcg gaaagaacca gctggggctc tagggggtat 2161 ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg 2221 accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc 2281 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga 2341 tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt 2401 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat 2461 agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat 2521 ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa 2581 tttaacgcga attaattctg tggaatgtgt gtcagttagg gtgtggaaag tccccaggct 2641 ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa 2701 agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa 2761 ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt 2821 ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc gcctctgcct 2881 ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaaaagc 2941 tcccgggagc ttgtatatcc attttcggat ctgatcaaga gacaggatga ggatcgtttc 3001 gcatgattga acaagatgga ttgcacgcag gttctccggc cgcttgggtg gagaggctat 3061 tcggctatga ctgggcacaa cagacaatcg gctgctctga tgccgccgtg ttccggctgt 3121 cagcgcaggg gcgcccggtt ctttttgtca agaccgacct gtccggtgcc ctgaatgaac 3181 tgcaggacga ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct tgcgcagctg 3241 tgctcgacgt tgtcactgaa gcgggaaggg actggctgct attgggcgaa gtgccggggc 3301 aggatctcct gtcatctcac cttgctcctg ccgagaaagt atccatcatg gctgatgcaa 3361 tgcggcggct gcatacgctt gatccggcta cctgcccatt cgaccaccaa gcgaaacatc 3421 gcatcgagcg agcacgtact cggatggaag ccggtcttgt cgatcaggat gatctggacg 3481 aagagcatca ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg cgcatgcccg 3541 acggcgagga tctcgtcgtg acccatggcg atgcctgctt gccgaatatc atggtggaaa 3601 atggccgctt ttctggattc atcgactgtg gccggctggg tgtggcggac cgctatcagg 3661 acatagcgtt ggctacccgt gatattgctg aagagcttgg cggcgaatgg gctgaccgct 3721 tcctcgtgct ttacggtatc gccgctcccg attcgcagcg catcgccttc tatcgccttc 3781 ttgacgagtt cttctgagcg ggactctggg gttcgaaatg accgaccaag cgacgcccaa 3841 cctgccatca cgagatttcg attccaccgc cgccttctat gaaaggttgg gcttcggaat 3901 cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc tggagttctt 3961 cgcccacccc aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac 4021 aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat 4081 caatgtatct tatcatgtct gtataccgtc gacctctagc tagagcttgg cgtaatcatg 4141 gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc 4201 cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc 4261 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat 4321 cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac 4381 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt 4441 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca 4501 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc 4561 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact 4621 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct 4681 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag 4741 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca 4801 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa 4861 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc 4921 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag 4981 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg 5041 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca 5101 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc 5161 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag 5221 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata 5281 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat 5341 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg 5401 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc 5461 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc 5521 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc 5581 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc 5641 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc 5701 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa 5761 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat 5821 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata 5881 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca 5941 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag 6001 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc 6061 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgcc //
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