质粒名称: | pEP-EGFPin |
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出品公司: | addgene |
目录编号: | 60961 |
存储实验室: | Nikolaus Osterrieder |
载体骨架基本信息 |
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载体名称: | pEGFP N1 |
空载体大小: | 4637 bp |
完整质粒大小: | 4989 bp |
载体类型: | 哺乳细胞表达 |
载体抗性: | Ampicillin and Kanamycin |
生长温度: | 37 ℃ |
宿主菌: | DH10B |
拷贝数: | 未知 |
插入基因信息 |
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插入基因名称: | GFP-Kana_I-SceI |
文献引用方法 |
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材料和方法部分: | pEP-EGFPin was a gift from Nikolaus Osterrieder (Addgene plasmid # 60961 ; http://n2t.net/addgene:60961 ; RRID:Addgene_60961) |
参考文献部分: | Two-step red-mediated recombination for versatile high-efficiency markerless DNA manipulation in Escherichia coli. Tischer BK, von Einem J, Kaufer B, Osterrieder N. Biotechniques. 2006 Feb;40(2):191-7. 10.2144/000112096 PubMed 16526409 |
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载体序列
LOCUS pEP-EGFPin 4637 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION PCR template for the insertion of eGFP by en passant (two-step Red-mediated) mutagenesis. ACCESSION . VERSION . KEYWORDS pEP-EGFPin SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 4637) AUTHORS Tischer BK, von Einem J, Kaufer B, Osterrieder N TITLE Two-step red-mediated recombination for versatile high-efficiency markerless DNA manipulation in Escherichia coli. JOURNAL Biotechniques. 2006 Feb;40(2):191-7. PUBMED 16526409 REFERENCE 2 (bases 1 to 4637) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.biofeng.com FEATURES Location/Qualifiers source 1..4637 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 2..18 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 2..18 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind 32..51 /label=T7 /note="T7 promoter, forward primer" promoter 32..50 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(112..133) /label=EGFP-C /note="EGFP, forward primer" primer_bind 461..480 /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" CDS complement(730..1545) /codon_start=1 /gene="aph(3')-Ia" /product="aminoglycoside phosphotransferase" /label=KanR /note="confers resistance to kanamycin in bacteria or G418 (Geneticin(R)) in eukaryotes" /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF" primer_bind 1453..1472 /label=Kan-R /note="Kanamycin resistance gene, reverse primer" primer_bind 1537..1556 /label=pENTR-R /note="pENTR vectors, reverse primer" primer_bind 1790..1811 /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(1888..1905) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(1888..1904) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(1897..1919) /label=M13/pUC Forward /note="In lacZ gene" rep_origin complement(2046..2502) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(2184..2205) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 2396..2415 /label=F1ori-R /note="F1 origin, reverse primer" promoter 2529..2633 /gene="bla" /label=AmpR promoter CDS 2634..3494 /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 complement(2852..2871) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 3665..4253 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 4154..4173 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 4407..4424 /label=L4440 /note="L4440 vector, forward primer" protein_bind 4541..4562 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 4577..4607 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 4615..4631 /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)." primer_bind 4620..5 /label=M13/pUC Reverse /note="In lacZ gene" ORIGIN 1 acaggaaaca gctatgacat gattacgaat ttaatacgac tcactatagg gaattcgagc 61 tcggtacccc ttgtacagct cgtccatgcc gagagtgatc ccggcggcgg tcacgaactc 121 cagcaggacc atgtgatcgc gcttctcgtt ggggtctttg ctcagggcgg actgggtgct 181 caggtagtgg ttgtcgggca gcagcacggg gccgtcgccg atgggggtgt tctgctggta 241 gtggtcggcg agctgcacgc tgccgtcctc gatgttgtgg cggatcttga agttcacctt 301 gatgccgttc ttctgcttgt cggccatgat atagacgttg tggctgttgt agttgtactc 361 cagcttgtgc cccaggatgt tgccgtcctc cttgaagtcg atgcccttca gctcgatgcg 421 gttcaccagg gtgtcgccct cgaacttcac ctcggcgcgg gtcttgtagt tgccgtcgtc 481 cttgaagaag atggtgcgct cctggacgta gccttcgggc atggcggact tgaagaagtc 541 gtgctgcttc atgtggtcgg ggtagcggct gaagcactgc acgccgtagg tcagggtggt 601 cacgagggtg ggccagggca cgggcagctt gccggtggtg cagatgaact tcagggtcag 661 cttgccgtag gtggcatcgc cctcgccctc gccgactagc cagtgttaca accaattaac 721 caattctgat tagaaaaact catcgagcat caaatgaaac tgcaatttat tcatatcagg 781 attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa actcaccgag 841 gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc gtccaacatc 901 aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga aatcaccatg 961 agtgacgact gaatccggtg agaatggcaa aagcttatgc atttctttcc agacttgttc 1021 aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac cgttattcat 1081 tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac aattacaaac 1141 aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat tttcacctga 1201 atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag tggtgagtaa 1261 ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca taaattccgt 1321 cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac ctttgccatg 1381 tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg tcgcacctga 1441 ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca tgttggaatt 1501 taatcgcggc ctcgagcaag acgtttcccg ttgaatatgg ctcataacac cccttgtatt 1561 actgtttatg taagcagaca gttttattgt tcatgatgat atatttttat cttgtgcaat 1621 gtaacatcag agattttgag acacaacgtg gctttgttga ataaatcgat taccctgtta 1681 tccctagcta gtggcagctt gccggtggtg cagatgaact tcagggtcag cttgccgtag 1741 gtggcatcgc cctcgccctc gccggacacg ctgaacttgt ggccgtttac gtcgccgtcc 1801 agctcgacca ggatgggcac caccccggtg aacagctcct cgcccttgct caccatggat 1861 cctcgactgc aggcatgcaa gcttggcact ggccgtcgtt ttacaacgtc gtgactggga 1921 aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg 1981 taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga 2041 atgggaaatt gtaaacgttt aatattttgt taaaattcgc gttaaatttt tgttaaatca 2101 gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga 2161 ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg 2221 actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat 2281 caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag 2341 ggatgccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga 2401 agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa 2461 ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtcaggtggc acttttcggg 2521 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc 2581 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta 2641 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg 2701 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg 2761 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac 2821 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg 2881 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt 2941 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg 3001 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac 3061 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt 3121 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag 3181 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc 3241 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc 3301 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta 3361 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg 3421 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga 3481 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac 3541 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa 3601 tcccttaacg tgagttttcg ttccacggcg cgccagaccc cgtagaaaag atcaaaggat 3661 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc 3721 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg 3781 gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc 3841 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg 3901 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg 3961 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa 4021 cgacctacac cgaactgaga tacctacagc gtgagcattg agaaagcgcc acgcttcccg 4081 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga 4141 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct 4201 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca 4261 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc 4321 ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg 4381 ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc 4441 caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca 4501 ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt tagctcactc 4561 attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt ggaattgtga 4621 gcggataaca atttcac //
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