質粒名稱: | pMH3 |
---|---|
出品公司: | 愛迪基因 |
目錄編號: | 52528 |
存儲實驗室: | Klaus Foerstemann |
載體骨架基本信息 |
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載體名稱: | pJet1.2 |
載體出品公司: | Thermor Fisher / Fermentas |
載體抗性: | 氨芐青霉素 |
空載體大小: | 2972 bp |
完整質粒大小: | 4477 bp |
載體修飾: | -- |
載體類型: | blunt cloning kit,鈍性克隆試劑盒 |
篩選標記: | 殺稻瘟菌素 |
高拷貝/低拷貝: | 高拷貝 |
生長條件: | 37 ℃ |
插入基因信息 |
|
插入基因名稱: | eGFP and Blasticigin resistance cassette |
物種來源: | Aequorea victoria |
插入大小: | -- |
基因突變: | -- |
GenBank編號 | -- |
啟動子 | -- |
標簽/融合蛋白: | -- |
克隆方法: | -- |
5' 克隆位點: | -- |
3' 克隆位點: | -- |
5' 測序引物 | taatacgactcactataggg |
3' 測序引物 | -- |
文獻引用方法 |
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材料和方法部分: | pMH3 was a gift from Klaus Foerstemann (Addgene plasmid # 52528 ; //n2t.net/addgene:52528 ; RRID:Addgene_52528) |
參考文獻部分: | Efficient chromosomal gene modification with CRISPR/cas9 and PCR-based homologous recombination donors in cultured Drosophila cells. Bottcher R, Hollmann M, Merk K, Nitschko V, Obermaier C, Philippou-Massier J, Wieland I, Gaul U, Forstemann K. Nucleic Acids Res. 2014 Apr 19. 10.1093/nar/gku289 PubMed 24748663 |
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pMH3載體質粒基本信息
pMH3質粒圖譜載體圖譜和pMH3載體序列質粒序列多克隆位點信息
pMH3質粒載體簡介
pMH3質粒序列載體序列
LOCUS pMH3 4477 bp ds-DNA circular SYN 06-AUG-2018 DEFINITION serves as PCR-template to generate HR donors for C-terminal GFP-tag. ACCESSION . VERSION . KEYWORDS pMH3 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 4477) AUTHORS Bottcher R, Hollmann M, Merk K, Nitschko V, Obermaier C, Philippou-Massier J, Wieland I, Gaul U, Forstemann K TITLE Efficient chromosomal gene modification with CRISPR/cas9 and PCR-based homologous recombination donors in cultured Drosophila cells. JOURNAL Nucleic Acids Res. 2014 Apr 19. PUBMED 24748663 REFERENCE 2 (bases 1 to 4477) AUTHORS . TITLE Direct Submission JOURNAL Exported Aug 6, 2018 from SnapGene Server 1.1.58 //www.snapgene.com FEATURES Location/Qualifiers source 1..4477 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 305..324 /label=T7 /note="T7 promoter, forward primer" promoter 305..323 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" CDS 358..1077 /codon_start=1 /product="enhanced GFP" /label=EGFP /note="mammalian codon-optimized" /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" primer_bind complement(403..424) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(664..683) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 1011..1032 /label=EGFP-C /note="EGFP, forward primer" protein_bind complement(1084..1117) /label=FRT (minimal) /bound_moiety="FLP recombinase from the Saccharomyces cerevisiae 2u plasmid" /note="supports FLP-mediated excision but not integration (Turan and Bode, 2011)" CDS 1394..1792 /codon_start=1 /gene="Aspergillus terreus BSD" /product="blasticidin S deaminase" /label=BSD /note="confers resistance to blasticidin" /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI KAIVKDSDGQPTAVGIRELLPSGYVWEG" protein_bind complement(1841..1874) /label=FRT (minimal) /bound_moiety="FLP recombinase from the Saccharomyces cerevisiae 2u plasmid" /note="supports FLP-mediated excision but not integration (Turan and Bode, 2011)" promoter complement(2309..2339) /label=lac UV5 promoter /note="E. coli lac promoter with an ""up"" mutation" protein_bind 2354..2375 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(2495..2512) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(2666..3254) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(2746..2765) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(3425..4285) /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 4048..4067 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(4286..4390) /gene="bla" /label=AmpR promoter primer_bind 4458..4476 /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" ORIGIN 1 gcccctgcag ccgaattata ttatttttgc caaataattt ttaacaaaag ctctgaagtc 61 ttcttcattt aaattcttag atgatacttc atctggaaaa ttgtcccaat tagtagcatc 121 acgctgtgag taagttctaa accatttttt tattgttgta ttatctctaa tcttactact 181 cgatgagttt tcggtattat ctctattttt aacttggagc aggttccatt cattgttttt 241 ttcatcatag tgaataaaat caactgcttt aacacttgtg cctgaacacc atatccatcc 301 ggcgtaatac gactcactat agggagagcg gccgccagat cttccggatg gctcgagatg 361 gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc 421 gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc 481 aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc 541 gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct accccgacca catgaagcag 601 cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc 661 aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg 721 aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag 781 ctggagtaca actacaacag ccacaacgtc tatatcatgg ccgacaagca gaagaacggc 841 atcaaggtga acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac 901 cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac 961 ctgagcaccc agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg 1021 ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagtaaact 1081 agtgaagttc ctatactttc tagagaatag gaacttcaaa taaacatatg ctgttggaat 1141 atactattca acctacaaaa ataacgttaa acaacactac tttatatttg atatgaatgg 1201 ccacaccttt tatgccataa aacatattgt aagagaatac cactcttttt attccttctt 1261 tccttcttgt acgttttttg ctgtgagtag gtcgtggtgc tggtgttgca gttgaaataa 1321 cttaaaatat aaatcataaa actcaaacat aaacttgact atttatttat ttattaagaa 1381 aggaggtacc ggtatggcca agcctttgtc tcaagaagaa tccaccctca ttgaaagagc 1441 aacggctaca atcaacagca tccccatctc tgaagactac agcgtcgcca gcgcagctct 1501 ctctagcgac ggccgcatct tcactggtgt caatgtatat cattttactg ggggaccttg 1561 cgcagaactc gtggtgctgg gcactgctgc tgctgcggca gctggcaacc tgacttgtat 1621 cgtcgcgatc ggaaatgaga acaggggcat cttgagcccc tgcggacggt gccgacaggt 1681 tcttctcgat ctgcatcctg ggatcaaagc catagtgaag gacagtgatg gacagccgac 1741 ggcagttggg attcgtgaat tgctgccctc tggttatgtg tgggagggct agaaattatt 1801 aaaaataaaa ctttaaaaat aaatttgttt ctaacatatg gaagttccta tactttctag 1861 agaataggaa cttcatcttt ctagaagatc tcctacaata ttctcagctg ccatggaaaa 1921 tcgatgttct tcttttattc tctcaagatt ttcaggctgt atattaaaac ttatattaag 1981 aactatgcta accacctcat caggaaccgt tgtaggtggc gtgggttttc ttggcaatcg 2041 actctcatga aaactacgag ctaaatattc aatatgttcc tcttgaccaa ctttattctg 2101 catttttttt gaacgaggtt tagagcaagc ttcaggaaac tgagacagga attttattaa 2161 aaatttaaat tttgaagaaa gttcagggtt aatagcatcc attttttgct ttgcaagttc 2221 ctcagcattc ttaacaaaag acgtctcttt tgacatgttt aaagtttaaa cctcctgtgt 2281 gaaattatta tccgctcata attccacaca ttatacgagc cggaagcata aagtgtaaag 2341 cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca ctgccaattg 2401 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga 2461 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg 2521 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag 2581 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc 2641 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca 2701 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt 2761 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc 2821 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc 2881 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc 2941 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact 3001 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg 3061 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta 3121 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca 3181 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa 3241 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg 3301 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc 3361 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg 3421 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat 3481 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg 3541 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa 3601 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca 3661 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc 3721 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt 3781 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa 3841 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat 3901 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct 3961 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga 4021 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag 4081 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga 4141 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca 4201 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg 4261 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc 4321 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag 4381 gggttccgcg cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc attattatca 4441 tgacattaac ctataaaaat aggcgtatca cgaggcc //
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