| 质粒名称: | pMH3 |
|---|---|
| 出品公司: | 爱迪基因 |
| 目录编号: | 52528 |
| 存储实验室: | Klaus Foerstemann |
|
载体骨架基本信息 |
|
| 载体名称: | pJet1.2 |
| 载体出品公司: | Thermor Fisher / Fermentas |
| 载体抗性: | 氨苄青霉素 |
| 空载体大小: | 2972 bp |
| 完整质粒大小: | 4477 bp |
| 载体修饰: | -- |
| 载体类型: | blunt cloning kit,钝性克隆试剂盒 |
| 筛选标记: | 杀稻瘟菌素 |
| 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: | eGFP and Blasticigin resistance cassette |
| 物种来源: | Aequorea victoria |
| 插入大小: | -- |
| 基因突变: | -- |
| GenBank编号 | -- |
| 启动子 | -- |
| 标签/融合蛋白: | -- |
| 克隆方法: | -- |
| 5' 克隆位点: | -- |
| 3' 克隆位点: | -- |
| 5' 测序引物 | taatacgactcactataggg |
| 3' 测序引物 | -- |
|
文献引用方法 |
|
| 材料和方法部分: | pMH3 was a gift from Klaus Foerstemann (Addgene plasmid # 52528 ; http://n2t.net/addgene:52528 ; RRID:Addgene_52528) |
| 参考文献部分: | 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 |
买家导航
载体基本信息
载体图谱质粒图谱和多克隆位点信息
载体简介
载体序列
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
http://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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