| 质粒名称: | AAT_g1 gRNA |
|---|---|
| 出品公司： | addgene |
| 目录编号: | 86005 |
| 存储实验室: | Tobias Cantz |
| 骨架基本信息 | |
| 载体名称: | pEX-A |
| 空载体大小: | 2450 bp |
| 完整质粒大小: | 2905 bp |
| 载体类型: | 哺乳细胞、CRISPR表达 |
| 载体抗性: | 氨苄霉素 |
| 生长温度: | 37 ℃ |
| 宿主菌: | DH5a |
| 拷贝数: | 高拷贝 |
|
插入基因信息 |
|
| 插入基因名称: | pU6-AAT_g1 sgRNA |
| 启动子: | pU6 |
| 克隆方法: | 未知 |
| 5’测序引物 | CGGATAACAATTTCACACAG |
| 3’测序引物 | SV40pA-R |
| 材料和方法部分: | AAT_g1 gRNA was a gift from Tobias Cantz (Addgene plasmid # 86005 ; http://n2t.net/addgene:86005 ; RRID:Addgene_86005) |
| 参考文献部分: | Improved bi-allelic modification of a transcriptionally silent locus in patient-derived iPSC by Cas9 nickase. Eggenschwiler R, Moslem M, Fraguas MS, Galla M, Papp O, Naujock M, Fonfara I, Gensch I, Wahner A, Beh-Pajooh A, Mussolino C, Tauscher M, Steinemann D, Wegner F, Petri S, Schambach A, Charpentier E, Cathomen T, Cantz T. Sci Rep. 2016 Dec 2;6:38198. doi: 10.1038/srep38198. 10.1038/srep38198 PubMed 27910942 |
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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
载体简介
载体序列
LOCUS AAT_g1 gRNA 2905 bp ds-DNA circular SYN 13-MAY-2021
DEFINITION plasmid vector encoding for U6-driven AAT_g1 gRNA.
ACCESSION .
VERSION .
KEYWORDS AAT_g1 gRNA
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 2905)
AUTHORS Eggenschwiler R, Moslem M, Fraguas MS, Galla M, Papp O, Naujock M,
Fonfara I, Gensch I, Wahner A, Beh-Pajooh A, Mussolino C, Tauscher
M, Steinemann D, Wegner F, Petri S, Schambach A, Charpentier E,
Cathomen T, Cantz T
TITLE Improved bi-allelic modification of a transcriptionally silent locus
in patient-derived iPSC by Cas9 nickase.
JOURNAL Sci Rep. 2016 Dec 2;6:38198. doi: 10.1038/srep38198.
PUBMED 27910942
REFERENCE 2 (bases 1 to 2905)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58
http://www.biofeng.com
FEATURES Location/Qualifiers
source 1..2905
/organism="synthetic DNA construct"
/mol_type="other DNA"
promoter 70..310
/label=U6 promoter
/note="RNA polymerase III promoter for human U6 snRNA"
primer_bind 70..90
/label=hU6-F
/note="Human U6 promoter, forward primer"
primer_bind 241..260
/label=LKO.1 5'
/note="Human U6 promoter, forward primer"
misc_RNA 339..414
/label=gRNA scaffold
/note="guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system"
protein_bind complement(426..450)
/gene="mutant version of attB"
/label=attB2
/bound_moiety="BP Clonase(TM)"
/note="recombination site for the Gateway(R) BP reaction"
primer_bind complement(485..507)
/label=M13/pUC Reverse
/note="In lacZ gene"
protein_bind 496..512
/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(520..550)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 565..586
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(703..720)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(874..1462)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(954..973)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS complement(1633..2493)
/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 2256..2275
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
promoter complement(2494..2598)
/gene="bla"
/label=AmpR promoter
primer_bind 2666..2684
/label=pBRforEco
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
primer_bind complement(2722..2744)
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
primer_bind 2844..2863
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
ORIGIN
1 tgtacaaaaa agcaggcttt aaaggaacca attcagtcga ctggatccgg taccaaggtc
61 gggcaggaag agggcctatt tcccatgatt ccttcatatt tgcatatacg atacaaggct
121 gttagagaga taattagaat taatttgact gtaaacacaa agatattagt acaaaatacg
181 tgacgtagaa agtaataatt tcttgggtag tttgcagttt taaaattatg ttttaaaatg
241 gactatcata tgcttaccgt aacttgaaag tatttcgatt tcttggcttt atatatcttg
301 tggaaaggac gaaacaccgc acagccttat gcacggccgt tttagagcta gaaatagcaa
361 gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt
421 tctagaccca gctttcttgt acaaagttgg cattaacctg cggccgcaag cttggatccg
481 aattcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat
541 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc
601 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
661 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct
721 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
781 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
841 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
901 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
961 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
1021 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
1081 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
1141 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
1201 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
1261 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaaggacagt
1321 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
1381 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
1441 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
1501 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
1561 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
1621 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
1681 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
1741 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
1801 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
1861 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
1921 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
1981 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
2041 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
2101 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
2161 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
2221 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
2281 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
2341 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
2401 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
2461 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
2521 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
2581 acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtct aagaaaccat
2641 tattatcatg acattaacct ataaaaatag gcgtatcacg aggccctttc gtctcgcgcg
2701 tttcggtgat gacggtgaaa acctctgaca catgcagctc ccggagacgg tcacagcttg
2761 tctgtaagcg gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg
2821 gtgtcggggc tggcttaact atgcggcatc agagcagatt gtactgagag tgcaccatat
2881 gggtaccgag ctcgcggccg caagc
//
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