| 质粒名称: | Cas9 sgRNA vector |
|---|---|
| 出品公司: | 爱迪基因 |
| 目录编号: | 68463 |
| 存储实验室: | Su-Chun Zhang |
|
载体骨架基本信息 |
|
| 载体名称: | gRNA cloning vector (Addgene plasmid #41824) |
| 载体出品公司: | -- |
| 载体抗性: | 卡那霉素 |
| 空载体大小: | 3900 bp |
| 完整质粒大小: | -- |
| 载体修饰: | 一个先前描述的嵌合引导RNA表达盒被排序为gBlocks并被克隆到gRNA克隆载体（Addgene质粒#41824）。新的sgRNA克隆载体（Cas9-sgRNA载体）包括两个BbsI限制性位点，用于sgRNA的快速克隆 |
| 载体类型: | 哺乳细胞表达,CRISPR |
| 筛选标记: | -- |
| 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: | -- |
| 物种来源: | -- |
| 插入大小: | -- |
| 基因突变: | -- |
| GenBank编号 | -- |
| 启动子 | U6 |
| 标签/融合蛋白: | -- |
| 克隆方法: | 限制性内切酶 |
| 5' 克隆位点: | -- |
| 3' 克隆位点: | -- |
| 5' 测序引物 | T7 |
| 3' 测序引物 | Sp6 |
|
文献引用方法 |
|
| 材料和方法部分: | Cas9 sgRNA vector was a gift from Su-Chun Zhang (Addgene plasmid # 68463 ; http://n2t.net/addgene:68463 ; RRID:Addgene_68463) |
| 参考文献部分: | Engineering Human Stem Cell Lines with Inducible Gene Knockout using CRISPR/Cas9. Chen Y, Cao J, Xiong M, Petersen AJ, Dong Y, Tao Y, Huang CT, Du Z, Zhang SC. Cell Stem Cell. 2015 Jul 1. pii: S1934-5909(15)00261-1. doi: 10.1016/j.stem.2015.06.001. 10.1016/j.stem.2015.06.001 PubMed 26145478 |
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 Cas9 sgRNA vector 3952 bp ds-DNA circular SYN 06-AUG-2018
DEFINITION Cas9 sgRNA vector for cloning.
ACCESSION .
VERSION .
KEYWORDS Cas9 sgRNA vector
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 3952)
AUTHORS Chen Y, Cao J, Xiong M, Petersen AJ, Dong Y, Tao Y, Huang CT, Du Z,
Zhang SC
TITLE Engineering Human Stem Cell Lines with Inducible Gene Knockout using
CRISPR/Cas9.
JOURNAL Cell Stem Cell. 2015 Jul 1. pii: S1934-5909(15)00261-1. doi:
10.1016/j.stem.2015.06.001.
PUBMED 26145478
REFERENCE 2 (bases 1 to 3952)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Aug 6, 2018 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..3952
/organism="synthetic DNA construct"
/mol_type="other DNA"
protein_bind 107..128
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 143..173
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 181..197
/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 186..208
/label=M13/pUC Reverse
/note="In lacZ gene"
primer_bind 205..221
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind 205..221
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
promoter 239..257
/label=SP6 promoter
/note="promoter for bacteriophage SP6 RNA polymerase"
primer_bind 239..256
/label=SP6
/note="SP6 promoter, forward primer"
protein_bind 342..366
/gene="mutant version of attB"
/label=attB2
/bound_moiety="BP Clonase(TM)"
/note="recombination site for the Gateway(R) BP reaction"
promoter complement(460..700)
/label=U6 promoter
/note="RNA polymerase III promoter for human U6 snRNA"
primer_bind complement(510..529)
/label=LKO.1 5'
/note="Human U6 promoter, forward primer"
primer_bind complement(680..700)
/label=hU6-F
/note="Human U6 promoter, forward primer"
primer_bind complement(839..858)
/label=T7
/note="T7 promoter, forward primer"
promoter complement(840..858)
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
primer_bind complement(865..882)
/label=M13 Forward
/note="In lacZ gene. Also called M13-F20 or M13 (-21)
Forward"
primer_bind complement(865..881)
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(874..896)
/label=M13/pUC Forward
/note="In lacZ gene"
CDS 1019..1321
/codon_start=1
/gene="ccdB"
/product="CcdB, a bacterial toxin that poisons DNA gyrase"
/label=ccdB
/note="Plasmids containing the ccdB gene cannot be
propagated in standard E. coli strains."
/translation="QFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDKV
SRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
primer_bind 1200..1219
/label=ccdB-fwd
/note="ccdB gene, forward primer"
CDS 1670..2464
/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
GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
primer_bind complement(1724..1743)
/label=Neo-R
/note="Neomycin resistance gene, reverse primer"
primer_bind 2334..2353
/label=Neo-F
/note="Neomycin resistance gene, forward primer"
regulatory 2665..2674
/regulatory_class="other"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
CDS 2671..3045
/codon_start=1
/gene="Sh ble from Streptoalloteichus hindustanus"
/product="antibiotic-binding protein"
/label=BleoR
/note="confers resistance to bleomycin, phleomycin, and
Zeocin(TM)"
/translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
EFALRDPAGNCVHFVAEEQD"
rep_origin 3183..3771
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 3672..3691
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
primer_bind 3925..3942
/label=L4440
/note="L4440 vector, forward primer"
ORIGIN
1 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
61 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc
121 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa
181 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagctat
241 ttaggtgaca ctatagaata ctcaagctat gcatcaagct tggtaccgag ctcggatcca
301 ctagtaacgg ccgccagtgt gctggaattc gccctttaat gccaactttg tacaagaaag
361 ctgggtctag aaaaaaagca ccgactcggt gccacttttt caagttgata acggactagc
421 cttattttaa cttgctattt cacgtcttct cgaagacccg tcctttccac aagatatata
481 aagccaagaa atcgaaatac tttcaagtta cggtaagcat atgatagtcc attttaaaac
541 ataattttaa aactgcaaac tacccaagaa attattactt tctacgtcac gtattttgta
601 ctaatatctt tgtgtttaca gtcaaattaa ttctaattat ctctctaaca gccttgtatc
661 gtatatgcaa atatgaagga atcatgggaa ataggccctc ttcctgcccg accttggtac
721 cggatccagt cgactgaatt ggttccttta aagcctgctt ttttgtacaa agggcgaatt
781 ctgcagatat ccatcacact ggcggccgct cgagcatgca tctagagggc ccaattcgcc
841 ctatagtgag tcgtattaca attcactggc cgtcgtttta caacgtcgtg actgggaaaa
901 ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca gctggcgtaa
961 tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctat acgtacggca
1021 gtttaaggtt tacacctata aaagagagag ccgttatcgt ctgtttgtgg atgtacagag
1081 tgatattatt gacacgccgg ggcgacggat ggtgatcccc ctggccagtg cacgtctgct
1141 gtcagataaa gtctcccgtg aactttaccc ggtggtgcat atcggggatg aaagctggcg
1201 catgatgacc accgatatgg ccagtgtgcc ggtctccgtt atcggggaag aagtggctga
1261 tctcagccac cgcgaaaatg acatcaaaaa cgccattaac ctgatgttct ggggaatata
1321 aatgtcaggc atgagattat caaaaaggat cttcacctag atccttttca cgtagaaagc
1381 cagtccgcag aaacggtgct gaccccggat gaatgtcagc tactgggcta tctggacaag
1441 ggaaaacgca agcgcaaaga gaaagcaggt agcttgcagt gggcttacat ggcgatagct
1501 agactgggcg gttttatgga cagcaagcga accggaattg ccagctgggg cgccctctgg
1561 taaggttggg aagccctgca aagtaaactg gatggctttc tcgccgccaa ggatctgatg
1621 gcgcagggga tcaagctctg atcaagagac aggatgagga tcgtttcgca tgattgaaca
1681 agatggattg cacgcaggtt ctccggccgc ttgggtggag aggctattcg gctatgactg
1741 ggcacaacag acaatcggct gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg
1801 cccggttctt tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc aagacgaggc
1861 agcgcggcta tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt
1921 cactgaagcg ggaagggact ggctgctatt gggcgaagtg ccggggcagg atctcctgtc
1981 atctcacctt gctcctgccg agaaagtatc catcatggct gatgcaatgc ggcggctgca
2041 tacgcttgat ccggctacct gcccattcga ccaccaagcg aaacatcgca tcgagcgagc
2101 acgtactcgg atggaagccg gtcttgtcga tcaggatgat ctggacgaag agcatcaggg
2161 gctcgcgcca gccgaactgt tcgccaggct caaggcgagc atgcccgacg gcgaggatct
2221 cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc
2281 tggattcatc gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc
2341 tacccgtgat attgctgaag agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta
2401 cggtatcgcc gctcccgatt cgcagcgcat cgccttctat cgccttcttg acgagttctt
2461 ctgaattatt aacgcttaca atttcctgat gcggtatttt ctccttacgc atctgtgcgg
2521 tatttcacac cgcatacagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
2581 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg
2641 cttcaataat agcacgtgag gagggccacc atggccaagt tgaccagtgc cgttccggtg
2701 ctcaccgcgc gcgacgtcgc cggagcggtc gagttctgga ccgaccggct cgggttctcc
2761 cgggacttcg tggaggacga cttcgccggt gtggtccggg acgacgtgac cctgttcatc
2821 agcgcggtcc aggaccaggt ggtgccggac aacaccctgg cctgggtgtg ggtgcgcggc
2881 ctggacgagc tgtacgccga gtggtcggag gtcgtgtcca cgaacttccg ggacgcctcc
2941 gggccggcca tgaccgagat cggcgagcag ccgtgggggc gggagttcgc cctgcgcgac
3001 ccggccggca actgcgtgca cttcgtggcc gaggagcagg actgacacgt gctaaaactt
3061 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc
3121 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
3181 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
3241 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
3301 ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac
3361 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
3421 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
3481 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
3541 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
3601 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
3661 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
3721 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
3781 aacgcggcct ttttacggtt cctgggcttt tgctggcctt ttgctcacat gttctttcct
3841 gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct
3901 cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga ag
//
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