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- 详细信息
- 文献和实验
- 技术资料
- 保存条件：
负20摄氏度
- 保质期：
3个月
- 英文名：
pXT7-Cas9
- 库存：
100
- 供应商：
LSMBIO
- 规格：
2ug
pXT7-Cas9
pXT7-Cas9 Description
PXT7-Cas9 is a Xenopus gene editing Cas9 expression plasmid.
pXT7-Cas9 Sequence
LOCUS Exported 7354 bp ds-DNA circular SYN 17-JAN-2018
DEFINITION synthetic circular DNA
FEATURES Location/Qualifiers
source 1..7354
/organism="synthetic DNA construct"
/mol_type="other DNA"
promoter 417..435
/label=SP6 promoter
/note="promoter for bacteriophage SP6 RNA polymerase"
3'UTR complement(541..666)
/label=Xenopus globin 3'-UTR
/note="3'-UTR of the major beta-globin gene of Xenopus
laevis"
CDS complement(702..749)
/codon_start=1
/product="bipartite nuclear localization signal from
nucleoplasmin"
/label=nucleoplasmin NLS
/translation="KRPAATKKAGQAKKKK"
CDS complement(750..4853)
/codon_start=1
/product="Cas9 (Csn1) endonuclease from the Streptococcus
pyogenes Type II CRISPR/Cas system"
/label=Cas9
/note="generates RNA-guided double strand breaks in DNA"
/translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
DATLIHQSITGLYETRIDLSQLGGD"
CDS complement(4878..4898)
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
regulatory 4901..4910
/regulatory_class="other"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
5'UTR complement(4923..4965)
/label=Xenopus globin 5'-UTR
/note="translational enhancer from the 5'-UTR of the major
beta-globin gene of Xenopus laevis"
promoter complement(4978..4996)
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
rep_origin complement(5417..6005)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(6176..7036)
/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"
promoter complement(7037..7141)
/gene="bla"
/label=AmpR promoter
ORIGIN
1 tagcgtatca gagtctcgtc cgtcggtcgt atgacggtga aacctctgac acatgcagct
61 cccggagacg gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg
121 cgcgtcagcg ggtgttggcg ggtgtcgggg ctggcttaac tatgcggcat cagagcagat
181 tgtactgaga gtgcaccata tgcggtgtga aataccgcac agatgcgtaa ggagaaaata
241 ccgcatcagg cgccattcgc cattcaggct gcgcaactgt tgggaagggc gatcggtgcg
301 ggcctcttcg ctattacgcc agctggcgaa agggggatgt gctgcaaggc gattaagttg
361 ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg acggccagtg aattggattt
421 aggtgacact atagaatacg aattaattcg agctcgcccg gggatcctct agagtcgacc
481 tgcagggggg gggggggggg gttttttttt tttttttttt tttttttttt tttttagaat
541 gtgaagaaac tttcttttta ttaggagcag atacgaatgg ctacattttg ggggacaaca
601 ttttgtaaag tgtaagttgg tattatgtag cttagagact ccattcgggt gttcttgagg
661 ctggtttagt ggtaaccaga tcctagtcag tcactagatt atttcttttt cttagcttga
721 ccagctttct tagtagcagc aggacgcttg tctccaccga gctgagagag gtcgattctt
781 gtttcataga gccccgtaat tgactgatga atcagtgtgg cgtccaggac ctcctttgta
841 gaggtgtacc gctttctgtc tatggtggtg tcgaagtact tgaaggctgc aggcgcgccc
901 aagttggtca gagtaaacaa gtggataatg ttttctgcct gctccctgat gggcttatcc
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文献和实验该产品被引用文献
Upregulation of Antioxidant Capacity and Nucleotide Precursor Availability Suffices for Oncogenic Transformation
Author：YangZhang1YiXu1WenyunLu23Jonathan M.Ghergurovich24LiliGuo56Ian A.Blair5Joshua D.Rabinowitz23XiaoluYang17
Received 22 January 2020, Revised 4 August 2020, Accepted 30 September 2020, Available online 6 November 2020.
Published: November 6, 2020
Cell Metabolism
Available online 6 November 2020 In Press
doi: doi.org/10.1016/j.cmet.2020.10.002
相关实验
最近 CRISPR/Cas9 基因敲除技术可谓风靡全世界的实验室，CRISPR 被称为规律成簇间隔短回文重复，其实际上就是一种基因编辑器，是细菌用以保护自身对抗病毒的一个系统。后来，研究人员发现，它似乎是一种精确的万能基因武器，可以用来删除、添加、激活或抑制其他生物体的目标基因。由于其相对于前几代基因敲除技术而言更为简便高效。所以一经问世便受到科研界的热捧。利用 Cas9 质粒建立 knock-out 细胞系实验过程主要有以下几步：1. 确定待敲除基因的靶位点；2. 设计识别靶位点识别的一对
CRISPR/Cas9 基因敲除实验-- sgRNA 及引物设计
来看一下。提交订单，等待引物到达：华大基因的速度非常快，大约在 2 - 3 个工作日即可拿到，在等待期间需要准备好：LB + 抗生素抗性平板；感受态细胞及相应培养基；无内毒素的 Cas9 载体质粒；BbsI 限制性内切酶；质粒小提试剂盒；常规限制性内切酶。
的表达质粒。A）PCR 扩增的方法U6：来源于人 U6 小核启动子，常用小 RNA 表达，转录本为 shRNA。将 sgRNA 放到 U6 后面，利用 U6 的启动来扩增 sgRNA。将这部分内容整合到 Cas9 expression plasmid pSpCas9（Cas9 表达质粒）中，一起共转。B）sgRNA 表达质粒的方法现在我要用这个方法来举例设计 p53 引物找到包括 sgRNA 序列的前后 1000 个碱基的序列（2000 bp）找到的序列如下：将上面复制的序列粘贴到 blast 中
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