质粒编号
质粒名称
规格
价格
V008715
pDD162 (Peft-3::Cas9 + Empty sgRNA)
5 μg (冻干粉)
我们所展示的质粒图谱主要是从文献和开放数据库中收集而来，主要是为了方便研究工作，其中一小部分质粒进行了质量控制，可供科学家使用。
所有的产品都严格仅供学术研究使用，不能应用于临床试验，包括人体摄入、注射或外用的药理学用途。
确保质粒的关键元件正确，但是我们并不能保证实验结果。页面展示的图谱序列为理论序列，可能与测序结果不一致，请自行比对后确定是否满足要求。(如果测过序，本页面一般会提供下载)
开放数据库中的大多数载体序列都没有被完全测序。如果实际序列与参考序列的相似度超过99%，则将其视为正确。
由于科学研究是在探索未知，具有很大的不确定性，在任何情况下，我们都不承担超出质粒本身的额外经济损失或责任。
Note:
- 载体名称:
- pDD162 (Peft-3::Cas9 + Empty sgRNA)
- 载体抗性:
- Ampicillin
- 载体长度:
- 8113 bp
- 载体类型:
- Worm Expression, CRISPR
- 复制子:
- ori
- 拷贝数:
- High Copy
- 启动子:
- eft-3
- 克隆方法:
- Ligation Independent Cloning
- 3'引物:
- ggtgtgaaataccgcacaga
pDD162 (Peft-3::Cas9 + Empty sgRNA) 载体图谱
质粒操作方法
1. 发货形式：质粒干粉(常温运输，存于-20度，请务必先转化提质粒后使用)
2. 收到质粒干粉后请先5000rpm离心1min，再加入20μl ddH2O溶解质粒;（质粒复测的浓度有时候与标称值差距较大，这可能是因为冻干质粒在管中的位置、复溶效率、测量偏差以及管壁的吸附导致，因此建议先转化提质粒后再使用）
3. 取1支100μl 感受态于冰上解冻10min，加入2μl质粒，再冰浴30min后，42℃热激60s，不要搅动，再冰浴2min；
4. 加入900μl无抗的LB液体培养基，180rpm震荡37℃培养45min (30℃培养1-1.5小时);
5. 6000rpm离心5min，仅留100μl上清液重悬细菌沉淀，并涂布至目标质粒抗性的LB平板上;
6. 将平板倒置37℃培养14h，如果要求是30℃则培养20h; (菌落过多则将质粒稀释后再转化；没有菌落则加入10μl质粒转化；建议不要直接转表达感受态， 要先转克隆感受态，重提质粒后再导入表达感受态);
7. 挑取单菌落至LB液体培养基中，加入对应抗生素，220rpm震荡培养14h，根据实验需要和质粒提取试剂盒说明书提取质粒。
pDD162 (Peft-3::Cas9 + Empty sgRNA) 载体序列
LOCUS 40924_14295 8113 bp DNA circular SYN 13-MAY-2021
DEFINITION Cas9 + sgRNA plasmid that can be modified to cleave any Cas9 target
site in the C. elegans genome..
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 8113)
AUTHORS Dickinson DJ, Ward JD, Reiner DJ, Goldstein B
TITLE Engineering the Caenorhabditis elegans genome using Cas9-triggered
homologous recombination.
JOURNAL Nat Methods. 2013 Sep 1. doi: 10.1038/nmeth.2641.
PUBMED 23995389
REFERENCE 2 (bases 1 to 8113)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 8113)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Nat
Methods. 2013 Sep 1. doi: 10.1038/nmeth.2641."
COMMENT SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..8113
/mol_type="other DNA"
/organism="synthetic DNA construct"
rep_origin 1..589
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 735..751
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
protein_bind 771..791
/label=attB4
/note="core recombination site for the Gateway(R) BP
reaction"
promoter 792..1391
/label=eft-3 promoter
/note="promoter for the C. elegans elongation factor
1-alpha
gene (Mitrovich and Anderson, 2000)"
CDS join(1408..2451,2503..3573,3625..4650,4702..5664)
/codon_start=1
/product="S. pyogenes Cas9, codon optimized for C. elegans
with synthetic introns"
/label=Cas9
/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 5680..5700
/codon_start=1
/label=SV40 NLS
/note="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/translation="PKKKRKV"
CDS 5701..5727
/codon_start=1
/label=HA
/note="HA (human influenza hemagglutinin) epitope tag"
/translation="YPYDVPDYA"
terminator 5742..6065
/label=tbb-2 terminator
/note="transcription terminator for the C. elegans tubulin
beta-2 gene"
protein_bind complement(6069..6089)
/label=attB3
/note="core recombination site for the Gateway(R) BP
reaction"
primer_bind complement(6097..6113)
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
promoter 6134..6483
/label=CeU6 promoter
/note="promoter for the C. elegans R07E5.16 U6 snRNA gene
(Dickinson et al., 2013)"
misc_RNA 6485..6560
/label=gRNA scaffold
/note="guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system"
primer_bind 6832..6854
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
primer_bind complement(6892..6910)
/label=pBRforEco
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
promoter 6978..7082
/label=AmpR promoter
CDS 7083..7940
/codon_start=1
/label=AmpR
/note="beta-lactamase"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGSDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"