质粒编号
质粒名称
规格
价格
V011750
tet-pLKO-sgRNA-puro
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科学研究使用，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。
- 载体名称:
- tet-pLKO-sgRNA-puro
- 载体抗性:
- Ampicillin
- 载体长度:
- 8880 bp
- 载体类型:
- Mammalian Expression, Lentiviral, CRISPR
- 复制子:
- ori
- 筛选标记:
- Puromycin
- 拷贝数:
- Low Copy
- 启动子:
- hPGK
- 克隆方法:
- Restriction Enzyme
- 5'引物:
- tet-pLKO-seq-1: 5’- GTTTCAGACCCACCTCCCAAC’-3
tet-pLKO-sgRNA-puro 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。
tet-pLKO-sgRNA-puro 载体序列
LOCUS V011750 8880 bp DNA circular SYN 13-MAY-2021
DEFINITION Exported.
ACCESSION V011750
VERSION V011750
KEYWORDS tet-pLKO-sgRNA-puro
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
.
REFERENCE 1 (bases 1 to 8880)
AUTHORS Huang HT, Seo HS, Zhang T, Wang Y, Jiang B, Li Q, Buckley DL, Nabet
B, Roberts JM, Paulk J, Dastjerdi S, Winter GE, McLauchlan H, Moran
J, Bradner JE, Eck MJ, Dhe-Paganon S, Zhao JJ, Gray NS
TITLE MELK is not necessary for the proliferation of basal-like breast
cancer cells.
JOURNAL Elife. 2017 Sep 19;6. pii: e26693. doi: 10.7554/eLife.26693.
PUBMED 28926338
REFERENCE 2 (bases 1 to 8880)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 8880)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Elife.";
date: "2017-09-19"; pages: "
10.7554/eLife.26693"
SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..8880
/mol_type="other DNA"
/organism="synthetic DNA construct"
promoter 7..507
/label="hPGK promoter"
/note="human phosphoglycerate kinase 1 promoter"
intron 516..1088
/label="beta-globin intron"
/note="intron from rabbit beta-globin gene"
primer_bind 1096..1115
/label="pCAG-F"
/note="Rabbit beta-globin intron, for pCAG plasmids,
forward primer"
promoter 1143..1161
/label="T7 promoter"
/note="promoter for bacteriophage T7 RNA polymerase"
CDS 1172..1792
/label="TetR"
/note="tetracycline repressor TetR"
misc_feature 1827..2400
/label="IRES"
/note="internal ribosome entry site (IRES) of the
encephalomyocarditis virus (EMCV)"
CDS 2420..3016
/label="PuroR"
/note="puromycin N-acetyltransferase"
LTR 3147..3380
/label="3' LTR (Delta-U3)"
/note="self-inactivating 3' long terminal repeat (LTR) from
HIV-1"
polyA_signal 3452..3586
/label="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"
rep_origin 3613..3748
/label="SV40 ori"
/note="SV40 origin of replication"
promoter complement(3769..3787)
/label="T7 promoter"
/note="promoter for bacteriophage T7 RNA polymerase"
primer_bind complement(3797..3813)
/label="M13 fwd"
/note="common sequencing primer, one of multiple similar
variants"
rep_origin 3955..4410
/label="f1 ori"
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 4436..4540
/label="AmpR promoter"
CDS 4541..5398
/label="AmpR"
/note="beta-lactamase"
rep_origin 5572..6160
/label="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 6314..6331
/label="L4440"
/note="L4440 vector, forward primer"
protein_bind 6448..6469
/label="CAP binding site"
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 6484..6514
/label="lac promoter"
/note="promoter for the E. coli lac operon"
protein_bind 6522..6538
/label="lac operator"
/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 6546..6562
/label="M13 rev"
/note="common sequencing primer, one of multiple similar
variants"
promoter 6583..6601
/label="T3 promoter"
/note="promoter for bacteriophage T3 RNA polymerase"
promoter 6629..6855
/label="RSV promoter"
/note="Rous sarcoma virus enhancer/promoter"
LTR 6856..7036
/label="5' LTR (truncated)"
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
misc_feature 7083..7208
/label="HIV-1 Psi"
/note="packaging signal of human immunodeficiency virus
type 1"
misc_feature 7701..7934
/label="RRE"
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
CDS 8119..8163
/label="gp41 peptide"
/note="antigenic peptide corresponding to amino acids 655
to 669 of the HIV envelope protein gp41 (Lutje Hulsik et
al., 2013)"
CDS 8312..8353
/note="Protein Tat from Human immunodeficiency virus type 1
group M subtype B (isolate WMJ22). Accession#: P12509"
/label="Protein Tat"
promoter 8452..8546
/label="H1-2O2 promoter"
/note="doxycycline-inducible variant of the human H1 RNA
promoter (Henriksen et al., 2007)"
misc_feature 8711..8828
/label="cPPT/CTS"
/note="central polypurine tract and central termination
sequence of HIV-1"
promoter 8877..8880
/label="hPGK promoter"
/note="human phosphoglycerate kinase 1 promoter"