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:
- pLVPT-tTR-KRAB
- 载体抗性:
- Ampicillin
- 载体长度:
- 11634 bp
- 载体类型:
- Mammalian Expression, Lentiviral
- 复制子:
- ori
- 拷贝数:
- High Copy
- 启动子:
- mPGK
- 克隆方法:
- Restriction Enzyme
- 5'引物:
- See map
pLVPT-tTR-KRAB 载体载体图谱
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。
pLVPT-tTR-KRAB 载体载体序列
LOCUS V006525 11634 bp DNA circular SYN 13-MAY-2021 DEFINITION Exported. ACCESSION V006525 VERSION V006525 KEYWORDS pLVPT-tTR-KRAB SOURCE synthetic DNA construct ORGANISM synthetic DNA construct . REFERENCE 1 (bases 1 to 11634) AUTHORS Szulc J, Wiznerowicz M, Sauvain MO, Trono D, Aebischer P TITLE A versatile tool for conditional gene expression and knockdown. JOURNAL Nat Methods. 2006 Feb . 3(2):109-16. PUBMED 16432520 REFERENCE 2 (bases 1 to 11634) TITLE Direct Submission REFERENCE 3 (bases 1 to 11634) AUTHORS . TITLE Direct Submission COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Nat Methods. 2006 Feb . 3(2):109-16." SGRef: number: 2; type: "Journal Article" FEATURES Location/Qualifiers source 1..11634 /mol_type="other DNA" /organism="synthetic DNA construct" CDS 176..217 /note="Protein Tat from Human immunodeficiency virus type 1 group M subtype B (isolate WMJ22). Accession#: P12509" /label="Protein Tat" misc_feature 294..411 /label="cPPT/CTS" /note="central polypurine tract and central termination sequence of HIV-1" promoter 450..949 /label="PGK promoter" /note="mouse phosphoglycerate kinase 1 promoter" CDS 1006..1722 /label="EGFP" /note="enhanced GFP" misc_feature 1805..2367 /label="IRES" /note="internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV)" CDS 2372..2992 /label="TetR" /note="tetracycline repressor TetR" CDS 2993..3013 /label="SV40 NLS" /note="nuclear localization signal of SV40 (simian virus 40) large T antigen" CDS 3116..3310 /label="KRAB" /note="Kruppel-associated box (KRAB) transcriptional repression domain from the human zinc finger protein ZNF10 (Margolin et al., 1994)" misc_feature 3480..4068 /label="WPRE" /note="woodchuck hepatitis virus posttranscriptional regulatory element" protein_bind 4206..4476 /label="tetracycline response element" /note="contains seven copies of the tetracycline operator tetO" LTR 4515..4695 /label="5' LTR (truncated)" /note="truncated 5' long terminal repeat (LTR) from HIV-1" promoter complement(4720..4738) /label="SP6 promoter" /note="promoter for bacteriophage SP6 RNA polymerase" promoter 5526..5630 /label="AmpR promoter" CDS 5631..6488 /label="AmpR" /note="beta-lactamase" rep_origin 6662..7250 /direction=RIGHT /label="ori" /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" promoter 7496..7825 /label="SV40 promoter" /note="SV40 enhancer and early promoter" intron 8959..9024 /label="small t intron" /note="SV40 (simian virus 40) small t antigen intron" CDS 9154..9174 /label="SV40 NLS" /note="nuclear localization signal of SV40 (simian virus 40) large T antigen" polyA_signal 9599..9733 /label="SV40 poly(A) signal" /note="SV40 polyadenylation signal" LTR 10277..10534 /label="5' LTR (truncated)" /note="truncated 5' long terminal repeat (LTR) from HIV-1" misc_feature 10581..10706 /label="HIV-1 Psi" /note="packaging signal of human immunodeficiency virus type 1" misc_feature 11199..11432 /label="RRE" /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." CDS join(11617..11634,1..27) /label="gp41 peptide" /note="antigenic peptide corresponding to amino acids 655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik et al., 2013)"