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。
pLVX-IRES-tdTomato is an HIV-1-based, lentiviral expression vector that allows the simultaneous expression of your protein of interest and tdTomato in virtually any mammalian cell type, including primary cells. tdTomato is a member of the family of fruit fluorescent proteins derived from the Discosoma sp. red fluorescent protein, DsRed (1). The vector expresses the two proteins from a bicistronic mRNA transcript, allowing tdTomato to be used as an indicator of transduction efficiency and a marker for selection by flow cytometry. Expression of the bicistronic transcript is driven by the constitutively active human cytomegalovirus immediate early promoter (PCMV IE) located just upstream of the MCS. An encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES), positioned between the MCS and tdTomato, facilitates cap-independent translation of tdTomato from an internal start site at the IRES/tdTomato junction (1). pLVX-IRES-tdTomato contains all of the viral processing elements necessary for the production of replication-incompetent lentivirus, as well as elements to improve viral titer, transgene expression, and overall vector function. The woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) promotes RNA processing events and enhances nuclear export of viral RNA (2), leading to increased viral titers from packaging cells. In addition, the vector includes a Rev-response element (RRE), which further increases viral titers by enhancing the transport of unspliced viral RNA out of the nucleus (3). Finally, pLVX-IRES-tdTomato also contains a central polypurine tract/central termination sequence element (cPPT/CTS). During target cell infection, this element creates a central DNA flap that increases nuclear import of the viral genome, resulting in improved vector integration and more efficient transduction (4). The vector also contains a pUC origin of replication and an E. coli ampicillin resistance gene (Ampr ) for propagation and selection in bacteria.
- 载体名称:
- pLVX-IRES-tdTomato
- 载体抗性:
- Ampicillin
- 载体长度:
- 8892 bp
- 载体类型:
- Viral Expression & Packaging Vectors
- 复制子:
- ori
- 载体来源:
- Clontech
- 筛选标记:
- tdTomato
- 拷贝数:
- High copy number
- 启动子:
- CMV
- 克隆方法:
- Enzyme Cut
- 表达方法:
- Constiutive, Stable
pLVX-IRES-tdTomato 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。
pLVX-IRES-tdTomato 载体序列
LOCUS V012721 8892 bp DNA circular SYN 08-APR-2021
DEFINITION Exported.
ACCESSION V012721
VERSION V012721
KEYWORDS pLVX-IRES-tdTomato
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
.
REFERENCE 1 (bases 1 to 8892)
TITLE Direct Submission
REFERENCE 2 (bases 1 to 8892)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..8892
/mol_type="other DNA"
/organism="synthetic DNA construct"
LTR 1..634
/label="3' LTR"
/note="3' long terminal repeat (LTR) from HIV-1"
misc_feature 681..806
/label="HIV-1 Psi"
/note="packaging signal of human immunodeficiency virus
type 1"
misc_feature 1303..1536
/label="RRE"
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
CDS 1721..1765
/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 1914..1955
/note="Protein Tat from Human immunodeficiency virus type 1
group M subtype B (isolate WMJ22). Accession#: P12509"
/label="Protein Tat"
misc_feature 2027..2144
/label="cPPT/CTS"
/note="central polypurine tract and central termination
sequence of HIV-1"
enhancer 2201..2504
/label="CMV enhancer"
/note="human cytomegalovirus immediate early enhancer"
promoter 2505..2708
/label="CMV promoter"
/note="human cytomegalovirus (CMV) immediate early
promoter"
misc_feature 2843..3420
/label="IRES2"
/note="internal ribosome entry site (IRES) of the
encephalomyocarditis virus (EMCV)"
CDS 3417..4844
/label="tdTomato"
/note="tandem dimeric (pseudo-monomeric) derivative of
DsRed (Shaner et al., 2004)"
misc_feature 4861..5449
/label="WPRE"
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
LTR 5656..6289
/label="5' LTR"
/note="5' long terminal repeat (LTR) from HIV-1"
primer_bind complement(6417..6433)
/label="M13 rev"
/note="common sequencing primer, one of multiple similar
variants"
protein_bind complement(6441..6457)
/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)."
promoter complement(6465..6495)
/label="lac promoter"
/note="promoter for the E. coli lac operon"
protein_bind complement(6510..6531)
/label="CAP binding site"
/note="CAP binding activates transcription in the presence
of cAMP."
rep_origin complement(6819..7407)
/direction=LEFT
/label="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(7581..8438)
/label="AmpR"
/note="beta-lactamase"
promoter complement(8439..8543)
/label="AmpR promoter"
polyA_signal 8591..8725
/label="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"