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。
The hEF1a promoter and the HTLV 5'LTR forms a composite promoter. hEF1a-HTLV promoter is a composite promoter comprising the human Elongation Factor-1α(EF-1α) core promoter and the R segment and part of the U5 sequence (R-U5’) of the Human T-Cell Leukemia Virus (HTLV) Type 1 Long Terminal Repeat. The EF-1α promoter exhibits a strong activity and yields long lasting expression of a transgene in vivo. The R-U5’ has been coupled to the EF-1α core promoter to enhance stability of RNA.
- 载体名称:
- pCDH-CMV-MCS-EF1-copGFP
- 载体抗性:
- Ampicillin
- 载体长度:
- 7546 bp
- 载体类型:
- Lentiviral vectors
- 复制子:
- ori
- 启动子:
- RSV
- 克隆方法:
- Enzyme digestion and ligation
- 5'引物:
- CMV-F: CGCAAATGGGCGGTAGGCGTG
- 载体标签:
- copGFP
pCDH-CMV-MCS-EF1-copGFP 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。
pCDH-CMV-MCS-EF1-copGFP 载体序列
LOCUS V007087 7546 bp DNA circular SYN 13-JAN-2022
DEFINITION Exported.
ACCESSION V007087
VERSION V007087
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
.
REFERENCE 1 (bases 1 to 7546)
TITLE Direct Submission
REFERENCE 2 (bases 1 to 7546)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..7546
/mol_type="other DNA"
/organism="synthetic DNA construct"
promoter 6..233
/label="RSV promoter"
/note="Rous sarcoma virus enhancer/promoter"
LTR 234..414
/label="5' LTR (truncated)"
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
misc_feature 458..583
/label="HIV-1 Psi"
/note="packaging signal of human immunodeficiency virus
type 1"
misc_feature 1076..1309
/label="RRE"
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
CDS 1493..1537
/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 1686..1727
/note="Protein Tat from Human immunodeficiency virus type 1
group M subtype B (isolate WMJ22). Accession#: P12509"
/label="Protein Tat"
misc_feature 1798..1915
/label="cPPT/CTS"
/note="central polypurine tract and central termination
sequence of HIV-1"
promoter 2005..2208
/label="CMV promoter"
/note="human cytomegalovirus (CMV) immediate early
promoter"
promoter 2349..2560
/label="EF-1-alpha core promoter"
/note="core promoter for human elongation factor
EF-1-alpha"
LTR 2573..2841
/label="5' LTR (truncated)"
/note="truncated 5' long terminal repeat (LTR) from human
T-cell leukemia virus (HTLV) type 1"
CDS 2897..3562
/label="CopGFP"
/note="green fluorescent protein 2 from Pontellina plumata,
also known as ppluGFP2 (Shagin et al., 2004)"
misc_feature 3641..4229
/label="WPRE"
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
LTR 4303..4536
/label="3' LTR (Delta-U3)"
/note="self-inactivating 3' long terminal repeat (LTR) from
HIV-1"
polyA_signal 4608..4742
/label="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"
rep_origin 4748..4883
/label="SV40 ori"
/note="SV40 origin of replication"
primer_bind complement(4921..4937)
/label="M13 rev"
/note="common sequencing primer, one of multiple similar
variants"
protein_bind complement(4945..4961)
/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(4969..4999)
/label="lac promoter"
/note="promoter for the E. coli lac operon"
protein_bind complement(5014..5035)
/label="CAP binding site"
/note="CAP binding activates transcription in the presence
of cAMP."
rep_origin complement(5323..5911)
/direction=LEFT
/label="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(6085..6942)
/label="AmpR"
/note="beta-lactamase"
promoter complement(6943..7047)
/label="AmpR promoter"
primer_bind 7521..7537
/label="M13 fwd"
/note="common sequencing primer, one of multiple similar
variants"