Note: pBABE-Puro (5.2 kb) is a retroviral vector for gene delivery. It contains LTRs (long terminal repeats) for viral integration, a MMLV Psi (Ψ) packaging signal, a truncated gag sequence, and an MCS for gene insertion. The vector also carries AmpR for bacterial selection, a pUC ori, and a SV40 promoter driving PuroR (puromycin resistance) for stable selection. It is widely used for creating stable cell lines via retroviral transduction.
基本信息
- 载体名称:
- pBABE-Puro
- 载体抗性:
- Ampicillin
- 载体长度:
- 5169 bp
- 载体类型:
- Viral Expression & Packaging Vectors
- 复制子:
- ori
- 载体来源:
- Morgenstern JP, Land H.
- 筛选标记:
- Puromycin
- 拷贝数:
- High copy number
- 克隆方法:
- Restriction Enzyme
- 5'引物:
- pBABE 5'
- 3'引物:
- pBABE 3'
- 感受态:
- Top10
产品信息
质粒编号:V010490
质粒名称:pBABE-Puro
规格:5 μg (冻干粉)
下载资源
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。
确保质粒的关键元件正确，但是我们并不能保证实验效果。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。
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。
参考文献
- Yang S, Brindley PJ, Zeng Q, Li Y, Zhou J, Liu Y, Liu B, Cai L, Zeng T, Wei Q, Lan L, McManus DP. Transduction of Schistosoma japonicum schistosomules with vesicular stomatitis virus glycoprotein pseudotyped murine leukemia retrovirus and expression of reporter human telomerase reverse transcriptase in the transgenic schistosomes. Mol Biochem Parasitol. 2010 Dec;174(2):109-16. doi: 10.1016/j.molbiopara.2010.07.007. Epub 2010 Aug 6. PMID: 20692298; PMCID: PMC3836731.
pBABE-Puro 质粒 (编号: V010490)序列
LOCUS pBABE-Puro. 5169 bp DNA circular SYN 01-JAN-1980
DEFINITION Retroviral expression vector derived from the Moloney murine
leukemia virus. Contains a puromycin resistance marker.
ACCESSION .
VERSION .
KEYWORDS pBABE-Puro
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 5169)
AUTHORS Morgenstern JP, Land H.
TITLE Advanced mammalian gene transfer: high titre retroviral vectors with
multiple drug selection markers and a complementary helper-free
packaging cell line.
JOURNAL Nucleic Acids Res. 1990;18:3587-96.
PUBMED 2194165
REFERENCE 2 (bases 1 to 5169)
AUTHORS Cell Biolabs
TITLE Direct Submission
REFERENCE 3 (bases 1 to 5169)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Nucleic
Acids Res."; date: "1990"; volume: "18"; pages: "3587-96"
COMMENT SGRef: number: 2; type: "Journal Article"
COMMENT Strong expression is driven by the viral promoter in the 5' LTR. The
commercially provided sequence was modified to include the splice
donor mutation.
FEATURES Location/Qualifiers
source 1..5169
/mol_type="other DNA"
/organism="synthetic DNA construct"
LTR 8..477
/label=LTR
/note="long terminal repeat from Moloney murine leukemia
virus"
misc_feature 541
/label=splice donor mutation
/note="splice donor mutation"
/note="T-to-C mutation that blocks RNA splicing"
misc_feature 542..741
/label=MMLV Psi
/note="packaging signal of Moloney murine leukemia virus
(MMLV)"
CDS 934..1350
/label=gag (truncated)
/note="truncated Moloney murine leukemia virus (MMLV) gag
gene lacking the start codon"
misc_feature 1355..1402
/label=MCS
/note="MCS"
/note="multiple cloning site"
promoter 1413..1742
/label=SV40 promoter
/note="SV40 enhancer and early promoter"
CDS 1752..2348
/label=PuroR
/note="puromycin N-acetyltransferase"
LTR 2450..3043
/label=LTR
/note="long terminal repeat from Moloney murine leukemia
virus"
rep_origin complement(3415..4003)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(4184..5041)
/label=AmpR
/note="beta-lactamase"
promoter complement(5042..5146)
/label=AmpR promoter