Note:
基本信息
- 载体名称:
- pTIT-P
- 载体抗性:
- Ampicillin
- 载体长度:
- 4026 bp
- 载体类型:
- Rabies virus-derived expression vector
- 复制子:
- ori
- 载体来源:
- Finke S, Conzelmann KK.
- 启动子:
- T3
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。
pTIT-P 质粒 (编号: V002627)序列
LOCUS V002627 4026 bp DNA circular SYN 18-DEC-2018
DEFINITION Exported.
ACCESSION V002627
VERSION V002627
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
.
REFERENCE 1 (bases 1 to 4026)
AUTHORS Finke S, Conzelmann KK.
TITLE Virus promoters determine interference by defective RNAs: selective
amplification of mini-RNA vectors and rescue from cDNA by a 3'
copy-back ambisense rabies virus
JOURNAL J. Virol. 73 (5), 3818-3825 (1999)
PUBMED 10196276
REFERENCE 2 (bases 1 to 4026)
AUTHORS Wickersham IR, Sullivan HA, Seung HS.
TITLE Production of glycoprotein-deleted rabies viruses for monosynaptic
tracing and high-level gene expression in neurons
JOURNAL Unpublished
REFERENCE 3 (bases 1 to 4026)
AUTHORS Wickersham IR.
TITLE Direct Submission
JOURNAL Submitted (06-DEC-2009) Brain and Cognitive Sciences, Howard Hughes
Medical Institute and Massachusetts Institute of Technology, 43
Vassar St., Cambridge, MA 02139, USA
REFERENCE 4 (bases 1 to 4026)
TITLE Direct Submission
REFERENCE 5 (bases 1 to 4026)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "J. Virol.";
date: "1999"; volume: "73"; issue: "5"; pages: "3818-3825"
SGRef: number: 2; type: "Journal Article"; journalName:
"Unpublished"
SGRef: number: 3; type: "Journal Article"; journalName: "Submitted
(06-DEC-2009) Brain and Cognitive Sciences, Howard Hughes Medical
Institute and Massachusetts Institute of Technology, 43 Vassar St.,
Cambridge, MA 02139, USA"
SGRef: number: 4; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..4026
/mol_type="other DNA"
/organism="synthetic DNA construct"
primer_bind 148..164
/label="M13 fwd"
/note="common sequencing primer, one of multiple similar
variants"
promoter 171..189
/label="T7 promoter"
/note="promoter for bacteriophage T7 RNA polymerase"
misc_feature 240..701
/label="IRES"
/note="internal ribosome entry site (IRES) of the
encephalomyocarditis virus (EMCV)"
CDS 706..1596
/gene="P"
/label="Phosphoprotein"
/note="Phosphoprotein from Rabies virus (strain SAD B19).
Accession#: P16286"
terminator 1712..1759
/label="T7 terminator"
/note="transcription terminator for bacteriophage T7 RNA
polymerase"
primer_bind complement(1782..1798)
/label="SK primer"
/note="common sequencing primer, one of multiple similar
variants"
promoter complement(1835..1853)
/label="T3 promoter"
/note="promoter for bacteriophage T3 RNA polymerase"
primer_bind complement(1874..1890)
/label="M13 rev"
/note="common sequencing primer, one of multiple similar
variants"
protein_bind complement(1898..1914)
/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(1922..1952)
/label="lac promoter"
/note="promoter for the E. coli lac operon"
protein_bind complement(1967..1988)
/label="CAP binding site"
/note="CAP binding activates transcription in the presence
of cAMP."
rep_origin complement(2276..2864)
/direction=LEFT
/label="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(3038..3895)
/label="AmpR"
/note="beta-lactamase"
promoter complement(3896..4000)
/label="AmpR promoter"