质粒编号
质粒名称
规格
价格
V010931
pETcoco-2
5 μg (冻干粉)
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。
pETcoco-2 serves as the backbone vector, with L-arabinose added to increase copy number. Following IPTG induction, T7 RNA polymerase is activated to initiate transcription of the target gene controlled by the T7 promoter, enabling the secretion and expression of two bacteriocins in E. coli Tuner™(DE3) strain.
- 载体名称:
- pETcoco-2
- 载体抗性:
- Ampicillin
- 载体长度:
- 12417 bp
- 载体类型:
- pET & Duet Vectors (Novagen)
- 复制子:
- ori2
- 载体来源:
- Novagen (EMD Millipore)
- 拷贝数:
- Low copy number
- 启动子:
- araBAD
- 感受态:
- EPI300
pETcoco-2 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。
pETcoco-2 载体序列
LOCUS pETcoco-2. 12417 bp DNA circular SYN 01-JAN-1980
DEFINITION Bacterial vector with an ampicillin resistance marker that allows
single-copy replication, on-demand amplification, and tightly
regulated expression.
ACCESSION .
VERSION .
KEYWORDS pETcoco-2
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 12417)
AUTHORS Novagen (EMD Millipore)
TITLE Direct Submission
REFERENCE 2 (bases 1 to 12417)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"
COMMENT The copy number can be increased to 20-50 copies per cell by adding
arabinose to the growth medium.
FEATURES Location/Qualifiers
source 1..12417
/mol_type="other DNA"
/organism="synthetic DNA construct"
protein_bind 3..27
/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)."
RBS 42..64
/label=RBS
/note="efficient ribosome binding site from bacteriophage
T7 gene 10 (Olins and Rangwala, 1989)"
CDS 72..74
/codon_start=1
/product="start codon"
/label=start codon
/note="ATG"
/translation="M"
CDS 75..92
/label=6xHis
/note="6xHis affinity tag"
CDS 102..146
/label=S-Tag
/note="affinity and epitope tag derived from pancreatic
ribonuclease A"
CDS 162..176
/label=enterokinase site
/note="enterokinase recognition and cleavage site"
CDS 249..281
/label=HSV tag
/note="HSV (herpes simplex virus) epitope tag"
CDS 326..343
/label=6xHis
/note="6xHis affinity tag"
terminator 410..457
/label=T7 terminator
/note="transcription terminator for bacteriophage T7 RNA
polymerase"
CDS complement(834..1709)
/label=araC
/note="L-arabinose regulatory protein"
promoter 1736..2020
/label=araBAD promoter
/note="promoter of the L-arabinose operon of E. coli; the
araC regulatory gene is transcribed in the opposite
direction (Guzman et al., 1995)"
CDS 2080..3225
/label=trfA
/note="trans-acting replication protein that binds to and
activates oriV"
CDS complement(3449..4306)
/label=AmpR
/note="beta-lactamase"
promoter complement(4307..4398)
/label=AmpR promoter
rep_origin complement(4965..5677)
/direction=LEFT
/label=oriV
/note="incP origin of replication"
rep_origin 5756..5975
/label=ori2
/note="secondary origin of replication for the bacterial F
plasmid; also known as oriS"
CDS 6066..6818
/label=repE
/note="replication initiation protein for the bacterial F
plasmid"
misc_feature 6824..7074
/label=incC
/note="incompatibility region of the bacterial F plasmid"
CDS 7400..8572
/label=sopA
/note="partitioning protein for the bacterial F plasmid"
CDS 8575..9543
/label=sopB
/note="partitioning protein for the bacterial F plasmid"
misc_feature 9619..10092
/gene="sopC"
/label=sopC
/note="sopC"
/note="centromere-like partitioning region of the bacterial
F plasmid"
misc_feature complement(10352..10750)
/label=cos
/note="lambda cos site; allows packaging into phage lambda
particles"
protein_bind complement(10768..10801)
/label=loxP
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (ATGTATGC) (Shaw et al., 2021)."
protein_bind complement(10894..10915)
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
CDS complement(10931..12010)
/label=lacI
/note="lac repressor"
promoter complement(12011..12088)
/label=lacI promoter