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。
pTWIN1 is an E. coli plasmid cloning vector designed for recombinant protein expression, labeling, and cyclization using the IMPACT-TWIN Kit (NEB #E6901) (1). It contains the pMB1 origin of replication from pBR322 and is maintained at a similar copy number to pBR322; in addition, pTWIN1 also contains an M13 origin of replication. The multiple cloning site (MCS) is positioned to allow translational fusion of an intein tag to the N-terminus, C-terminus, or both, of the cloned target protein. The mini-inteins encoded by pTWIN1, the Ssp DnaB intein and the Mxe GyrA intein, cleave the peptide bond at their C- and N-termini, respectively (1-4). The chitin binding domain (CBD) from B. circulans, fused to each intein, facilitates purification of the intein-target protein precursor. Transcription of the intein tags is controlled by the inducible T7 promoter, requiring E. coli strains containing integrated copies of the T7 RNA polymerase gene [e.g., NEB #C2566, #C2833 or BL21(DE3)] for expression. Basal expression from the T7 promoter is minimized by the binding of the Lac repressor, encoded by the lacI gene, to the lac operator immediately downstream of the T7 promoter (5). Translation of the intein tags utilizes the translation initiation signal (Shine Dalgarno sequence) from the strongly expressed T7 gene 10 protein (φ10).
- 载体名称:
- pTWIN1
- 载体抗性:
- Ampicillin
- 载体长度:
- 7375 bp
- 载体类型:
- Bacterial expression vector
- 复制子:
- ori
- 载体来源:
- NEB
- 拷贝数:
- High copy number
- 启动子:
- T7
- 克隆方法:
- Enzyme Cut
- 载体标签:
- intein
- 表达方法:
- IPTG induced
pTWIN1 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。
pTWIN1 载体序列
LOCUS pTWIN1. 7375 bp DNA circular SYN 09-DEC-2020
DEFINITION synthetic circular DNA.
ACCESSION .
VERSION .
KEYWORDS pTWIN1
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7375)
TITLE Direct Submission
REFERENCE 2 (bases 1 to 7375)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..7375
/mol_type="other DNA"
/organism="synthetic DNA construct"
promoter 35..139
/label=AmpR promoter
CDS 140..997
/label=AmpR
/note="beta-lactamase"
rep_origin complement(1042..1555)
/direction=LEFT
/label=M13 ori
/note="M13 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
rep_origin 1666..2254
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(2626..2814)
/label=rop
/note="Rop protein, which maintains plasmids at low copy
number"
protein_bind complement(3337..3358)
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
CDS complement(3374..4453)
/label=lacI
/note="lac repressor"
promoter complement(4454..4531)
/label=lacI promoter
terminator 4684..4770
/label=rrnB T1 terminator
/note="transcription terminator T1 from the E. coli rrnB
gene"
terminator 4867..4953
/gene="Escherichia coli rrnB"
/label=Escherichia coli rrnB terminator
/note="rrnB T1 terminator"
/note="transcription terminator T1 from the E. coli rrnB
gene"
terminator 5050..5136
/gene="Escherichia coli rrnB"
/label=Escherichia coli rrnB terminator
/note="rrnB T1 terminator"
/note="transcription terminator T1 from the E. coli rrnB
gene"
terminator 5233..5319
/gene="Escherichia coli rrnB"
/label=Escherichia coli rrnB terminator
/note="rrnB T1 terminator"
/note="transcription terminator T1 from the E. coli rrnB
gene"
terminator complement(5419..5462)
/label=rrnB T1 terminator
/note="transcription terminator T1 from the E. coli rrnB
gene"
promoter 5637..5655
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
protein_bind 5656..5680
/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 5695..5717
/label=RBS
/note="efficient ribosome binding site from bacteriophage
T7 gene 10 (Olins and Rangwala, 1989)"
CDS 5749..5901
/label=CBD
/note="chitin binding domain from chitinase A1 (Watanabe et
al., 1994)"
CDS 5941..6402
/codon_start=1
/label=Ssp DnaB intein
/note="Ssp DnaB intein"
/translation="AISGDSLISLASTGKRVSIKDLLDEKDFEIWAINEQTMKLESAKV
SRVFCTGKKLVYILKTRLGRTIKATANHRFLTIDGWKRLDELSLKEHIALPRKLESSSL
QLSPEIEKLSQSDIYWDSIVSITETGVEEVFDLTVPGPHNFVANDIIVHN"
CDS 6445..7038
/label=Mxe GyrA intein
/note="modified GyrA intein (Southworth et al., 1999)"
CDS 7069..7224
/codon_start=1
/gene="Bacillus circulans chiA"
/product="chitin binding domain from chitinase A1 (Watanabe
et al., 1994)"
/label=Bacillus circulans chiA
/note="CBD"
/translation="TTNPGVSAWQVNTAYTAGQLVTYNGKTYKCLQPHTSLAGWEPSNV
PALWQLQ"
terminator 7303..7350
/label=T7 terminator
/note="transcription terminator for bacteriophage T7 RNA
polymerase"