质粒编号
质粒名称
规格
价格
V010577
pNIC28-Bsa4
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学术研究使用，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。
Note:pNIC28-Bsa4, a core vector for SGC, enables high-efficiency expression/purification of human proteins via T7-LacO system, N-terminal His6-TEV tag, and LIC cloning, supporting crystallization studies.
- 载体名称:
- pNIC28-Bsa4
- 载体抗性:
- Kanamycin
- 载体长度:
- 7284 bp
- 载体类型:
- Structural Genomics Vectors
- 复制子:
- ori
- 载体来源:
- Savitsky P, Bray J, Cooper CD, Marsden BD, Mahajan P, Burgess-Brown
- 拷贝数:
- High copy number
- 启动子:
- sacB
pNIC28-Bsa4 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。
pNIC28-Bsa4 载体序列
LOCUS 40924_33252 7284 bp DNA circular SYN 18-DEC-2018
DEFINITION Expression vector pNIC28-Bsa4, complete sequence.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7284)
AUTHORS Stols L, Gu M, Dieckman L, Raffen R, Collart FR, Donnelly MI.
TITLE A new vector for high-throughput, ligation-independent cloning
encoding a tobacco etch virus protease cleavage site
JOURNAL Protein Expr. Purif. 25 (1), 8-15 (2002)
PUBMED 12071693
REFERENCE 2 (bases 1 to 7284)
AUTHORS Keates T, Cooper CD, Savitsky P, Allerston CK, Phillips C,
Hammarstrom M, Daga N, Berridge G, Mahajan P, Burgess-Brown NA,
Muller S, Graslund S, Gileadi O.
TITLE Expressing the human proteome for affinity proteomics: optimising
expression of soluble protein domains and in vivo biotinylation
JOURNAL N Biotechnol (2011) In press
PUBMED 22027370
REFERENCE 3 (bases 1 to 7284)
AUTHORS Gileadi O, Burgess-Brown N, Loppnau P.
TITLE Direct Submission
JOURNAL Submitted (25-DEC-2006) Structural Genomics Consortium, University
of Oxford, Botnar Research Centre, Oxford OX3 7LD, UK
REFERENCE 4 (bases 1 to 7284)
TITLE Direct Submission
REFERENCE 5 (bases 1 to 7284)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Protein
Expr. Purif."; date: "2002"; volume: "25"; issue: "1"; pages: "8-15"
COMMENT SGRef: number: 2; type: "Journal Article"; journalName: "N
Biotechnol (2011) In press"
COMMENT SGRef: number: 3; type: "Journal Article"; journalName: "Submitted
(25-DEC-2006) Structural Genomics Consortium, University of Oxford,
Botnar Research Centre, Oxford OX3 7LD, UK"
COMMENT SGRef: number: 4; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..7284
/mol_type="other DNA"
/organism="synthetic DNA construct"
promoter 1..19
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
protein_bind 20..44
/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 59..81
/label=RBS
/note="efficient ribosome binding site from bacteriophage
T7 gene 10 (Olins and Rangwala, 1989)"
CDS 92..109
/label=6xHis
/note="6xHis affinity tag"
CDS 134..154
/label=TEV site
/note="tobacco etch virus (TEV) protease recognition and
cleavage site"
promoter 170..615
/label=sacB promoter
/note="sacB promoter and control region"
CDS 616..2034
/label=SacB
/note="secreted levansucrase that renders bacterial growth
sensitive to sucrose"
misc_feature complement(2083..2097)
/note="3'-LIC sequence; add complement to downstream PCR
primer"
CDS 2145..2162
/label=6xHis
/note="6xHis affinity tag"
terminator 2229..2276
/label=T7 terminator
/note="transcription terminator for bacteriophage T7 RNA
polymerase"
rep_origin 2313..2768
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
CDS complement(2864..3676)
/label=KanR
/note="aminoglycoside phosphotransferase"
rep_origin 3798..4386
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
misc_feature complement(4572..4714)
/label=bom
/note="basis of mobility region from pBR322"
CDS complement(4819..5007)
/label=rop
/note="Rop protein, which maintains plasmids at low copy
number"
protein_bind complement(5782..5803)
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
CDS complement(5819..6898)
/label=lacI
/note="lac repressor"
promoter complement(6899..6976)
/label=lacI promoter