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。
- 载体名称:
- pMCSG77
- 载体抗性:
- Kanamycin
- 载体长度:
- 5445 bp
- 载体类型:
- Structural Genomics Vectors
- 复制子:
- p15A ori
- 载体来源:
- Eschenfeldt WH, Makowska-Grzyska M, Stols L, Donnelly MI,
- 拷贝数:
- Medium copy number
pMCSG77 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。
pMCSG77 载体序列
LOCUS pMCSG77. 5445 bp DNA circular SYN 01-JAN-1980
DEFINITION Bacterial vector with a p15A origin encoding tRNA genes for rare Arg
and Ile codons, for expressing a protein with a 6xHis-TEV leader
plus a second untagged protein.
ACCESSION .
VERSION .
KEYWORDS pMCSG77
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 5445)
AUTHORS Eschenfeldt WH, Makowska-Grzyska M, Stols L, Donnelly MI,
Jedrzejczak R, Joachimiak A.
TITLE New LIC vectors for production of proteins from genes containing
rare codons.
JOURNAL J. Struct. Funct. Genomics 2013;14:135-44.
PUBMED 24057978
REFERENCE 2 (bases 1 to 5445)
AUTHORS Midwest Center for Structural Genomics
TITLE Direct Submission
REFERENCE 3 (bases 1 to 5445)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "J. Struct.
Funct. Genomics"; date: "2013"; volume: "14"; pages: "135-44"
COMMENT SGRef: number: 2; type: "Journal Article"
COMMENT There are two sites for ligation-independent cloning (LIC).
To express a 6xHis-TEV-tagged protein, linearize with SspI and treat
with T4 DNA polymerase plus dGTP.
To express a second untagged protein, linearize with SmaI and treat
with T4 DNA polymerase plus dATP.
FEATURES Location/Qualifiers
source 1..5445
/mol_type="other DNA"
/organism="synthetic DNA construct"
terminator complement(26..73)
/label=T7 terminator
/note="transcription terminator for bacteriophage T7 RNA
polymerase"
CDS complement(140..157)
/label=6xHis
/note="6xHis affinity tag"
RBS 243..248
/note="ribosome binding site"
CDS complement(279..299)
/label=TEV site
/note="tobacco etch virus (TEV) protease recognition and
cleavage site"
CDS complement(324..341)
/label=6xHis
/note="6xHis affinity tag"
CDS complement(342..344)
/codon_start=1
/product="start codon"
/label=start codon
/note="ATG"
/translation="M"
RBS complement(352..374)
/label=RBS
/note="efficient ribosome binding site from bacteriophage
T7 gene 10 (Olins and Rangwala, 1989)"
protein_bind complement(389..413)
/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(414..432)
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
tRNA complement(707..783)
/label=argU
tRNA complement(1053..1128)
/label=ileX
promoter 1245..1322
/label=lacIq promoter
/note="In the lacIq allele, a single base change in the
promoter boosts expression of the lacI gene about 10-fold."
CDS 1323..2402
/label=lacI
/note="lac repressor"
protein_bind 2418..2439
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
terminator 2607..2641
/label=lambda t0 terminator
/note="minimal transcription terminator from phage lambda
(Scholtissek and Grosse, 1987)"
CDS complement(3353..4165)
/label=KanR
/note="aminoglycoside phosphotransferase"
rep_origin complement(4760..5305)
/direction=LEFT
/label=p15A ori
/note="Plasmids containing the medium-copy-number p15A
origin of replication can be propagated in E. coli cells
that contain a second plasmid with the ColE1 origin."