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:
- pMLBAD_GlycoTagGFP
- 载体抗性:
- Trimethoprim
- 载体长度:
- 7583 bp
- 载体类型:
- Expression vector
- 复制子:
- pBBR1 oriV
- 载体来源:
- Keys TG, Wetter M, Hang I, Rutschmann C, Russo S, Mally M, Steffen M, Zuppiger M, Muller F, Schneider J, Faridmoayer A, Lin CW, Aebi M.
- 启动子:
- araBAD
pMLBAD_GlycoTagGFP 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。
pMLBAD_GlycoTagGFP 载体序列
LOCUS 40924_31100 7583 bp DNA circular SYN 18-DEC-2018
DEFINITION Expression vector pMLBAD_GlycoTagGFP, complete sequence.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7583)
AUTHORS Keys TG, Wetter M, Hang I, Rutschmann C, Russo S, Mally M, Steffen
M, Zuppiger M, Muller F, Schneider J, Faridmoayer A, Lin CW, Aebi M.
TITLE A biosynthetic route for polysialylating proteins in Escherichia
coli
JOURNAL Metab. Eng. (2017) In press
PUBMED 29101090
REFERENCE 2 (bases 1 to 7583)
AUTHORS Keys TG.
TITLE Direct Submission
JOURNAL Submitted (21-NOV-2016) Institute of Microbiology, ETH Zurich,
Vladimir-Prelog-Weg 4, Zurich, ZH 8093, Switzerland
REFERENCE 3 (bases 1 to 7583)
TITLE Direct Submission
REFERENCE 4 (bases 1 to 7583)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Metab. Eng.
(2017) In press"
COMMENT SGRef: number: 2; type: "Journal Article"; journalName: "Submitted
(21-NOV-2016) Institute of Microbiology, ETH Zurich,
Vladimir-Prelog-Weg 4, Zurich, ZH 8093, Switzerland"
COMMENT SGRef: number: 3; type: "Journal Article"
COMMENT ##Assembly-Data-START##
Sequencing Technology :: Sanger dideoxy sequencing
##Assembly-Data-END##
FEATURES Location/Qualifiers
source 1..7583
/mol_type="other DNA"
/organism="synthetic DNA construct"
promoter 55..83
/label=Pc promoter
/note="class 1 integron promoter"
CDS 410..643
/codon_start=1
/label=TpR
/note="E. coli plasmid-associated dihydrofolate reductase"
/translation="MGQSSDEANAPVAGQFALPLSATFGLGDRVRKKSGAAWQGQVVGW
YCTKLTPEGYAVESESHPGSVQIYPVAALERVA"
CDS complement(872..1747)
/codon_start=1
/label=araC
/note="L-arabinose regulatory protein"
/translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
EKVNDVAVKLS"
promoter 1774..2054
/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)"
RBS 2063..2085
/label=RBS
/note="efficient ribosome binding site from bacteriophage
T7 gene 10 (Olins and Rangwala, 1989)"
CDS 2101..2127
/codon_start=1
/label=9xHis
/note="9xHis affinity tag"
/translation="HHHHHHHHH"
CDS 2137..2823
/codon_start=1
/label=superfolder GFP
/note="GFP variant that folds robustly even when fused to
poorly folded proteins (Nager et al., 2011)"
/translation="SKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKF
ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGT
YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKAN
FKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLEF
VTAAGIT"
misc_feature 2833..2889
/label=NAT-tag (C-terminal glycosylation-tag)
/note="NAT-tag (C-terminal glycosylation-tag)"
CDS 2890..2913
/codon_start=1
/label=Strep-Tag II
/note="peptide that binds Strep-Tactin(R), an engineered
form
of streptavidin"
/translation="WSHPQFEK"
terminator 3150..3236
/label=rrnB T1 terminator
/note="transcription terminator T1 from the E. coli rrnB
gene"
terminator 3328..3355
/label=rrnB T2 terminator
/note="transcription terminator T2 from the E. coli rrnB
gene"
promoter complement(4210..4312)
/label=cat promoter
/note="promoter of the E. coli cat gene encoding
chloramphenicol acetyltransferase"
CDS complement(4462..5481)
/codon_start=1
/gene="mob"
/product="Mob"
/function="required for plasmid mobilization"
/label=mob
/protein_id="ATN32196.1"
/translation="MAAYAIMRCKKLAKMGNVAASLKHAYRERETPNADASRTPENEHW
AASSTDEAMGRLRELLPEKRRKDAVLAVEYVMTASPEWWKSASQEQQAAFFEKAHKWLA
DKYGADRIVTASIHRDETSPHMTAFVVPLTQDGRLSAKEFIGNKAQMTRDQTTFAAAVA
DLGLQRGIEGSKARHTRIQAFYEALERPPVGHVTISPQAVEPRAYAPQGLAEKLGISKR
VETPEAVADRLTKAVRQGYEPALQAAAGAREMRKKADQAQETARDLRERLKPVLDALGP
LNRDMQAKAAAIIKAVGEKLLTEQREVQRQKQAQRQQERGRAHFPEKCHLTSKKPLLS"
gene complement(4462..5481)
/gene="mob"
/label=mob
rep_origin 5705..6474
/label=pBBR1 oriV
/note="replication origin of the broad-host-range plasmid
pBBR1 from Bordetella bronchiseptica; requires the pBBR1
Rep protein for replication"
CDS 6475..7134
/codon_start=1
/label=pBBR1 Rep
/note="replication protein for the broad-host-range plasmid
pBBR1 from Bordetella bronchiseptica"
/translation="MATQSREIGIQAKNKPGHWVQTERKAHEAWAGLIARKPTAAMLLH
HLVAQMGHQNAVVVSQKTLSKLIGRSLRTVQYAVKDLVAERWISVVKLNGPGTVSAYVV
NDRVAWGQPRDQLRLSVFSAAVVVDHDDQDESLLGHGDLRRIPTLYPGEQQLPTGPGEE
PPSQPGIPGMEPDLPALTETEEWERRGQQRLPMPDEPCFLDDGEPLEPPTRVTLPRR"