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:
- pWM91-L6-PT3-T4
- 载体抗性:
- Ampicillin
- 载体长度:
- 13274 bp
- 载体类型:
- Cloning vector
- 复制子:
- R6K γ ori
- 载体来源:
- Lee JW, Gyorgy A, Cameron DE, Pyenson N, Choi KR, Way JC, Silver PA, Del Vecchio D, Collins JJ.
- 启动子:
- sacB
pWM91-L6-PT3-T4 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。
pWM91-L6-PT3-T4 载体序列
LOCUS 40924_46598 13274 bp DNA circular SYN 18-DEC-2018
DEFINITION Cloning vector pWM91-L6-PT3-T4, complete sequence.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 13274)
AUTHORS Lee JW, Gyorgy A, Cameron DE, Pyenson N, Choi KR, Way JC, Silver PA,
Del Vecchio D, Collins JJ.
TITLE Creating Single-Copy Genetic Circuits
JOURNAL Mol. Cell 63 (2), 329-336 (2016)
PUBMED 27425413
REFERENCE 2 (bases 1 to 13274)
AUTHORS Lee JW, Gyorgy A, Cameron DE, Pyenson N, Choi KR, Way JC, Silver PA,
Del Vecchio D, Collins JJ.
TITLE Direct Submission
JOURNAL Submitted (18-MAY-2016) Biological Engineering, MIT, 45 Carleton St,
MIT Building E25-Rm302, Cambridge, MA 02142, USA
REFERENCE 3 (bases 1 to 13274)
TITLE Direct Submission
REFERENCE 4 (bases 1 to 13274)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Mol. Cell";
date: "2016"; volume: "63"; issue: "2"; pages: "329-336"
COMMENT SGRef: number: 2; type: "Journal Article"; journalName: "Submitted
(18-MAY-2016) Biological Engineering, MIT, 45 Carleton St, MIT
Building E25-Rm302, Cambridge, MA 02142, USA"
COMMENT SGRef: number: 3; type: "Journal Article"
COMMENT ##Assembly-Data-START##
Sequencing Technology :: Sanger dideoxy sequencing
##Assembly-Data-END##
FEATURES Location/Qualifiers
source 1..13274
/mol_type="other DNA"
/organism="synthetic DNA construct"
rep_origin complement(60..515)
/direction=LEFT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind 657..673
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
promoter 683..701
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
primer_bind 727..743
/label=KS primer
/note="common sequencing primer, one of multiple similar
variants"
misc_feature 776..1194
/label=similar to lacZ (upstream)
/note="similar to lacZ (upstream)"
terminator complement(1237..1331)
/label=lambda t0 terminator
/note="transcription terminator from phage lambda"
CDS complement(1394..2107)
/label=yeGFP
/note="yeast-enhanced green fluorescent protein"
regulatory complement(2108..2131)
/label=G5
/note="G5"
/regulatory_class="ribosome_binding_site"
RBS 2113..2121
/label=Shine-Dalgarno sequence
/note="full consensus sequence for ribosome-binding sites
upstream of start codons in E. coli; complementary to a
region in the 3' end of the 16S rRNA (Chen et al., 1994)"
promoter complement(2149..2222)
/label=PLtetO-1 promoter
/note="modified phage lambda PL promoter with tet operator
sites (Lutz and Bujard, 1997)"
misc_feature complement(2265..2348)
/label=mfLon-specific tag pdt2
/note="mfLon-specific tag pdt2"
CDS complement(2349..3428)
/label=lacI
/note="lac repressor"
RBS 3434..3442
/label=Shine-Dalgarno sequence
/note="full consensus sequence for ribosome-binding sites
upstream of start codons in E. coli; complementary to a
region in the 3' end of the 16S rRNA (Chen et al., 1994)"
promoter complement(3474..3547)
/label=PLtetO-1 promoter
/note="modified phage lambda PL promoter with tet operator
sites (Lutz and Bujard, 1997)"
protein_bind 3566..3585
/label=lac operator (symmetric)
/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). The
symmetric lac operator was optimized for tight binding of
lac repressor."
protein_bind 3602..3621
/label=lac operator (symmetric)
/bound_moiety="lac repressor encoded by lacI"
/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). The
symmetric lac operator was optimized for tight binding of
lac repressor."
promoter 3622..3651
/label=trc promoter
/note="strong E. coli promoter; hybrid between the trp and
lac UV5 promoters"
protein_bind complement(3658..3677)
/label=lac operator (symmetric)
/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). The
symmetric lac operator was optimized for tight binding of
lac repressor."
regulatory 3690..3721
/label=T4
/note="T4"
/regulatory_class="ribosome_binding_site"
RBS 3708..3716
/label=Shine-Dalgarno sequence
/note="full consensus sequence for ribosome-binding sites
upstream of start codons in E. coli; complementary to a
region in the 3' end of the 16S rRNA (Chen et al., 1994)"
CDS 3722..4342
/label=TetR
/note="tetracycline repressor TetR"
regulatory 4388..4462
/label=Ptrc-2
/note="Ptrc-2"
/regulatory_class="promoter"
promoter 4401..4430
/label=trc promoter
/note="strong E. coli promoter; hybrid between the trp and
lac UV5 promoters"
protein_bind 4438..4454
/label=lac operator
/bound_moiety="lac repressor encoded by lacI"
/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)."
prim_transcript 4438
/label=transcription start
/note="transcription start"
regulatory 4470..4496
/label=M2
/note="M2"
/regulatory_class="ribosome_binding_site"
RBS 4483..4491
/label=Shine-Dalgarno sequence
/note="full consensus sequence for ribosome-binding sites
upstream of start codons in E. coli; complementary to a
region in the 3' end of the 16S rRNA (Chen et al., 1994)"
CDS 4497..5204
/label=mCherry
/note="monomeric derivative of DsRed fluorescent protein
(Shaner et al., 2004)"
terminator 5267..5353
/label=rrnB T1 terminator
/note="transcription terminator T1 from the E. coli rrnB
gene"
misc_feature 5422..5821
/label=similar to lacZ (downstream)
/note="similar to lacZ (downstream)"
promoter complement(5840..5858)
/label=T3 promoter
/note="promoter for bacteriophage T3 RNA polymerase"
primer_bind complement(5879..5895)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
protein_bind 5903..5919
/label=lac operator
/bound_moiety="lac repressor encoded by lacI"
/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(5927..5957)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind complement(5972..5993)
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
CDS complement(6697..7065)
/label=traJ
/note="oriT-recognizing protein"
oriT complement(7098..7207)
/direction=LEFT
/label=oriT
/note="incP origin of transfer"
promoter 8374..8819
/label=sacB promoter
/note="sacB promoter and control region"
CDS 8820..10238
/label=SacB
/note="secreted levansucrase that renders bacterial growth
sensitive to sucrose"
CDS complement(11811..12668)
/label=AmpR
/note="beta-lactamase"
promoter complement(12669..12773)
/label=AmpR promoter
rep_origin complement(12883..13271)
/direction=LEFT
/label=R6K gamma ori
/note="gamma replication origin from E. coli plasmid R6K;
requires the R6K initiator protein pi for replication"