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:
- pT19_T19_72bp-mKate2
- 载体抗性:
- Ampicillin
- 载体长度:
- 7287 bp
- 载体类型:
- Cloning vector
- 复制子:
- ori
- 载体来源:
- Li Y, Jiang Y, Chen H, Liao W, Li Z, Weiss R, Xie Z.
pT19_T19_72bp-mKate2 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。
pT19_T19_72bp-mKate2 载体序列
LOCUS 40924_42134 7287 bp DNA circular SYN 18-DEC-2018
DEFINITION Cloning vector pT19_T19_72bp-mKate2, complete sequence.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7287)
AUTHORS Li Y, Jiang Y, Chen H, Liao W, Li Z, Weiss R, Xie Z.
TITLE Modular construction of mammalian gene circuits using TALE
transcriptional repressors
JOURNAL Unpublished
REFERENCE 2 (bases 1 to 7287)
AUTHORS Li Y, Jiang Y, Chen H, Liao W.
TITLE Direct Submission
JOURNAL Submitted (03-SEP-2014) Bioinformatics Division/Center for Synthetic
REFERENCE 3 (bases 1 to 7287)
TITLE Direct Submission
REFERENCE 4 (bases 1 to 7287)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName:
"Unpublished"
COMMENT SGRef: number: 2; type: "Journal Article"; journalName: "Submitted
(03-SEP-2014) Bioinformatics Division/Center for Synthetic "
COMMENT SGRef: number: 3; type: "Journal Article"
COMMENT ##Assembly-Data-START##
Sequencing Technology :: Sanger dideoxy sequencing
##Assembly-Data-END##
FEATURES Location/Qualifiers
source 1..7287
/mol_type="other DNA"
/organism="synthetic DNA construct"
protein_bind 16..36
/label=attB1
/note="core recombination site for the Gateway(R) BP
reaction"
CDS 78..95
/codon_start=1
/label=6xHis
/note="6xHis affinity tag"
/translation="HHHHHH"
CDS 96..791
/codon_start=1
/label=mKate2
/note="monomeric far-red fluorescent protein (Shcherbo et
al., 2009)"
/translation="MVSELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMRIK
AVEGGPLPFAFDILATSFMYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLT
ATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEASTETLYPADGGLEGRADMAL
KLVGGGHLICNLKTTYRSKKPAKNLKMPGVYYVDRRLERIKEADKETYVEQHEVAVARY
CDLPSKLGHK"
protein_bind complement(834..858)
/label=attB2
/note="recombination site for the Gateway(R) BP reaction"
polyA_signal 1013..1068
/label=beta-globin poly(A) signal
/note="rabbit beta-globin polyadenylation signal (Gil and
Proudfoot, 1987)"
primer_bind complement(1429..1445)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
protein_bind 1453..1469
/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(1477..1507)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 1522..1543
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
misc_feature 1716..2552
/label=HA-R
/note="right homology arm from the adeno-associated virus
integration site (AAVS1) within intron 1 of the human
PPP1R12C gene"
promoter complement(2582..2600)
/label=T3 promoter
/note="promoter for bacteriophage T3 RNA polymerase"
primer_bind complement(2621..2637)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
protein_bind complement(2645..2661)
/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(2669..2699)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind complement(2714..2735)
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
rep_origin complement(3023..3611)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(3785..4642)
/codon_start=1
/label=AmpR
/note="beta-lactamase"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
promoter complement(4643..4747)
/label=AmpR promoter
rep_origin complement(4773..5228)
/direction=LEFT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind 5370..5386
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
promoter 5396..5414
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
misc_feature 5429..6232
/label=HA-L
/note="left homology arm from the adeno-associated virus
integration site (AAVS1) within intron 1 of the human
PPP1R12C gene"
misc_feature 6311..6336
/label=SA
/note="splice acceptor site"
protein_bind 7001..7021
/label=attB4
/note="core recombination site for the Gateway(R) BP
reaction"
protein_bind 7069..7163
/label=5X UAS
/note="five tandem copies of the 'ScaI site' 17-mer
CGGAGTACTGTCCTCCG, an upstream activating sequence (UAS)
that efficiently binds yeast Gal4 (Webster et al., 1988;
Pfeiffer et al., 2010)"
protein_bind 7177..7195
/label=TALER19 bs binding site
/bound_moiety="TALER19 bs"
misc_feature 7202..7261
/label=CMVmini
/note="CMVmini"
protein_bind 7268..7286
/label=TALER19 bs binding site
/bound_moiety="TALER19 bs"