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pUC-TTrepT 载体 (V002435)

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:
pUC-TTrepT
载体抗性:
Ampicillin
载体长度:
5056 bp
载体类型:
Cloning vector
复制子:
ori
载体来源:
Vieira J, Messing J.

pUC-TTrepT 载体图谱

pUC-TTrepT5056 bp6001200180024003000360042004800CAP binding sitelac promoterlac operatorM13 revpolylinker HindIII-NotI-SalI-NruI-AflII-EcoRV-Acc65I-EcoRIM13 fwdderived from pYK225 (derivative of pTT8)AmpR promoterAmpRori

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。

pUC-TTrepT 载体序列

LOCUS       40924_44928        5056 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pUC-TTrepT DNA, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5056)
  AUTHORS   Vieira J, Messing J.
  TITLE     The pUC plasmids, an M13mp7-derived system for insertion mutagenesis
            and sequencing with synthetic universal primers
  JOURNAL   Gene 19 (3), 259-268 (1982)
  PUBMED    6295879
REFERENCE   2  (bases 1 to 5056)
  AUTHORS   Fujita A, Misumi Y, Koyama Y.
  TITLE     Two versatile shuttle vectors for Thermus thermophilus-Escherichia 
            coli containing multiple cloning sites, lacZalpha gene and kanamycin
            or hygromycin resistance marker
  JOURNAL   Plasmid 67 (3), 272-275 (2012)
  PUBMED    22252135
REFERENCE   3  (bases 1 to 5056)
  AUTHORS   Fujita A.
  TITLE     Direct Submission
  JOURNAL   Submitted (08-SEP-2011) Contact:Atsushi Fujita National Institute of
            Advanced Science and Technology, Biomedical Research Institute; 
            1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan URL 
            :http://www.aist.go.jp/
REFERENCE   4  (bases 1 to 5056)
  TITLE     Direct Submission
REFERENCE   5  (bases 1 to 5056)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Gene"; 
            date: "1982"; volume: "19"; issue: "3"; pages: "259-268"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Plasmid"; 
            date: "2012"; volume: "67"; issue: "3"; pages: "272-275"
COMMENT     SGRef: number: 3; type: "Journal Article"; journalName: "Submitted 
            (08-SEP-2011) Contact:Atsushi Fujita National Institute of Advanced 
            Science and Technology, Biomedical Research Institute; 1-1-1 
            Higashi, Tsukuba, Ibaraki 305-8566, Japan URL 
            :http://www.aist.go.jp/"
COMMENT     SGRef: number: 4; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..5056
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    106..127
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     promoter        142..172
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    180..196
                     /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)."
     primer_bind     204..220
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    233..283
                     /note="polylinker 
                     HindIII-NotI-SalI-NruI-AflII-EcoRV-Acc65I-EcoRI"
     primer_bind     complement(284..300)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    690..3062
                     /label=derived from pYK225 (derivative of pTT8)
                     /note="derived from pYK225 (derivative of pTT8)"
     CDS             complement(1197..2360)
                     /codon_start=1
                     /gene="repA"
                     /product="putative RepA protein"
                     /label=repA
                     /protein_id="BAL70402.1"
                     /translation="MVLRAYAALRGLSPEALRAHLLAPPLRPERAREAFQRPYLAHFAQ
                     TLPRYPYATDDPKEGVRIYKRENALKRVHVQVGHYPHAVLRLVVDVDLPWPQVEERIHA
                     LPPSLVLVNPRSGHFHAWYELDPIPLTPPPGREGSLKGALALLAEVEALLEAYYGADPG
                     YNGLLSRNPFLHPPEWTWGGGKRWSLRDLHRELRGLLPSGTRRRVDPGLASYGRNNALF
                     DRLRAEAYAHVALFRGVPGGEEAFRAWVEQRAHALNQSLFRDHPKGPLDPREVHHTAKS
                     VAKWTYRNYRGARVYPVSSTGRPDRSRLSPQARALIPPLQGQELQEAVREGGRRRGSRR
                     RQEAEEKLTEALKRLQARGERVTARALAREAGVKPHTASKWLKRMRE"
     gene            complement(1197..2360)
                     /gene="repA"
                     /label=repA
     promoter        3150..3254
                     /label=AmpR promoter
     CDS             3255..4112
                     /label=AmpR
                     /note="beta-lactamase"
     rep_origin      4286..4874
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"