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pAPG2 载体 (V009468)

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:
pAPG2
载体抗性:
Kanamycin
载体长度:
10154 bp
载体类型:
Expression vector
复制子:
p15A ori
载体来源:
Kang MK, Eom JH, Kim Y, Um Y, Woo HM.
启动子:
tac

pAPG2 载体图谱

pAPG210154 bp50010001500200025003000350040004500500055006000650070007500800085009000950010000tac promoterlac operatorPinene synthase, chloroplasticRBSgeranyl diphosphate synthaserrnB T1 terminatorrrnB T2 terminatorAmpR promoterKanRp15A ori

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。

pAPG2 载体序列

LOCUS       V009468                10154 bp    DNA     circular SYN 18-DEC-2018
DEFINITION  Exported.
ACCESSION   V009468
VERSION     V009468
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
            .
REFERENCE   1  (bases 1 to 10154)
  AUTHORS   Kang MK, Eom JH, Kim Y, Um Y, Woo HM.
  TITLE     Biosynthesis of pinene from glucose using metabolically-engineered
            Corynebacterium glutamicum
  JOURNAL   Biotechnol. Lett. (2014) In press
   PUBMED   24930112
REFERENCE   2  (bases 1 to 10154)
  AUTHORS   Woo HM.
  TITLE     Direct Submission
  JOURNAL   Submitted (13-MAY-2014) Clean Energy Research Center, Korea
            Institute of Science and Technology, Hwarangno 14-gil 5,
            Seongbuk-gu, Seoul 136-791, Korea
REFERENCE   3  (bases 1 to 10154)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 10154)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     ##Assembly-Data-START##
            Sequencing Technology :: Sanger dideoxy sequencing
            ##Assembly-Data-END##
            SGRef: number: 1; type: "Journal Article"; journalName: "Biotechnol.
            Lett. (2014) In press"
            SGRef: number: 2; type: "Journal Article"; journalName: "Submitted
            (13-MAY-2014) Clean Energy Research Center, Korea Institute of
            Science and Technology, Hwarangno 14-gil 5, Seongbuk-gu, Seoul
            136-791, Korea"
            SGRef: number: 3; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..10154
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        266..294
                     /label="tac promoter"
                     /note="strong E. coli promoter; hybrid between the trp and
                     lac UV5 promoters"
     protein_bind    302..318
                     /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)."
     CDS             370..2253
                     /gene="ag3"
                     /label="Pinene synthase, chloroplastic"
                     /note="Pinene synthase, chloroplastic from Abies grandis.
                     Accession#: O24475"
     RBS             2263..2274
                     /note="strong bacterial ribosome binding site (Elowitz and
                     Leibler, 2000)"
     CDS             2281..3426
                     /codon_start=1
                     /product="geranyl diphosphate synthase"
                     /label="geranyl diphosphate synthase"
                     /note="AgGPPS2; codon-optimized for C. glutamicum"
                     /protein_id="AIF29189.1"
                     /translation="MAYSAMATMGYNGMAASCHTLHPTSPLKPFHGASTSLEAFNGEHM
                     GLLRGYSKRKLSSYKNPASRSSNATVAQLLNPPQKGKKAVEFDFNKYMDSKAMTVNEAL
                     NKAIPLRYPQKIYESMRYSLLAGGKRVRPVLCIAACELVGGTEELAIPTACAIEMIHTM
                     SLMHDDLPCIDNDDLRRGKPTNHKIFGEDTAVTAGNALHSYAFEHIAVSTSKTVGADRI
                     LRMVSELGRATGSEGVMGGQMVDIASEGDPSIDLQTLEWIHIHKTAMLLECSVVCGAII
                     GGASEIVIERARRYARCVGLLFQVVDDILDVTKSSDELGKTAGKDLISDKATYPKLMGL
                     EKAKEFSDELLNRAKGELSCFDPVKAAPLLGLADYVAFRQN"
     terminator      3653..3739
                     /label="rrnB T1 terminator"
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     terminator      3831..3858
                     /label="rrnB T2 terminator"
                     /note="transcription terminator T2 from the E. coli rrnB
                     gene"
     promoter        3877..3968
                     /label="AmpR promoter"
     CDS             complement(7916..8728)
                     /label="KanR"
                     /note="aminoglycoside phosphotransferase"
     rep_origin      complement(9323..9868)
                     /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."