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:
- pwFRT-pheS-gat
- 载体抗性:
- Ampicillin
- 载体长度:
- 4531 bp
- 载体类型:
- Cloning vector
- 复制子:
- ori
- 载体来源:
- Kang Y, Norris MH, Hoang TT.
pwFRT-pheS-gat 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。
pwFRT-pheS-gat 载体序列
LOCUS V002137 4531 bp DNA circular SYN 18-DEC-2018
DEFINITION Exported.
ACCESSION V002137
VERSION V002137
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
.
REFERENCE 1 (bases 1 to 4531)
AUTHORS Kang Y, Norris MH, Hoang TT.
TITLE Knock-out and pull-out recombineering protocols for natural
transformable Burkholderia thailandensis and Burkholderia
pseudomallei
JOURNAL Unpublished
REFERENCE 2 (bases 1 to 4531)
AUTHORS Kang Y, Norris MH, Hoang TT.
TITLE Direct Submission
JOURNAL Submitted (09-OCT-2009) Molecular Biosciences and Biological
Engineering, University of Hawaii at Manoa, 2538 McCarthy Mall,
Snyder Hall 207, Honolulu, HI 96822, USA
REFERENCE 3 (bases 1 to 4531)
TITLE Direct Submission
REFERENCE 4 (bases 1 to 4531)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName:
"Unpublished"
SGRef: number: 2; type: "Journal Article"; journalName: "Submitted
(09-OCT-2009) Molecular Biosciences and Biological Engineering,
University of Hawaii at Manoa, 2538 McCarthy Mall, Snyder Hall 207,
Honolulu, HI 96822, USA"
SGRef: number: 3; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..4531
/mol_type="other DNA"
/organism="synthetic DNA construct"
promoter 96..200
/label="AmpR promoter"
CDS 201..1058
/label="AmpR"
/note="beta-lactamase"
rep_origin 1232..1820
/label="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
protein_bind 2108..2129
/label="CAP binding site"
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 2144..2174
/label="lac promoter"
/note="promoter for the E. coli lac operon"
protein_bind 2182..2198
/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 2206..2222
/label="M13 rev"
/note="common sequencing primer, one of multiple similar
variants"
misc_feature 2289..2356
/note="wild type FRT; Flp recognition target"
protein_bind complement(2306..2353)
/label="FRT"
/bound_moiety="FLP recombinase from the Saccharomyces
cerevisiae 2u plasmid"
/note="FLP-mediated recombination occurs in the 8-bp core
sequence TCTAGAAA (Turan and Bode, 2011)."
regulatory 2393..2434
/note="PCS12; rpsL promoter from Burkholderia cenocepacia"
/regulatory_class="promoter"
RBS 2445..2467
/label="RBS"
/note="efficient ribosome binding site from bacteriophage
T7 gene 10 (Olins and Rangwala, 1989)"
CDS 2478..3488
/gene="pheS"
/label="Phenylalanine--tRNA ligase alpha subunit"
/note="Phenylalanine--tRNA ligase alpha subunit from
Burkholderia pseudomallei (strain 1710b). Accession#:
Q3JT08"
CDS 3527..3967
/codon_start=1
/gene="gat"
/product="glyphosate acetyl transferase"
/label="gat"
/note="confers resistance to glyphosate"
/protein_id="ADO63837.1"
/translation="MIEVKPINAEDTYDLRHRVLRPNQPIEACMFESDLTRSAFHLGGF
YGGKLISVASFHQAEHSELQGKKQYQLRGVATLEGYREQKAGSSLVKHAEEILRKRGAD
MIWCNARTSASGYYRKLGFSEQGEVFDTPPVGPHILMYKRIT"
gene 3527..3967
/gene="gat"
/label="gat"
protein_bind complement(4023..4070)
/label="FRT"
/note="FLP-mediated recombination occurs in the 8-bp core
sequence TCTAGAAA (Turan and Bode, 2011)."
primer_bind complement(4137..4153)
/label="M13 fwd"
/note="common sequencing primer, one of multiple similar
variants"