Note:
基本信息
- 载体名称:
- pGDR11-KOD
- 载体抗性:
- Ampicillin
- 载体长度:
- 7081 bp
- 载体类型:
- Expression vector
- 复制子:
- ori
- 载体来源:
- Dunn MR, Otto CE, Fenton KE, Chaput JC.
下载资源
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。
确保质粒的关键元件正确，但是我们并不能保证实验效果。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。
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。
pGDR11-KOD 质粒 (编号: V006069)序列
LOCUS 40924_21270 7081 bp DNA circular SYN 18-DEC-2018
DEFINITION Expression vector pGDR11-KOD, complete sequence.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7081)
AUTHORS Dunn MR, Otto CE, Fenton KE, Chaput JC.
TITLE Transference as a Mechanism for Expanding and Improving Polymerase
Function
JOURNAL Unpublished
REFERENCE 2 (bases 1 to 7081)
AUTHORS Dunn MR, Otto CE, Fenton KE, Chaput JC.
TITLE Direct Submission
JOURNAL Submitted (20-JAN-2015) The Biodesign Institute, Arizona State
University, 727 E Tyler Street, Tempe, AZ 85287, USA
REFERENCE 3 (bases 1 to 7081)
TITLE Direct Submission
REFERENCE 4 (bases 1 to 7081)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName:
"Unpublished"
COMMENT SGRef: number: 2; type: "Journal Article"; journalName: "Submitted
(20-JAN-2015) The Biodesign Institute, Arizona State University, 727
E Tyler Street, Tempe, AZ 85287, 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..7081
/mol_type="other DNA"
/organism="synthetic DNA construct"
CDS 1..2325
/codon_start=1
/gene="pol"
/product="exonuclease-deficient DNA polymerase"
/label=pol
/note="from Thermococcus kodakarensis"
/protein_id="ALL53339.1"
/translation="MILDTDYITEDGKPVIRIFKKENGEFKIEYDRTFEPYFYALLKDD
SAIEEVKKITAERHGTVVTVKRVEKVQKKFLGRPVEVWKLYFTHPQDVPAIRDKIREHP
AVIDIYEYDIPFAKRYLIDKGLVPMEGDEELKMLAFAIATLYHEGEEFAEGPILMISYA
DEEGARVITWKNVDLPYVDVVSTEREMIKRFLRVVKEKDPDVLITYNGDNFDFAYLKKR
CEKLGINFALGRDGSEPKIQRMGDRFAVEVKGRIHFDLYPVIRRTINLPTYTLEAVYEA
VFGQPKEKVYAEEITTAWETGENLERVARYSMEDAKVTYELGKEFLPMEAQLSRLIGQS
LWDVSRSSTGNLVEWFLLRKAYERNELAPNKPDEKELARRRQSYEGGYVKEPERGLWEN
IVYLDFRSLYPSIIITHNVSPDTLNREGCKEYDVAPQVGHRFCKDFPGFIPSLLGDLLE
ERQKIKKKMKATIDPIERKLLDYRQRAIKILANSYYGYYGYARARWYCKECAESVTAWG
REYITMTIKEIEEKYGFKVIYSDTDGFFATIPGADAETVKKKAMEFLKYINAKLPGALE
LEYEGFYKRGFFVTKKKYAVIDEEGKITTRGLEIVRRDWSEIAKETQARVLEALLKDGD
VEKAVRIVKEVTEKLSKYEVPPEKLVIHEQITRDLKDYKATGPHVAVAKRLAARGVKIR
PGTVISYIVLKGSGRIGDRAIPFDEFDPTKHKYDAEYYIENQVLPAVERILRAFGYRKE
DLRYQKTRQVGLSAWLKPKGT"
gene 1..2325
/gene="pol"
/label=pol
terminator 2361..2455
/label=lambda t0 terminator
/note="transcription terminator from phage lambda"
CDS 2499..3155
/label=CmR
/note="chloramphenicol acetyltransferase"
terminator 3223..3309
/label=rrnB T1 terminator
/note="transcription terminator T1 from the E. coli rrnB
gene"
protein_bind complement(3370..3391)
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
CDS complement(3407..4486)
/label=lacI
/note="lac repressor"
promoter complement(4485..4562)
/label=lacIq promoter
/note="In the lacIq allele, a single base change in the
promoter boosts expression of the lacI gene about 10-fold."
misc_feature 4754..4894
/label=bom
/note="basis of mobility region from pBR322"
rep_origin complement(5080..5668)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(5842..6699)
/label=AmpR
/note="beta-lactamase"
promoter complement(6700..6804)
/label=AmpR promoter
promoter 6914..6958
/label=T5 promoter
/note="bacteriophage T5 promoter for E. coli RNA
polymerase, with embedded lac operator"
operon 6964..6984
/operon="LacO"
/label=LacO operon
protein_bind 6966..6982
/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)."
RBS 7001..7012
/note="strong bacterial ribosome binding site (Elowitz and
Leibler, 2000)"
CDS 7031..7048
/label=6xHis
/note="6xHis affinity tag"
CDS 7064..7081
/label=thrombin site
/note="thrombin recognition and cleavage site"