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:
- pGWB685
- 载体抗性:
- Streptomycin
- 载体长度:
- 11441 bp
- 载体类型:
- Gateway binary vector
- 复制子:
- ori
- 宿主:
- Plants
- 载体来源:
- Momtaz S, Dutta A, Tanaka Y, Aboulela M, Nishimura K, Sugiura S, Niwa T, Maeo K, Goto-Yamada S, Kimura T, Ishiguro S, Mano S, Nakagawa T.
- 启动子:
- NOS
pGWB685 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。
pGWB685 载体序列
LOCUS 40924_23639 11441 bp DNA circular SYN 18-DEC-2018
DEFINITION Gateway binary vector pGWB685 DNA, complete sequence.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 11441)
AUTHORS Momtaz S, Dutta A, Tanaka Y, Aboulela M, Nishimura K, Sugiura S,
Niwa T, Maeo K, Goto-Yamada S, Kimura T, Ishiguro S, Mano S,
Nakagawa T.
TITLE Gateway Binary Vectors with Organelle-Targeted Fluorescent Proteins
for Highly Sensitive Reporter Assay in Gene Expression Analysis of
Plants
JOURNAL Unpublished
REFERENCE 2 (bases 1 to 11441)
AUTHORS Momtaz S, Dutta A, Tanaka Y, Aboulela M, Nishimura K, Sugiura S,
Niwa T, Maeo K, Goto-Yamada S, Kimura T, Ishiguro S, Mano S,
Nakagawa T.
TITLE Direct Submission
JOURNAL Submitted (17-OCT-2018) Contact:Tsuyoshi Nakagawa Shimane
University, Interdisciplinary Center for Science Research; 1060
Nishikawatsu, Matsue, Shimane 690-8504, Japan
REFERENCE 3 (bases 1 to 11441)
TITLE Direct Submission
REFERENCE 4 (bases 1 to 11441)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName:
"Unpublished"
COMMENT SGRef: number: 2; type: "Journal Article"; journalName: "Submitted
(17-OCT-2018) Contact:Tsuyoshi Nakagawa Shimane University,
Interdisciplinary Center for Science Research; 1060 Nishikawatsu,
Matsue, Shimane 690-8504, Japan"
COMMENT SGRef: number: 3; type: "Journal Article"
COMMENT constructed using pPZP221.
FEATURES Location/Qualifiers
source 1..11441
/mol_type="other DNA"
/organism="synthetic DNA construct"
misc_feature complement(54..78)
/label=RB T-DNA repeat
/note="right border repeat from nopaline C58 T-DNA"
primer_bind 281..297
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
protein_bind 338..462
/label=attR1
/note="recombination site for the Gateway(R) LR reaction"
promoter 499..529
/label=lac UV5 promoter
/note="E. coli lac promoter with an 'up' mutation"
CDS 583..1239
/codon_start=1
/label=CmR
/note="chloramphenicol acetyltransferase"
/translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
CDS 1584..1886
/codon_start=1
/label=ccdB
/note="CcdB, a bacterial toxin that poisons DNA gyrase"
/translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
protein_bind complement(1930..2054)
/label=attR2
/note="recombination site for the Gateway(R) LR reaction"
CDS 2071..2091
/codon_start=1
/label=SV40 NLS
/note="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/translation="PKKKRKV"
CDS 2092..2802
/codon_start=1
/label=TagRFP
/note="monomeric derivative of red fluorescent protein from
Entacmaea quadricolor (Merzlyak et al., 2007)"
/translation="MVSKGEELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTM
RIKVVEGGPLPFAFDILATSFMYGSRTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGG
VLTATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEANTEMLYPADGGLEGRSD
MALKLVGGGHLICNFKTTYRSKKPAKNLKMPGVYYVDHRLERIKEADKETYVEQHEVAV
ARYCDLPSKLGHKLN"
terminator 2827..3079
/label=NOS terminator
/note="nopaline synthase terminator and poly(A) signal"
primer_bind complement(3113..3129)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
protein_bind 3137..3153
/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(3161..3191)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind complement(3206..3227)
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
terminator complement(3263..3515)
/label=NOS terminator
/note="nopaline synthase terminator and poly(A) signal"
CDS complement(3678..4226)
/codon_start=1
/label=BlpR
/note="phosphinothricin acetyltransferase"
/translation="MSPERRPADIRRATEADMPAVCTIVNHYIETSTVNFRTEPQEPQE
WTDDLVRLRERYPWLVAEVDGEVAGIAYAGPWKARNAYDWTAESTVYVSPRHQRTGLGS
TLYTHLLKSLEAQGFKSVVAVIGLPNDPSVRMHEALGYAPRGMLRAAGFKHGNWHDVGF
WQLDFSLPVPPRPVLPVTEI"
promoter complement(4278..4457)
/label=NOS promoter
/note="nopaline synthase promoter"
misc_feature complement(4963..4987)
/label=LB T-DNA repeat
/note="left border repeat from nopaline C58 T-DNA"
CDS 5508..6296
/codon_start=1
/label=SmR
/note="aminoglycoside adenylyltransferase (Murphy, 1985)"
/translation="MGEAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
rep_origin 6545..7133
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
misc_feature complement(7319..7459)
/label=bom
/note="basis of mobility region from pBR322"
rep_origin complement(7803..7997)
/direction=LEFT
/label=pVS1 oriV
/note="origin of replication for the Pseudomonas plasmid
pVS1 (Heeb et al., 2000)"
CDS complement(8066..9136)
/codon_start=1
/label=pVS1 RepA
/note="replication protein from the Pseudomonas plasmid
pVS1 (Heeb et al., 2000)"
/translation="VSGRKPSGPVQIGAALGDDLVEKLKAAQAAQRQRIEAEARPGESW
QAAADRIRKESRQPPAAGAPSIRKPPKGDEQPDFFVPMLYDVGTRDSRSIMDVAVFRLS
KRDRRAGEVIRYELPDGHVEVSAGPAGMASVWDYDLVLMAVSHLTESMNRYREGKGDKP
GRVFRPHVADVLKFCRRADGGKQKDDLVETCIRLNTTHVAMQRTKKAKNGRLVTVSEGE
ALISRYKIVKSETGRPEYIEIELADWMYREITEGKNPDVLTVHPDYFLIDPGIGRFLYR
LARRAAGKAEARWLFKTIYERSGSAGEFKKFCFTVRKLIGSNDLPEYDLKEEAGQAGPI
LVMRYRNLIEGEASAGS"
CDS complement(9568..10194)
/codon_start=1
/label=pVS1 StaA
/note="stability protein from the Pseudomonas plasmid pVS1
(Heeb et al., 2000)"
/translation="MKVIAVLNQKGGSGKTTIATHLARALQLAGADVLLVDSDPQGSAR
DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP
VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI
LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI"