NovoBuilder 质粒设计

pCAMBIA2300 质粒 (编号: V010873)

pCAMBIA23008744 bp4008001200160020002400280032003600400044004800520056006000640068007200760080008400lac promoterlac operatorlacZ (truncated)RB T-DNA repeatpVS1 StaApVS1 RepApVS1 oriVbomoriaphA-3LB T-DNA repeatCaMV poly(A) signalNeoR/KanRCaMV 35S promoter (enhanced)
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基本信息

备注：pCAMBIA2300 is a binary vector​ for Agrobacterium-mediated plant transformation. Based on pVS1/pBR322 replicons, it carries KanR/NPTII​ selection and a reporter-free T-DNA​ with a pUC18-derived polylinker​ (EcoRI–HindIII), allowing users to clone their own promoter–gene–terminator cassette without GUS/Luc background interference.
载体名称
pCAMBIA2300
载体抗性
Kanamycin
载体长度
8744 bp
载体类型
Plant Vectors
复制子
ori
宿主
Plants
载体来源
Cambia
筛选标记
Neomycin/G418(Geneticin)
拷贝数
High copy number
启动子
CaMV 35S (enhanced)
5'引物
M13 fwd
3'引物
M13 rev
感受态
stbl3
培养温度
37℃

质粒操作方法

质粒复溶方法

  1. 发货形式：质粒干粉，常温运输，收到后请于 -20 ℃ 保存，先转化提质粒再使用。
  2. 收到干粉后先 5000 rpm 离心 1 min，加入 20 μl ddH₂O 溶解。质粒复测浓度与标称值可能有差距，这通常来自干粉在管中的位置、复溶效率、测量偏差与管壁吸附，建议先转化提质粒后再使用。
  3. 取 1 支 100 μl 感受态细胞冰上解冻 10 min，加入 2 μl 质粒，冰浴 30 min，42 ℃ 热激 60 s（不要摇动），再冰浴 2 min。
  4. 加入 900 μl 无抗生素 LB 液体培养基，180 rpm、37 ℃ 振荡培养 45 min（30 ℃ 培养 1–1.5 h）。
  5. 6000 rpm 离心 5 min，保留约 100 μl 上清重悬菌体，涂布到对应抗性的 LB 平板。
  6. 平板倒置 37 ℃ 培养 14 h（30 ℃ 则 20 h）。菌落过多可稀释质粒后再转化；无菌落可增加至 10 μl 质粒。建议先转克隆感受态、重提质粒后再导入表达感受态。
  7. 挑取单菌落接种 LB 液体培养基，加入对应抗生素，220 rpm 振荡培养 14 h，按试剂盒说明提取质粒。

通用质粒转化方法

  1. 取100 μL感受态细胞置于冰上解冻10 min，加入2 μL质粒，冰浴30 min；随后42 ℃热激60 s，期间不要晃动，再冰浴2 min。
  2. 加入900 μL无抗LB液体培养基，37 ℃、180 rpm振荡培养45 min（30 ℃条件下则培养1–1.5 h）。
  3. 6000 rpm离心5 min，弃上清，仅保留100 μL上清重悬菌体沉淀，涂布于对应质粒抗性的LB平板上。
  4. 平板倒置培养，37 ℃培养14 h，或30 ℃培养20 h。
  5. 挑取单菌落至LB液体培养基中，加入对应抗生素，220 rpm振荡培养14 h；根据实验需求及质粒提取试剂盒说明书提取质粒。

参考文献

  • Sahab S, Taylor N. Studies on Pure Mlb® (Multiple Left Border) Technology and Its Impact on Vector Backbone Integration in Transgenic Cassava. Front Plant Sci. 2022 Feb 4;13:816323. doi: 10.3389/fpls.2022.816323. PMID: 35185986; PMCID: PMC8855067.
  • Leclercq J, Szabolcs T, Martin F, Montoro P. Development of a new pCAMBIA binary vector using Gateway® technology. Plasmid. 2015 Sep;81:50-4. doi: 10.1016/j.plasmid.2015.07.003. Epub 2015 Jul 23. PMID: 26210260.
  • Matheka J, Tripathi JN, Merga I, Gebre E, Tripathi L. A simple and rapid protocol for the genetic transformation of Ensete ventricosum. Plant Methods. 2019 Nov 8;15:130. doi: 10.1186/s13007-019-0512-y. PMID: 31719836; PMCID: PMC6839154.

pCAMBIA2300 质粒 (编号: V010873) 注释

LOCUS       Exported                8744 bp DNA     circular SYN 10-SEP-2025
DEFINITION  Agrobacterium binary vector for plant transformation, with 
            kanamycin-resistance genes.
ACCESSION   AF234315
VERSION     .
KEYWORDS    pCAMBIA2300
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8744)
  AUTHORS   Cambia
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 8744)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 8744)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     SGRef: number: 2; type: "Journal Article"
COMMENT     The GenBank record was corrected by inserting a G at position 2281.
FEATURES             Location/Qualifiers
     source          1..8744
                     /lab_host="Plant Cells"
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     source          join(641..8744,1..640)
                     /lab_host="Plant Cells"
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        167..197
                     /note="lac promoter"
                     /note="promoter for the E. coli lac operon"
     protein_bind    205..221
                     /label=lac repressor encoded by lacI binding site
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="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     229..245
                     /label=M13 rev
                     /note="M13 rev"
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             241..474
                     /codon_start=1
                     /gene="lacZ (truncated)"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ (truncated)
                     /note="lacZ-alpha"
                     /translation="MTMITNSSSVPGDPLESTCRHASLALAVVLQRRDWENPGVTQLNR
                     LAAHPPFASWRNSEEARTDRPSQQLRSLNGEC"
     misc_feature    255..311
                     /label=MCS
                     /note="MCS"
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(315..331)
                     /label=M13 fwd
                     /note="M13 fwd"
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    534..558
                     /label=RB T-DNA repeat
                     /note="RB T-DNA repeat"
                     /note="right border repeat from nopaline C58 T-DNA"
     CDS             1859..2488
                     /codon_start=1
                     /product="stability protein from Pseudomonas plasmid pVS1"
                     /label=stability protein from Pseudomonas plasmid pVS1
                     /note="pVS1 StaA"
                     /translation="MKVIAVLNQKGGSGKTTIATHLARALQLAGADVLLVDSDPQGSAR
                     DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP
                     VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI
                     LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI"
     CDS             2917..3990
                     /codon_start=1
                     /product="replication protein from Pseudomonas plasmid
                     pVS1"
                     /label=replication protein from Pseudomonas plasmid
                     pVS1
                     /note="pVS1 RepA"
                     /translation="MSGRKPSGPVQIGAALGDDLVEKLKAAQAAQRQRIEAEARPGESW
                     QAAADRIRKESRQPPAAGAPSIRKPPKGDEQPDFFVPMLYDVGTRDSRSIMDVAVFRLS
                     KRDRRAGEVIRYELPDGHVEVSAGPAGMASVWDYDLVLMAVSHLTESMNRYREGKGDKP
                     GRVFRPHVADVLKFCRRADGGKQKDDLVETCIRLNTTHVAMQRTKKAKNGRLVTVSEGE
                     ALISRYKIVKSETGRPEYIEIELADWMYREITEGKNPDVLTVHPDYFLIDPGIGRFLYR
                     LARRAAGKAEARWLFKTIYERSGSAGEFKKFCFTVRKLIGSNDLPEYDLKEEAGQAGPI
                     LVMRYRNLIEGEASAGS"
     rep_origin      4056..4250
                     /label=pVS1 oriV
                     /note="pVS1 oriV"
                     /note="origin of replication for the Pseudomonas plasmid
                     pVS1 (Heeb et al., 2000)"
     misc_feature    4594..4734
                     /label=bom
                     /note="bom"
                     /note="basis of mobility region from pBR322"
     rep_origin      complement(4920..5508)
                     /direction=LEFT
                     /label=ori
                     /note="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(5595..6389)
                     /codon_start=1
                     /gene="aphA-3"
                     /product="aminoglycoside phosphotransferase"
                     /label=aphA-3
                     /note="KanR"
                     /note="confers resistance to kanamycin"
                     /translation="MAKMRISPELKKLIEKYRCVKDTEGMSPAKVYKLVGENENLYLKM
                     TDSRYKGTTYDVEREKDMMLWLEGKLPVPKVLHFERHDGWSNLLMSEADGVLCSEEYED
                     EQSPEKIIELYAECIRLFHSIDISDCPYTNSLDSRLAELDYLLNNDLADVDCENWEEDT
                     PFKDPRELYDFLKTEKPEEELVFSHGDLGDSNIFVKDGKVSGFIDLGRSGRADKWYDIA
                     FCVRSIREDIGEEQYVELFFDLLGIKPDWEKIKYYILLDELF"
     misc_feature    6814..6838
                     /label=LB T-DNA repeat
                     /note="LB T-DNA repeat"
                     /note="left border repeat from nopaline C58 T-DNA"
     polyA_signal    6916..7090
                     /label=CaMV poly(A) signal
                     /note="CaMV poly(A) signal"
                     /note="cauliflower mosaic virus polyadenylation signal"
     CDS             complement(7147..7944)
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=aph(3')-II (or nptII)
                     /note="NeoR/KanR"
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MGIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGR
                     PVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLL
                     SSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEH
                     QGLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDI
                     ALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     promoter        complement(8007..8684)
                     /note="CaMV 35S promoter (enhanced)"
                     /note="cauliflower mosaic virus 35S promoter with a
                     duplicated enhancer region"