首页技术支持质粒载体

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。

pComb3X is a new version of the pComb vectors. Improvements over pComb3 include increased stability and the introduction of an asymmetric SfiI cassette for directional cloning of full Fab, scFv, peptide, and other proteins for phage display. 6xHis and HA tags allow for purification and detection. An amber stop codon was introduced to turn off the expression of the pIII fusion protein by switching to a non-suppressor strain of E. coli allowing the production of soluble protein without subcloning.

载体名称:
pComb3X
载体抗性:
Ampicillin
载体长度:
3451 bp
载体类型:
Phagemid cloning vector
复制子:
ori
载体来源:
Rader C, Barbas CF III.
感受态:
DH10B
培养温度:
37℃

pComb3X 载体图谱

pComb3X3451 bp6001200180024003000CAP binding sitelac promoterlac operatorOmpA signal peptidepelB signal sequence6xHisHAM13 PIIIf1 oriAmpR promoterAmpRori

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。

pComb3X 载体序列

LOCUS       Exported                3451 bp DNA     circular SYN 18-NOV-2024
DEFINITION  Phagemid cloning vector pComb3X, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3451)
  AUTHORS   Rader C, Barbas CF III.
  JOURNAL   (in) PHAGE DISPLAY, A LABORATORY MANUAL. Cold Spring Harbor 
            Laboratory Press, Cold Spring Harbor, NY, USA (2000), In press
REFERENCE   2  (bases 1 to 3451)
  AUTHORS   Rader C, Barbas CF III.
  TITLE     Direct Submission
  JOURNAL   Submitted (16-MAY-2000) Department of Molecular Biology, BCC-526, 
            Scripps Research Institute, 10550 N. Torrey Pines Rd., La Jolla, CA 
            92037, USA
REFERENCE   3  (bases 1 to 3451)
  AUTHORS   Rader C, Fuller R, Barbas CF III.
  TITLE     Direct Submission
  JOURNAL   Submitted (05-SEP-2003) Department of Molecular Biology, BCC-526, 
            Scripps Research Institute, 10550 N. Torrey Pines Rd., La Jolla, CA 
            92037, USA
REFERENCE   4  (bases 1 to 3451)
  TITLE     Direct Submission
REFERENCE   5  (bases 1 to 3451)
  TITLE     Direct Submission
REFERENCE   6  (bases 1 to 3451)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "(in) PHAGE
            DISPLAY, A LABORATORY MANUAL. Cold Spring Harbor Laboratory Press,
            Cold Spring Harbor, NY, USA (2000), In press"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted
            (16-MAY-2000) Department of Molecular Biology, BCC-526, Scripps
            Research Institute, 10550 N. Torrey Pines Rd., La Jolla, CA 92037,
            USA"
COMMENT     SGRef: number: 3; type: "Journal Article"; journalName: "Submitted
            (05-SEP-2003) Department of Molecular Biology, BCC-526, Scripps
            Research Institute, 10550 N. Torrey Pines Rd., La Jolla, CA 92037,
            USA"
COMMENT     SGRef: number: 4; type: "Journal Article"
COMMENT     SGRef: number: 5; type: "Journal Article"
COMMENT     On Sep 5, 2003 this sequence version replaced AF268281.1.
FEATURES             Location/Qualifiers
     source          1..3451
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    109..130
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     promoter        145..175
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    183..199
                     /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)."
     sig_peptide     222..284
                     /label=OmpA signal peptide
                     /note="signal peptide from the E. coli outer membrane
                     protein OmpA"
     sig_peptide     329..394
                     /label=pelB signal sequence
                     /note="leader peptide for secretion"
     CDS             440..457
                     /codon_start=1
                     /label=6xHis
                     /note="6xHis affinity tag"
                     /translation="HHHHHH"
     CDS             464..490
                     /codon_start=1
                     /label=HA
                     /note="HA (human influenza hemagglutinin) epitope tag"
                     /translation="YPYDVPDYA"
     CDS             497..1030
                     /codon_start=1
                     /label=M13 PIII
                     /translation="EGGGSEGGGSEGGGSEGGGSGGGSGSGDFDYEKMANANKGAMTEN
                     ADENALQSDAKGKLDSVATDYGAAIDGFIGDVSGLANGNGATGDFAGSNSQMAQVGDGD
                     NSPLMNNFRQYLPSLPQSVECRPFVFSAGKPYEFSIDCDKINLFRGVFAFLLYVATFMY
                     VFSTFANILRNKES"
     rep_origin      complement(1066..1521)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        1548..1652
                     /label=AmpR promoter
     CDS             1653..2510
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      2684..3272
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"