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。
pComb3XSS is recommended for preparation of vector for library cloning. The "SS" refers to the double stuffer, a 1180bp stuffer in the Fab light chain cloning region bounded by SacI and XbaI restriction sites and a 360bp stuffer in Fab heavy chain cloning region bound by XhoI and SpeI restriction sites. Also, the 1670bp double stuffer (both stuffers plus the leader sequence between the Fab light chain and heavy chain cloning regions) can be removed by SfiI digest so that non-Fab genes of interest can be cloned. Please use TG1 competent cells, 2x YT medium, 100 μg/mL ampicillin. Use SacI/XbaI and XhoI/SpeI to construct Fab library and SfiI to construct scFv library
- 载体名称:
- pComb3XSS
- 载体抗性:
- Ampicillin
- 载体长度:
- 4992 bp
- 载体类型:
- Bacterial Expression ; phage display
- 复制子:
- ori
- 拷贝数:
- High Copy
- 启动子:
- lacZ
- 克隆方法:
- Restriction Enzyme
- 5'引物:
- 5'-ACC TAT TGC CTA CGG CAG CCG-3'
- 3'引物:
- 5'-AGA AGC GTA GTC CGG AAC GTC-3'
- 载体标签:
- 6xHis and HA tags
- 感受态:
- TG1
pComb3XSS 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。
pComb3XSS 载体序列
LOCUS Exported 4992 bp DNA circular SYN 19-NOV-2024
DEFINITION 3rd generation plasmid for phage display on modified geneIII,
contains stuffer fragment.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 4992)
AUTHORS Andris-Widhopf J, Rader C, Steinberger P, Fuller R, Barbas CF 3rd
TITLE Methods for the generation of chicken monoclonal antibody fragments
by phage display.
JOURNAL J Immunol Methods. 2000 Aug 28;242(1-2):159-81.
PUBMED 10986398
REFERENCE 2 (bases 1 to 4992)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 4992)
TITLE Direct Submission
REFERENCE 4 (bases 1 to 4992)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "J Immunol
Methods."; date: "2000-08-28"; volume: "242(1-2)"; pages: "159-81"
COMMENT SGRef: number: 2; type: "Journal Article"
COMMENT SGRef: number: 3; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..4992
/mol_type="other DNA"
/organism="synthetic DNA construct"
primer_bind 71..90
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
primer_bind 324..341
/label=L4440
/note="L4440 vector, forward primer"
protein_bind 458..479
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 494..524
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 532..548
/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)."
CDS 571..633
/codon_start=1
/label=OmpA signal peptide
/note="signal peptide from the E. coli outer membrane
protein OmpA"
/translation="MKKTAIAIAVALAGFATVAQA"
CDS complement(643..708)
/codon_start=1
/label=pelB signal sequence
/note="leader peptide for secretion"
/translation="MKYLLPTAAAGLLLLAAQPAMA"
primer_bind complement(756..772)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
protein_bind complement(780..796)
/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)."
promoter complement(804..834)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind complement(849..870)
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
CDS complement(924..941)
/codon_start=1
/label=6xHis
/note="6xHis affinity tag"
/translation="HHHHHH"
CDS complement(942..1253)
/codon_start=1
/label=hIg-CH
/translation="KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPATVSWNSGALTS
GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTS
"
CDS 1280..1498
/codon_start=1
/label=VL
/translation="WYQQKPGQAPRLLIYGTSSRATGIPDRFSGSGSGTDFTLTISRLE
PEDFAVYYCQQYGGSPWFGQGTKVELKR"
CDS 1499..1816
/codon_start=1
/label=hIg-kappa-CL
/note="Human immunoglobulin kappa light chain constant
region"
/translation="TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNA
LQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSLPVTKSFNRG
EC"
CDS 1850..1915
/codon_start=1
/label=pelB signal sequence
/note="leader peptide for secretion"
/translation="MKYLLPTAAAGLLLLAAQPAMA"
CDS 1934..2260
/codon_start=1
/label=TrxA
/note="E. coli thioredoxin"
/translation="MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDE
IADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFL
DANLA"
CDS 2312..2329
/codon_start=1
/label=6xHis
/note="6xHis affinity tag"
/translation="HHHHHH"
CDS 2336..2362
/codon_start=1
/label=HA
/note="HA (human influenza hemagglutinin) epitope tag"
/translation="YPYDVPDYA"
CDS 2369..2902
/codon_start=1
/product="M13 pIII
"
/label=pIII
/translation="EGGGSEGGGSEGGGSEGGGSGGGSGSGDFDYEKMANANKGAMTEN
ADENVLQSDAKGKLDSVATDYGAAIDGFIGDVSGLANGNGATGDFAGSNSQMAQVGDGD
NSPLMNNFRQYLPSLPQSVECRPFVFGAGKPYEFSIDCDKINLFRGVFAFLLYVATFMY
VFSTFANILRNKES"
rep_origin complement(2956..3411)
/direction=LEFT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 3438..3542
/label=AmpR promoter
CDS 3543..4400
/codon_start=1
/label=AmpR
/note="beta-lactamase"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRR
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
rep_origin 4574..4992
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"