Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V001413 | pComb3XSS | In stock, instant shipping |
Buy one, get one free! |
Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.
Basic Vector Information
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
- Vector Name:
- pComb3XSS
- Antibiotic Resistance:
- Ampicillin
- Length:
- 4992 bp
- Type:
- Bacterial Expression ; phage display
- Replication origin:
- ori
- Copy Number:
- High Copy
- Promoter:
- lacZ
- Cloning Method:
- Restriction Enzyme
- 5' Primer:
- 5'-ACC TAT TGC CTA CGG CAG CCG-3'
- 3' Primer:
- 5'-AGA AGC GTA GTC CGG AAC GTC-3'
- Fusion Tag:
- 6xHis and HA tags
- Growth Strain(s):
- TG1
pComb3XSS vector Map
Plasmid Protocol
1. Centrifuge at 5,000×g for 5 min.
2. Carefully open the tube and add 20 μl of sterile water to dissolve the DNA.
3. Close the tube and incubate for 10 minutes at room temperature.
4. Briefly vortex the tube and then do a quick spin to concentrate the liquid at the bottom. Speed is less than 5000×g.
5. Store the plasmid at -20 ℃.
6. The concentration of plasmid re-measurement sometimes differs from the nominal value, which may be due to the position of the lyophilized plasmid in the tube, the efficiency of the re-dissolution, the measurement bias, and adsorption on the wall of the tube, therefore, it is recommended to transform and extract the plasmid before using it
General Plasmid Transform Protocol
1. Take one 100μl of the competent cells and thaw it on ice for 10min, add 2μl of plasmid, then ice bath for 30min, then heat-shock it at 42℃ for 60s, do not stir, and then ice bath for 2min.
2. Add 900μl of LB liquid medium without antibiotics, and incubate at 37℃ for 45min (30℃ for 1-1.5 hours) with 180rpm shaking.
3. Centrifuge at 6000rpm for 5min, leave only 100μl of supernatant to resuspend the bacterial precipitate and spread it onto the target plasmid-resistant LB plate.
4. Invert the plate and incubate at 37℃ for 14h, or at 30℃ for 20h.
5. Pick a single colony into LB liquid medium, add the corresponding antibiotics, incubate at 220rpm for 14h, and extract the plasmid according to the experimental needs and the instructions of the plasmid extraction kit.
References
- Chai D, Wang G, Fang L, Li H, Liu S, Zhu H, Zheng J. The optimization system for preparation of TG1 competent cells and electrotransformation. Microbiologyopen. 2020 Jul;9(7):e1043.
- Ji Y, Chen L, Wang Y, Zhang K, Wu H, Liu Y, Wang Y, Wang J. Development of a Double Nanobody-Based Sandwich Immunoassay for the Detecting Staphylococcal Enterotoxin C in Dairy Products. Foods. 2021 Oct 13;10(10):2426.
pComb3XSS vector Sequence
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"