Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V008299 | pComb3X | 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
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.
- Vector Name:
- pComb3X
- Antibiotic Resistance:
- Ampicillin
- Length:
- 3451 bp
- Type:
- Phagemid cloning vector
- Replication origin:
- ori
- Source/Author:
- Rader C, Barbas CF III.
pComb3X vector 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
- Schladetsch MA, Wiemer AJ. Generation of Single-Chain Variable Fragment (scFv) Libraries for Use in Phage Display. Curr Protoc. 2021 Jul;1(7):e182. doi: 10.1002/cpz1.182. PMID: 34232564
pComb3X vector Sequence
LOCUS 40924_12730 3451 bp DNA circular SYN 17-DEC-2018
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)
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 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"
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"