pComb3X vector (V008299)

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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.

Growth Strain(s):: DH10B

Growth Temperature:: 37℃

pComb3X vector Map

Copy Sequence View 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       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"