pComb3XSS vector (V001413)

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 a 3rd generation plasmid for phage display on modified geneIII, containing stuffer fragment. Please use TG1 competent cells, 2x YT medium, 100 μg/mL ampicillin.

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 Vector Map

View Map in NovoBuilder

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

Copy Sequence

Download GeneBank File(.gb)

LOCUS       40924_12740        4992 bp DNA     circular SYN 13-MAY-2021
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)
  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"
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)."
     sig_peptide     571..633
                     /label=OmpA signal peptide
                     /note="signal peptide from the E. coli outer membrane
                     protein OmpA"
     sig_peptide     complement(643..708)
                     /label=pelB signal sequence
                     /note="leader peptide for secretion"
     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             1499..1816
                     /codon_start=1
                     /label=hIg-kappa-CL
                     /note="Human immunoglobulin kappa light chain constant
                     region"
                     /translation="TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNA
                     LQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSLPVTKSFNRG
                     EC"
     sig_peptide     1850..1915
                     /label=pelB signal sequence
                     /note="leader peptide for secretion"
     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"
     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"