pCANTAB-5E Plasmid

pCANTAB-5E

Catalog No. PVT10573
Packing 2ug

 

pCANTAB-5E Description

This system is used for antibody gene expression and preparation of recombinant antibody. The host strain TG1 was replicated and expressed by plasmid vector pCANTAB5e, and was cultured on 2 *YT medium at 37 C. The vector pCANTAB5e is used to construct recombinant scFv. It has ampicillin resistance and the size is 4522 bp. The auxiliary phage M13K07 is used to rescue the phage particles. It is Kana-resistant and can be replicated by the auxiliary phage and expressed on the phage surface in the form of fusion. There is a sequence encoding Tag tail peptide (E-Tag) behind the scFv gene. There is an amber termination codon behind the tail peptide. It is located between the scFv gene and the cpIII gene. In the inhibitory bacteria TG1, only 20% of the amber codon is effective, so it can be read through the protein translation process to form scFv-cp. In non-inhibitory strains, such as HB2151, the terminator is recognized, and the scFv gene terminates before the cpIII gene in the translation process, forming an independent antibody protein that remains in the cell membrane gap, and leaks into the culture medium after a long period of culture to form soluble expression.

 

[Operation method]

This method comes from the network. This platform has not been confirmed by experiments, for reference only.

 

1. preparation of auxiliary phage virus species

 

1) the auxiliary bacteriophage M13K07 was crossed on the 2 * YT agar plate.

 

2) Prepare 2 *YT semi-solid agar (0.7% agar) as top Agar, cool to 50 ~C, take 4 mL Top Agar and add 0.5 mL overnight culture of fresh TG1 bacteria (OD660 up to 0.8), fully mix, along the line concentration from low to high direction, pour Top Agar;

 

3) Cultured at 37 C for 6-12 hours, single plaque was selected by inoculation needle, inoculated at 30-200 mL 2 YT-K (kanamycin 70 UG / mL), and cultured at 37 C for 10-14 hours.

 

4) 8000 rpm centrifugation 15 min, 4 C;

 

5) Take the supernatant carefully, and suggest that 0.45 micron filter membrane be used to filter, then pack aseptic tubules and store them at 4 C for at least half a year.

 

2. titration of bacteriophages

 

1) prepare 2 x YT culture plates without any antibiotics; 5-6.

2) 0.7% agar plate was prepared with 2 *YT medium, namely Top Agar, which was cooled to 42 C and stored at 42 C.

3) the above bacteriophage solution was diluted with 10-6, 10-7, 10-8, 10-9 and 10-10 with the medium.

4) Take the overnight culture of TG1 bacterial solution (OD660 = 1 or so), pack small test tube, 500 mu L / small test tube, marked 10-6 to 10-10 dilution;

Remarks: preparation of host bacteria should be carried out according to the instructions of TG1 strain.

5) Add 100 mu L of the phage diluted successively in step 3 above to each standard dilution tube, mix well, and oscillate gently at 37 C for 30 min.

6) Add Top Agar 3 mL to each tube, pour the prepared dishes immediately, and incubate at 37 C overnight.

7) calculate the number of plaque on the plate, multiplied by the corresponding dilution multiple, and the general concentration can reach 1012pfu/mL.

[note]

1. bacteriophage virus species should be titrated according to the recommended method before using.

2. phage virus species can be stored at 4 degrees centigrade, not frozen at -20 or lower temperatures.

 

pCANTAB-5E Information
Function E.coli Cloning plasmid
Promoter: Lac/lac promoter

Plasmid size: 5216bp

Plasmid tagging: N-g3 signal, C-E tag

Prokaryotic resistance: ampicillin Amp

Clone strain: Escherichia coli Stbl3

Culture conditions: 37 C, aerobic, LB

 

pCANTAB-5E Sequence

LOCUS       Exported                5216 bp ds-DNA     circular SYN 27-OCT-2017

DEFINITION  synthetic circular DNA

KEYWORDS    pCANTAB-5E (Non empty vector)

SOURCE      synthetic DNA construct

  ORGANISM  synthetic DNA construct

REFERENCE   1  (bases 1 to 5216)

  AUTHORS   .

  TITLE     Direct Submission

           

FEATURES             Location/Qualifiers

     source          1..5216

                     /organism="synthetic DNA construct"

                     /mol_type="other DNA"

     promoter        96..200

                     /gene="bla"

                     /label=AmpR promoter

     CDS             201..1061

                     /codon_start=1

                     /gene="bla"

                     /product="beta-lactamase"

                     /label=AmpR

                     /note="confers resistance to ampicillin, carbenicillin, and

                     related antibiotics"

                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI

                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS

                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW

                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA

                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS

                     LIKHW"

     rep_origin      1232..1820

                     /direction=RIGHT

                     /label=ori

                     /note="high-copy-number colE1/pMB1/pBR322/pUC origin of 

                     replication"

     promoter        2144..2174

                     /label=lac promoter

                     /note="promoter for the E. coli lac operon"

     protein_bind    2182..2198

                     /label=lac operator

                     /bound_moiety="lac repressor encoded by lacI"

                     /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)."

     misc_feature    2218..2315

                     /label=g3 signal

     misc_feature    2329..3066

                     /label=Stuffer(闂堢偟鈹栨潪鏂ょ礆

     misc_feature    3075..3120

                     /label=E tag

     CDS             3520..4338

                     /codon_start=1

                     /label=fd gene 3

                     /translation="MFQNNRFRNRQGALTVYTGTVTQGTDPVKTYYQYTPVSSKAMYDA

                     YWNGKFRDCAFHSGFNEDPFVCEYQGQSSDLPQPPVNAGGGSGGGSGGGSEGGGSEGGG

                     SEGGGSEGGGSGGGSGSGDFDYEKMANANKGAMTENADENALQSDAKGKLDSVATDYGA

                     AIDGFIGDVSGLANGNGATGDFAGSNSQMAQVGDGDNSPLMNNFRQYLPSLPQSVECRP

                     YVFGAGKPYEFSIDCDKINLFRGVFAFLLYVATFMYVFSTFANILRNKES"

     rep_origin      complement(4560..5015)

                     /direction=LEFT

                     /label=f1 ori

                     /note="f1 bacteriophage origin of replication; arrow 

                     indicates direction of (+) strand synthesis"

ORIGIN

        1 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt

       61 cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt

      121 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat

      181 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt

      241 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg

      301 ctgaagatca gttgggtgct cgagtgggtt acatcgaact ggatctcaac agcggtaaga

      361 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc

      421 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac

      481 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg

      541 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca

      601 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg

      661 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg

      721 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg

      781 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag

      841 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg

      901 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct

      961 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac

     1021 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact

     1081 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga

     1141 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt

     1201 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct

     1261 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc

     1321 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc

     1381 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc

     1441 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg

     1501 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt

     1561 cgtgcataca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg

     1621 agcattgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg

     1681 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt

     1741 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag

     1801 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt

     1861 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta

     1921 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt

     1981 cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc

     2041 cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca

     2101 acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc

     2161 cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg

     2221 accatgatta cgccaagctt tggagccttt tttttggaga ttttcaacgt gaaaaaatta

     2281 ttattcgcaa ttcctttagt tgttcctttc tatgcggccc agccggccat ggcccaggtg

     2341 aagctgcagc agtcaggacc tggcctggtg gcgccctcac agagcctgtc catcacatgc

     2401 accgtctcag ggttttcatt aaccagctat ggtgtacact gggttcgcca gcctccagga

     2461 aagggtctgg agtggctggg agtaatatgg gctggtggaa gcacaaacta taattcagct

     2521 ctcaaatcca gactgaacat cagcaaggac aactccaaga gccaagtttt cttaaaaatg

     2581 aacagtctcc aaactgatga cacagccatg tactactgtg ccagaaactg gggcagctac

     2641 tggtacttcg atgtctgggg ccaaggccac ggtcaccgtc tcctcagtgg aggcggttca

     2701 ggcggaggtg gcggaggtgg ctctggcggt ggcggatcgg acattgagct cacccagtct

     2761 ccagcaatca tgtctgcatc tccaggggaa aaggtcacca tgacctgcag ggccagctca

     2821 agtataagtt ccagttactt gcactggtac cagcagaagt caggcgcttc ccccaaaccc

     2881 ttgattcata ggacatccaa cctggcttct ggagtcccag ctcgcttcag tggcagtggg

     2941 tctgggacct cttactctct cacaatcagc agcgtggagg ctgaagatga tgcaacttat

     3001 tactgccagc agtggagtgg ttacccattc acgttcggtg ctgggaccaa gctcgagatc

     3061 aaacgggcgg ccgcaggtgc gccggtgccg tatccggatc cgctggaacc gcgtgccgca

     3121 tagactgttg aaagttgttt agcaaaacct catacagaaa attcatttac taacgtctgg

     3181 aaagacgaca aaactttaga tcgttacgct aactatgagg gctgtctgtg gaatgctaca

     3241 ggcgttgtgg tttgtactgg tgacgaaact cagtgttacg gtacatgggt tcctattggg

     3301 cttgctatcc ctgaaaatga gggtggtggc tctgagggtg gcggttctga gggtggcggt

     3361 tctgagggtg gcggtactaa acctcctgag tacggtgata cacctattcc gggctatact

     3421 tatatcaacc ctctcgacgg cacttatccg cctggtactg agcaaaaccc cgctaatcct

     3481 aatccttctc ttgaggagtc tcagcctctt aatactttca tgtttcagaa taataggttc

     3541 cgaaataggc agggtgcatt aactgtttat acgggcactg ttactcaagg cactgacccc

     3601 gttaaaactt attaccagta cactcctgta tcatcaaaag ccatgtatga cgcttactgg

     3661 aacggtaaat tcagagactg cgctttccat tctggcttta atgaggatcc attcgtttgt

     3721 gaatatcaag gccaatcgtc tgacctgcct caacctcctg tcaatgctgg cggcggctct

     3781 ggtggtggtt ctggtggcgg ctctgagggt ggcggctctg agggtggcgg ttctgagggt

     3841 ggcggctctg agggtggcgg ttccggtggc ggctccggtt ccggtgattt tgattatgaa

     3901 aaaatggcaa acgctaataa gggggctatg accgaaaatg ccgatgaaaa cgcgctacag

     3961 tctgacgcta aaggcaaact tgattctgtc gctactgatt acggtgctgc tatcgatggt

     4021 ttcattggtg acgtttccgg ccttgctaat ggtaatggtg ctactggtga ttttgctggc

     4081 tctaattccc aaatggctca agtcggtgac ggtgataatt cacctttaat gaataatttc

     4141 cgtcaatatt taccttcttt gcctcagtcg gttgaatgtc gcccttatgt ctttggcgct

     4201 ggtaaaccat atgaattttc tattgattgt gacaaaataa acttattccg tggtgtcttt

     4261 gcgtttcttt tatatgttgc cacctttatg tatgtatttt cgacgtttgc taacatactg

     4321 cgtaataagg agtcttaata agaattcact ggccgtcgtt ttacaacgtc gtgactggga

     4381 aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg

     4441 taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga

     4501 atggcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatata

     4561 aattgtaaac gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt

     4621 ttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agcccgagat

     4681 agggttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa

     4741 cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac catcacccaa

     4801 atcaagtttt ttggggtcga ggtgccgtaa agcactaaat cggaacccta aagggagccc

     4861 ccgatttaga gcttgacggg gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc

     4921 gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg taaccaccac

     4981 acccgccgcg cttaatgcgc cgctacaggg cgcgtactat ggttgctttg acgggtgcag

     5041 tctcagtaca atctgctctg atgccgcata gttaagccag ccccgacacc cgccaacacc

     5101 cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac

     5161 cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcga

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