pCANTAB-5E, 2 ug

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    pCANTAB-5E


    Catalog No. PVT10573                                                    
    Packing 2ug

     

    pCANTAB-5E Information

    Plasmid size: 5216bp

    Plasmid Tags: n-g3 signal, C-E tag

    Prokaryotic resistance: Amp

    Clone strain: TG1

    Culture conditions: 37℃

    Plasmid host: Escherichia coli

    Plasmid use: gene template

    Fragment type:

    Prokaryotic resistance: Amp

     

    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 4517 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 Sequence

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

    DEFINITION  synthetic circular DNA

    KEYWORDS    pCANTAB-5E

    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

         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

    //
     

     

    Caution:
    1.  This product is FOR RESEARCH USE ONLY!
    2.  The item is lyophilized form, Please take the powder plasmid by centrifugation at 5000rpm/min for 1min. Add 20μl ddH2O in to the tube of plasmid.