f88-4 vector (V009988)

Basic Vector Information

      • Vector Name:
      • f88-4
      • Antibiotic Resistance:
      • Tetracycline
      • Length:
      • 9234 bp
      • Type:
      • Filamentous phage display vector
      • Source/Author:
      • Scott JK, Smith GP.

f88-4 vector Vector Map

f88-49234 bp400800120016002000240028003200360040004400480052005600600064006800720076008000840088009200pVpVIIpVIIIfd terminatorpVIpItacrecombinant major coat protein precursortrpATcRtetR/tetA promotersTetRpII

Plasmid Resuspension 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 ℃.

f88-4 vector Sequence

Copy Sequence

Download GeneBank File(.gb)

LOCUS       Exported                9234 bp ds-DNA     circular SYN 17-DEC-2018
DEFINITION  Filamentous phage display vector f88-4, complete sequence.
ACCESSION   AF218363
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9234)
  AUTHORS   Scott JK, Smith GP.
  TITLE     Searching for peptide ligands with an epitope library
  JOURNAL   Science 249 (4967), 386-390 (1990)
  PUBMED    1696028
REFERENCE   2  (bases 1 to 9234)
  AUTHORS   Smith GP, Choukri S.
  TITLE     Direct Submission
  JOURNAL   Submitted (21-DEC-1999) Division of Biological Sciences, University 
            of Missouri, Tucker Hall, Columbia, MO 65211-7400, USA
REFERENCE   3  (bases 1 to 9234)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     This phage display vector is derived by splicing a 379-bp 
            semi-synthetic major coat protein gene into filamentous phage vector
            fd-tet. The resulting phage has two major coat protein genes: the 
            wild-type gene VIII and the artificial one rVIII. The artificial 
            gene is driven by a tac promoter and is therefore inducible by IPTG;
            under fully induced conditions (1 mM IPTG) roughly 300 of the 3900 
            coat protein subunits stem from the artificial gene rVIII, the 
            remainder stemming from the wild-type gene VIII. The artificial gene
            rVIII has unique HindIII and PstI cloning sites separated by a 21-bp
            'stuffer.'  Replacement of the stuffer with an appropriate synthetic
            or natural DNA insert can result in display of up to 300 copies of a
            peptide encoded by the insert on the virion surface, fused to the 
            rVIII-derived coat protein subunits. This surface display requires 
            that the first 9 bp of the insert should encode the last three amino
            acids of the signal peptide, and that the reading frame of the 
            insert's coding sequence be correctly fused to the reading frame of 
            the remainder of the rVIII coding sequence downstream of the PstI 
            clonging site.
FEATURES             Location/Qualifiers
     source          join(1..5647,6027..9234)
                     /organism="Filamentous phage cloning vector fd-tet"
                     /mol_type="genomic DNA"
                     /label=presented in GenBank Accession Number AF217317
                     /note="presented in GenBank Accession Number AF217317"
                     /db_xref="taxon:127516"
     source          1..9234
                     /organism="Filamentous phage display vector f88-4"
                     /focus
                     /lab_host="Escherichia coli"
                     /mol_type="genomic DNA"
                     /db_xref="taxon:127586"
     CDS             843..1106
                     /codon_start=1
                     /gene="V"
                     /product="pV"
                     /label=M13 gene V
                     /note="replication"
                     /translation="MIKVEIKPSQAQFTTRSGVSRQGKPYSLNEQLCYVDLGNEYPVLV
                     KITLDEGQPAYAPGLYTVHLSSFKVGQFGSLMIDRLRLVPAK"
     CDS             1108..1209
                     /codon_start=1
                     /gene="VII"
                     /product="pVII"
                     /label=M13 gene VII
                     /note="minor coat protein"
                     /translation="MEQVADFDTIYQAMIQISVVLCFALGIIAGGQR"
     CDS             1206..1304
                     /codon_start=1
                     /gene="IX"
                     /product="pIX"
                     /label=M13 gene IX
                     /note="minor coat protein"
                     /translation="MSVLVYSFASFVLGWCLRSGITYFTRLMETSS"
     CDS             1301..1522
                     /codon_start=1
                     /gene="VIII"
                     /product="pVIII"
                     /label=M13 gene VIII
                     /note="major coat protein"
                     /translation="MKKSLVLKASVAVATLVPMLSFAAEGDDPAKAAFDSLQASATEYI
                     GYAWAMVVVIVGATIGIKLFKKFTSKAS"
     terminator      1526..1574
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     CDS             2856..3194
                     /codon_start=1
                     /gene="VI"
                     /product="pVI"
                     /label=M13 gene VI
                     /note="minor coat protein"
                     /translation="MPVLLGIPLLLRFLGFLLVTLFGYLLTFLKKGFGKIAIAISLFLA
                     LIIGLNSILVGYLSDISAQLPSDFVQGVQLILPSNALPCFYVILSVKAAIFIFDVKQKI
                     VSYLDWDK"
     CDS             3197..4243
                     /codon_start=1
                     /gene="I"
                     /product="pI"
                     /label=M13 gene I
                     /note="phage assembly"
                     /translation="MAVYFVTGKLGSGKTLVSVGKIQDKIVAGCKIATNLDLRLQNLPQ
                     VGRFAKTPRVLRIPDKPSISDLLAIGRGNDSYDENKNGLLVLDECGTWFNTRSWNDKER
                     QPIIDWFLHARKLGWDIIFLVQDLSIVDKQARSALAEHVVYCRRLDRITLPFVGTLYSL
                     VTGSKMPLPKLHVGVVKYGDSQLSPTVERWLYTGKNLYNAYDTKQAFSSNYDSGVYSYL
                     TPYLSHGRYFKPLNLGQKMKLTKIYLKKFSRVLCLAIGFASAFTYSYITQPKPEVKKVV
                     SQTYDFDKFTIDSSQRLNLSYRYVFKDSKGKLINSDDLQKQGYSITYIDLCTVSIKKGN
                     SNEIVKCN"
     regulatory      5666..5730
                     /regulatory_class="promoter"
                     /note="tac"
     promoter        5674..5702
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    5710..5726
                     /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)."
     CDS             5748..5978
                     /codon_start=1
                     /product="recombinant major coat protein precursor"
                     /label=recombinant major coat protein precursor
                     /protein_id="AAF78530.1"
                     /translation="MKKSLVLKASVAVATLVPMLSFANVPAEGDDPAKAAFDSLQASAT
                     EYIGYAWAMVVVIVGATIGIKLFKKFTSKAS"
     sig_peptide     5748..5816
     misc_feature    5807..5812
                     /label=HindIII site
                     /note="HindIII site"
     misc_feature    5808..5828
                     /label=21-bp stuffer
                     /note="21-bp stuffer"
     mat_peptide     5823..5975
                     /product="recombinant major coat protein"
     misc_feature    5824..5829
                     /label=PstI site
                     /note="PstI site"
     regulatory      5981..6021
                     /regulatory_class="terminator"
                     /note="trpA"
     variation       5983
                     /replace="a"
     CDS             complement(6290..7495)
                     /codon_start=1
                     /gene="tetA from transposon Tn10"
                     /product="tetracycline efflux protein"
                     /label=TcR
                     /note="confers resistance to tetracycline"
                     /translation="MNSSTKIALVITLLDAMGIGLIMPVLPTLLREFIASEDIANHFGV
                     LLALYALMQVIFAPWLGKMSDRFGRRPVLLLSLIGASLDYLLLAFSSALWMLYLGRLLS
                     GITGATGAVAASVIADTTSASQRVKWFGWLGASFGLGLIAGPIIGGFAGEISPHSPFFI
                     AALLNIVTFLVVMFWFRETKNTRDNTDTEVGVETQSNSVYITLFKTMPILLIIYFSAQL
                     IGQIPATVWVLFTENRFGWNSMMVGFSLAGLGLLHSVFQAFVAGRIATKWGEKTAVLLG
                     FIADSSAFAFLAFISEGWLVFPVLILLAGGGIALPALQGVMSIQTKSHQQGALQGLLVS
                     LTNATGVIGPLLFAVIYNHSLPIWDGWIWIIGLAFYCIIILLSMTFMLTPQAQGSKQET
                     SA"
     promoter        7503..7558
                     /gene="tetR"
                     /label=tetR/tetA promoters
                     /note="overlapping promoters for bacterial tetR and tetA"
     protein_bind    7504..7522
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7534..7552
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O1 for the tetR and tetA genes"
     CDS             7577..8200
                     /codon_start=1
                     /gene="tetR from transposon Tn10"
                     /product="tetracycline repressor TetR"
                     /label=TetR
                     /note="TetR binds to the tetracycline operator tetO to 
                     inhibit transcription. This inhibition can be relieved by 
                     adding tetracycline or doxycycline."
                     /translation="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     CDS             8833..831
                     /codon_start=1
                     /gene="II"
                     /product="pII"
                     /label=M13 gene II
                     /note="replication"
                     /translation="MIDMLVLRLPFIDSLVCSRLSGNDLIAFVDLSKIATLSGMNLSAR
                     TVEYHIDGDLTVSGLSHPFESLPTHYSGIAFKIYEGSKNFYPCVEIKASPAKVLQGHNV
                     FGTTDLALCSEALLLNFANSLPCLYDLLDVNATTISRIDATFSARAPNENIAKQVIDHL
                     RNVSNGQTKSTRSQNWESTVTWNETSRHRTLVAYLKHVELQHQIQQLSSKPSAKMTSYQ
                     KEQLKVLSNPDLLEFASGLVRFEARIETRYLKSFGLPLNLFDAIRFASDYNRQGKDLIF
                     DLWSFSFSELFKAFEGDSMNIYDDSAVLDAIQSKHFTITPSGKTSFAKASRYFGFYRRL
                     VNEGYDSVALTMPRNSFWRYVSALVECGIPKSQLMNLSTCNNVVPLVRFINVDFSSQRP
                     DWYNEPVLKIA"

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