LOCUS       Exported                5769 bp ds-DNA     circular SYN 17-DEC-2018
DEFINITION  Expression vector pColdTF DNA, complete sequence.
ACCESSION   AB213654
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5769)
  AUTHORS   Shodai T, Takakura H.
  TITLE     Expression vector pColdTF
  JOURNAL   Published Only in Database (2005)
REFERENCE   2  (bases 1 to 5769)
  AUTHORS   Shodai T, Takakura H.
  TITLE     Direct Submission
  JOURNAL   Submitted (17-MAY-2005) Toshihiro Shodai, TAKARA BIO INC.; 
            Seta3-4-1, OTSU, SHIGA 520-2193, Japan 
            (E-mail:shiyoudait@takara-bio.co.jp, Tel:81-77-543-7229, 
            Fax:81-77-543-7238)
REFERENCE   3  (bases 1 to 5769)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..5769
                     /organism="Expression vector pColdTF"
                     /lab_host="Escherichia coli"
                     /mol_type="other DNA"
                     /note="expresses an open reading frame as a trigger factor 
                     fusion, containing the translation enhancing element, His6,
                     protease cleavage site for HRV 3C protease, thrombin and 
                     factor Xa"
                     /db_xref="taxon:329577"
     gene            15..270
                     /gene="cspA"
                     /label=cspA
     regulatory      15..81
                     /regulatory_class="promoter"
                     /gene="cspA"
     promoter        15..81
                     /gene="cspA"
                     /label=cspA promoter
                     /note="promoter of the E. coli cold shock protein cspA gene
                     (Mitta et al., 1997)"
     misc_feature    82..121
                     /gene="cspA"
                     /label=synthetic lac operator sequence
                     /note="synthetic lac operator sequence"
     protein_bind    84..100
                     /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    122..255
                     /gene="cspA"
                     /label=modified cspA 5UTR
                     /note="modified cspA 5UTR"
     5UTR           122..255
                     /gene="cspA"
                     /label=cspA 5UTR
                     /note="5UTR of the E. coli cold shock protein cspA gene 
                     (Mitta et al., 1997)"
     CDS             256..1725
                     /codon_start=1
                     /gene="tig"
                     /product="trigger factor tag"
                     /label=tig
                     /protein_id="BAD98926.1"
                     /translation="MNHKVHHHHHHMQVSVETTQGLGRRVTITIAADSIETAVKSELVN
                     VAKKVRIDGFRKGKVPMNIVAQRYGASVRQDVLGDLMSRNFIDAIIKEKINPAGAPTYV
                     PGEYKLGEDFTYSVEFEVYPEVELQGLEAIEVEKPIVEVTDADVDGMLDTLRKQQATWK
                     EKDGAVEAEDRVTIDFTGSVDGEEFEGGKASDFVLAMGQGRMIPGFEDGIKGHKAGEEF
                     TIDVTFPEEYHAENLKGKAAKFAINLKKVEERELPELTAEFIKRFGVEDGSVEGLRAEV
                     RKNMERELKSAIRNRVKSQAIEGLVKANDIDVPAALIDSEIDVLRRQAAQRFGGNEKQA
                     LELPRELFEEQAKRRVVVGLLLGEVIRTNELKADEERVKGLIEEMASAYEDPKEVIEFY
                     SKNKELMDNMRNVALEEQAVEAVLAKAKVTEKETTFNELMNQQASAGLEVLFQGPSAGL
                     VPRGSGGIEGRHMELGTLEGSEFKLVDLQSR"
     gene            256..1725
                     /gene="tig"
                     /label=tig
     misc_feature    256..270
                     /gene="cspA"
                     /label=TEE (translation enhancing element)
                     /note="TEE (translation enhancing element)"
     CDS             256..270
                     /codon_start=1
                     /gene="cspA"
                     /product="translation enhancing element for E. coli (Qing 
                     et al., 2004)"
                     /label=TEE
                     /translation="MNHKV"
     misc_feature    271..288
                     /gene="tig"
                     /label=His6 tag
                     /note="His6 tag"
     CDS             271..288
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             289..1593
                     /codon_start=1
                     /label=Trigger Factor
                     /translation="MQVSVETTQGLGRRVTITIAADSIETAVKSELVNVAKKVRIDGFR
                     KGKVPMNIVAQRYGASVRQDVLGDLMSRNFIDAIIKEKINPAGAPTYVPGEYKLGEDFT
                     YSVEFEVYPEVELQGLEAIEVEKPIVEVTDADVDGMLDTLRKQQATWKEKDGAVEAEDR
                     VTIDFTGSVDGEEFEGGKASDFVLAMGQGRMIPGFEDGIKGHKAGEEFTIDVTFPEEYH
                     AENLKGKAAKFAINLKKVEERELPELTAEFIKRFGVEDGSVEGLRAEVRKNMERELKSA
                     IRNRVKSQAIEGLVKANDIDVPAALIDSEIDVLRRQAAQRFGGNEKQALELPRELFEEQ
                     AKRRVVVGLLLGEVIRTNELKADEERVKGLIEEMASAYEDPKEVIEFYSKNKELMDNMR
                     NVALEEQAVEAVLAKAKVTEKETTFNELMNQQASAG"
     misc_feature    1594..1617
                     /gene="tig"
                     /label=HRV 3C Protease site
                     /note="HRV 3C Protease site"
     CDS             1594..1617
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     misc_feature    1627..1644
                     /gene="tig"
                     /label=Thrombin site
                     /note="Thrombin site"
     CDS             1627..1644
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     misc_feature    1651..1662
                     /gene="tig"
                     /label=Factor Xa site
                     /note="Factor Xa site"
     CDS             1651..1662
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     misc_feature    1663..1722
                     /gene="tig"
                     /note="MCS = Multiple cloning sites contain the follow 
                     restriction sites: NdeI, SacI, KpnI, XhoI, BamHI, EcoRI, 
                     HindIII, SalI, PstI, XbaI"
     misc_feature    1730..1874
                     /gene="cspA"
                     /label=cspA 3UTR
                     /note="cspA 3UTR"
     gene            1730..1874
                     /gene="cspA"
                     /label=cspA
     3UTR           1730..1874
                     /gene="cspA"
                     /label=cspA 3UTR
                     /note="3UTR of the E. coli cold shock protein cspA gene 
                     (Mitta et al., 1997)"
     misc_feature    complement(2073..2546)
                     /label=M13 intergenic region
                     /note="M13 intergenic region"
     rep_origin      complement(2073..2528)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     promoter        2628..2732
                     /gene="bla"
                     /label=AmpR promoter
     CDS             2733..3593
                     /codon_start=1
                     /gene="Amp"
                     /product="beta-lactamase"
                     /label=Amp
                     /protein_id="BAD98927.1"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     gene            2733..3593
                     /gene="Amp"
                     /label=Amp
     rep_origin      3753..4367
                     /label=ColE1 origin
                     /note="ColE1 origin"
     rep_origin      3764..4352
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(4524..5606)
                     /codon_start=1
                     /gene="lacI"
                     /product="lactose operon repressor"
                     /label=lacI
                     /protein_id="BAD98928.1"
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     gene            complement(4524..5606)
                     /gene="lacI"
                     /label=lacI
     promoter        complement(5607..5684)
                     /gene="lacI"
                     /label=lacI promoter