LOCUS       Exported                4806 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Expression vector pKIL-HIS3, complete sequence.
ACCESSION   AF050464
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4806)
  AUTHORS   Van Reeth T, Dreze PL, Szpirer J, Szpirer C, Gabant P.
  TITLE     Positive selection vectors to generate fused genes for the 
            expression of his-tagged proteins
  JOURNAL   BioTechniques 25 (5), 898-904 (1998)
  PUBMED    9821593
REFERENCE   2  (bases 1 to 4806)
  AUTHORS   Van Reeth T, Gabant P, Dreze PL, Szpirer J, Szpirer C.
  TITLE     Direct Submission
  JOURNAL   Submitted (24-FEB-1998) Departement de Biologie Moleculaire, 
            Universite Libre de Bruxelles, 67, rue des Chevaux, Rhode St. Genese
            1640, Belgium
REFERENCE   3  (bases 1 to 4806)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..4806
                     /organism="Expression vector pKIL-HIS3"
                     /host="Escherichia coli"
                     /mol_type="genomic DNA"
                     /db_xref="taxon:74458"
     gene            28..1002
                     /gene="ccdB"
                     /label=ccdB
     regulatory      28..58
                     /regulatory_class="promoter"
                     /gene="ccdB"
     promoter        30..59
                     /label=trc promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    67..83
                     /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    100..169
                     /label=rrnG antiterminator
                     /note="antiterminator from the E. coli rrnG leader region 
                     (Berg et al., 1989)"
     CDS             220..246
                     /codon_start=1
                     /product="synthetic cistron containing a ribosome binding 
                     site (Shine-Dalgarno sequence), for enhancing the bacterial
                     expression of a downstream cistron (Schoner, 1997)"
                     /label=minicistron
                     /note="This first cistron should terminate 3 bp upstream of
                     the ATG for the second cistron."
                     /translation="MYRLNKEE"
     CDS             250..774
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB protein"
                     /label=ccdB
                     /note="CcdB fusion protein"
                     /protein_id="AAC78290.1"
                     /translation="MSRRRRQAWFSSSSSSLIEGSVDLAVVLQRRDWENPGVTQLNRLA
                     AHPPFASWRNSEEARTDRPSQQLRSLYVRQFKVYTYKRESRYRLFVDVQSDIIDTPGRR
                     MVIPLASARLLSDKVSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDI
                     KNAINLMFWGI"
     misc_feature    253..259
                     /gene="ccdB"
                     /note="unique NruI (blunt) cloning site that can be used 
                     for generation of C-terminal His-tagged protein"
     CDS             257..271
                     /codon_start=1
                     /product="enterokinase recognition and cleavage site"
                     /label=enterokinase site
                     /translation="DDDDK"
     CDS             281..298
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             472..774
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="QFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDKV
                     SRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     regulatory      876..1002
                     /regulatory_class="terminator"
                     /gene="ccdB"
     terminator      996..1082
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1174..1201
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        1221..1312
                     /gene="bla"
                     /label=AmpR promoter
     CDS             1313..2173
                     /codon_start=1
                     /gene="ampR"
                     /product="beta-lactamase"
                     /label=ampR
                     /protein_id="AAC78291.1"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     gene            1313..2173
                     /gene="ampR"
                     /label=ampR
                     /note="ampicillin resistance gene"
     rep_origin      2319..2992
     rep_origin      2344..2932
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     misc_feature    3118..3258
                     /label=bom
                     /note="basis of mobility region from pBR322"
     promoter        3444..3521
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     CDS             3522..4604
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPSTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     CDS             3645..4604
                     /codon_start=1
                     /gene="lacIq"
                     /product="LacIq"
                     /function="repressor of lac promoter"
                     /label=lacIq
                     /protein_id="AAC78292.1"
                     /translation="MAELNYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSR
                     ADQLGASVVVSMVERSGVEACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPA
                     LFLDVSDQTPINSIIFSHEDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKY
                     LTRNQIQPIAEREGDWSAMSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLR
                     VGADISVVGYDDTEDSSCYIPPSTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVS
                     LVKRKTTLAPNTQTASPRALADSLMQLARQVSRLESGQ"
     gene            3645..4604
                     /gene="lacIq"
                     /label=lacIq