LOCUS       Exported                3616 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pKILHIS-2, complete sequence.
ACCESSION   AF028838
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3616)
  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 3616)
  AUTHORS   Van Reeth T, Gabant P, Dreze P, Szpirer J, Szpirer C.
  TITLE     Direct Submission
  JOURNAL   Submitted (07-OCT-1997) Departement de Biologie Moleculaire 
            Laboratoire de Biologie du Developpement, Universite Libre de 
            Bruxelles, 67, Rue des Chevaux, 1640 Rhode Saint-Genese, Belgium
REFERENCE   3  (bases 1 to 3616)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..3616
                     /organism="Cloning vector pKILHIS-2"
                     /host="Escherichia coli"
                     /mol_type="genomic DNA"
                     /db_xref="taxon:74554"
     rep_origin      17..396
                     /direction=RIGHT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     rep_origin      18..429
                     /label=f1
                     /note="f1"
     CDS             complement(444..1238)
                     /codon_start=1
                     /product="kanamycin marker protein"
                     /label=kanamycin marker protein
                     /protein_id="AAC78280.1"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     rep_origin      1625..2297
                     /label=ColE1
                     /note="ColE1"
     rep_origin      1650..2238
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     terminator      2393..2420
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      2512..2598
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     regulatory      2561..2718
                     /regulatory_class="terminator"
     CDS             complement(2820..3380)
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB protein"
                     /label=ccdB
                     /note="CcdB fusion protein"
                     /protein_id="AAC78281.1"
                     /translation="MGGSHHHHHHGMASMTGGQQMGRDLYDDDDIGSVDLAVVLQRRDW
                     ENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLYVRQFKVYTYKRESRYRLFVD
                     VQSDIIDTPGRRMVIPLASARLLSDKVSRELYPVVHIGDESWRMMTTDMASVPVSVIGE
                     EVADLSHRENDIKNAINLMFWGI"
     gene            complement(2820..3380)
                     /gene="ccdB"
                     /label=ccdB
     CDS             complement(2820..3122)
                     /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"
     misc_feature    3291..3300
                     /label=cloning site
                     /note="cloning site"
     CDS             complement(3315..3347)
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     CDS             complement(3351..3368)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(3384..3410)
                     /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"
     misc_feature    3461..3530
                     /label=rrnG antiterminator
                     /note="antiterminator from the E. coli rrnG leader region 
                     (Berg et al., 1989)"
     protein_bind    3547..3563
                     /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)."
     promoter        complement(3571..3600)
                     /label=trc promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"