LOCUS       40924_26729        3616 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pKILHIS-2, complete sequence.
ACCESSION   .
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)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 3616)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "BioTechniques"; date: "1998"; volume: "25"; issue: "5"; pages: 
            "898-904"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "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"
COMMENT     SGRef: number: 3; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..3616
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      17..396
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     CDS             complement(447..1238)
                     /codon_start=1
                     /label=NeoR/KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     rep_origin      1650..2238
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     terminator      complement(2393..2420)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB
                     gene"
     terminator      complement(2512..2598)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     CDS             complement(2823..3122)
                     /codon_start=1
                     /label=ccdB
                     /note="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /translation="QFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDKV
                     SRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     misc_feature    3291..3300
                     /label=cloning site
                     /note="cloning site"
     CDS             complement(3315..3347)
                     /codon_start=1
                     /label=T7 tag (gene 10 leader)
                     /note="leader peptide from bacteriophage T7 gene 10"
                     /translation="MASMTGGQQMG"
     CDS             complement(3351..3368)
                     /codon_start=1
                     /label=6xHis
                     /note="6xHis affinity tag"
                     /translation="HHHHHH"
     CDS             complement(3387..3410)
                     /codon_start=1
                     /label=minicistron
                     /note="synthetic cistron containing a ribosome binding site
                     (Shine-Dalgarno sequence), for enhancing the bacterial 
                     expression of a downstream cistron (Schoner, 1997)"
                     /translation="MYRLNKEE"
     misc_feature    complement(3461..3530)
                     /label=rrnG antiterminator
                     /note="antiterminator from the E. coli rrnG leader region
                     (Berg et al., 1989)"
     protein_bind    complement(3547..3563)
                     /label=lac operator
                     /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"
ORIGIN
        1 cctgaatggc gaatggacgc gccctgtagc ggcgcattaa gcgcgcgggt gtggtggtta
       61 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
      121 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
      181 tagggttccg atttagagct ttacggcacc tcgaccgcaa aaaacttgat ttgggtgatg
      241 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
      301 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atcgcggtct
      361 attcttttga tttataaggg atgttgccga tttcggccta ttggttaaaa aatgagctga
      421 tttaacaaaa attttaacaa aattcagaag aactcgtcaa gaaggcgata gaaggcgatg
      481 cgctgcgaat cgggagcggc gataccgtaa agcacgagga agcggtcagc ccattcgccg
      541 ccaagctctt cagcaatatc acgggtagcc aacgctatgt cctgatagcg gtccgccaca
      601 cccagccggc cacagtcgat gaatccagaa aagcggccat tttccaccat gatattcggc
      661 aagcaggcat cgccatgggt cacgacgaga tcctcgccgt cgggcatgct cgccttgagc
      721 ctggcgaaca gttcggctgg cgcgagcccc tgatgctctt cgtccagatc atcctgatcg
      781 acaagaccgg cttccatccg agtacgtgct cgctcgatgc gatgtttcgc ttggtggtcg
      841 aatgggcagg tagccggatc aagcgtatgc agccgccgca ttgcatcagc catgatggat
      901 actttctcgg caggagcaag gtgagatgac aggagatcct gccccggcac ttcgcccaat
      961 agcagccagt cccttcccgc ttcagtgaca acgtcgagca cagctgcgca aggaacgccc
     1021 gtcgtggcca gccacgatag ccgcgctgcc tcgtcttgca gttcattcag ggcaccggac
     1081 aggtcggtct tgacaaaaag aaccgggcgc ccctgcgctg acagccggaa cacggcggca
     1141 tcagagcagc cgattgtctg ttgtgcccag tcatagccga atagcctctc cacccaagcg
     1201 gccggagaac ctgcgtgcaa tccatcttgt tcaatcatgc gaaacgatcc tcatcctgtc
     1261 tcttgatcag agcttgatcc cctgcgccat cagatccttg gcggcgagaa agccatccag
     1321 tttactttgc agggcttccc aaccttacca gagggcgccc cagctggcaa ttccggttcg
     1381 cttgctgtcc ataaaaccgc ccagtctagc tatcgccatg taagcccact gcaagctacc
     1441 tgctttctct ttgcgcttgc gttttccctt gtccagatag cccagtagct gacattcatc
     1501 cggggtcagc accgtttctg cggactggct ttctacgtga aaaggatcta ggtgaagatc
     1561 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     1621 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     1681 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     1741 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt
     1801 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     1861 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     1921 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     1981 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     2041 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     2101 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     2161 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     2221 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct gggcttttgc
     2281 tggccttttg ctcacatgat tattgaagca tttatcaggg ttattgtctc atgagcggat
     2341 acatatttga atgtatttag aaaaataaac aaaaagagtt tgtagaaacg caaaaaggcc
     2401 atccgtcagg atggccttct gcttaatttg atgcctggca gtttatggcg ggcgtcctgc
     2461 ccgccaccct ccgggccgtt gcttcgcaac gttcaaatcc gctcccggcg gatttgtcct
     2521 actcaggaga gcgttcaccg acaaacaaca gataaaacga aaggcccagt ctttcgactg
     2581 agcctttcgt tttatttgat gcctggcagt tccctactct cgcatgggga gaccccacac
     2641 taccatcggc gctacggcgt ttcacttctg agttcggcat ggggtcaggt gggaccaccg
     2701 cgctactgcc gccaggcaaa ttctgtttta tcagaccgct tctgcgttct gatttaatct
     2761 gtatcaggct gaaaatcttc tctcatccgc caaaacagcc aagcttcgaa ttctgacatt
     2821 tatattcccc agaacatcag gttaatggcg tttttgatgt cattttcgcg gtggctgaga
     2881 tcagccactt cttccccgat aacggagacc ggcacactgg ccatatcggt ggtcatcatg
     2941 cgccagcttt catccccgat atgcaccacc gggtaaagtt cacgggagac tttatctgac
     3001 agcagacgtg cactggccag ggggatcacc atccgtcgcc ccggcgtgtc aataatatca
     3061 ctctgtacat ccacaaacag acgataacgg ctctctcttt tataggtgta aaccttaaac
     3121 tgccgtacgt ataggctgcg caactgttgg gaagggcgat cggtgcgggc ctcttcgcta
     3181 ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat taagttgggt aacgccaggg
     3241 ttttcccagt cacgacgttg taaaacgacg gccaggtcga cggatccgat atcgtcatcg
     3301 tcgtacagat cccgacccat ttgctgtcca ccagtcatgc tagccatacc atgatgatga
     3361 tgatgatgag aaccccccat ggtttattcc tccttattta atcgatacat taatatatac
     3421 ctctttaatt tttaataata aagttaatcg ataattccgg tcgagtgccc acacagattg
     3481 tctgataaat tgttaaagag cagtgccgct tcgctttttc tcagcggcgc tgtttcctgt
     3541 gtgaaattgt tatccgctca caattccaca cattatacga gccggatgat taattgtcaa
     3601 cagctcattt cagaat
//
