LOCUS       40924_19846        4307 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Flexi(R) vector with a kanamycin resistance marker, for bacterial or
            cell-free expression of a protein with a cleavable C-terminal 
            HaloTag(R)-6xHis dual tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4307)
  AUTHORS   Promega
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 4307)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Subclone a coding sequence between the SgfI/AsiSI and 
            EcoICRI/Eco53kI sites to remove the lethal barnase gene.
FEATURES             Location/Qualifiers
     source          1..4307
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        21..39
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             92..424
                     /codon_start=1
                     /label=barnase
                     /note="ribonuclease from Bacillus amyloliquefaciens"
                     /translation="MAQVINTFDGVADYLQTYHKLPDNYITKSEAQALGWVASKGNLAD
                     VAPGKSIGGDIFSNREGKLPGKSGRTWREADINYTSGFRNSDRILYSSDWLIYKTTDHY
                     QTFTKIR"
     CDS             467..487
                     /codon_start=1
                     /label=TEV site
                     /note="tobacco etch virus (TEV) protease recognition and 
                     cleavage site"
                     /translation="EDLYFQS"
     CDS             500..1387
                     /codon_start=1
                     /label=HaloTag(R)
                     /note="modified bacterial dehalogenase that forms covalent
                     bonds with chloroalkane derivatives"
                     /translation="SEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSY
                     VWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIH
                     DWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLIID
                     QNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVE
                     EYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLI
                     GSEIARWLSTLEISG"
     CDS             1388..1405
                     /codon_start=1
                     /label=6xHis
                     /note="6xHis affinity tag"
                     /translation="HHHHHH"
     terminator      1430..1477
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             1858..2649
                     /codon_start=1
                     /label=NeoR/KanR
                     /note="aminoglycoside phosphotransferase from Tn5"
                     /translation="MLEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     rep_origin      complement(2824..3412)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     misc_feature    3528..3811
                     /label=cer region
                     /note="ColE1-derived recombination site that helps to
                     maintain plasmids as monomers"
     terminator      4041..4127
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     terminator      4219..4246
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB
                     gene"
ORIGIN
        1 ggatctcgat cccgcgaaat taatacgact cactataggg tattttaact ttactaagga
       61 gcgatcgcca tggaataagt aaggaatcca catggcacag gttatcaaca cgtttgacgg
      121 ggttgcggat tatcttcaga catatcataa gctacctgat aattacatta caaaatcaga
      181 agcacaagcc ctcggctggg tggcatcaaa agggaacctt gcagacgtcg ctccggggaa
      241 aagcatcggc ggagacatct tctcaaacag ggaaggcaaa ctcccgggca aaagcggacg
      301 aacatggcgt gaagcggata ttaactatac atcaggcttc agaaattcag accggattct
      361 ttactcaagc gactggctga tttacaaaac aacggaccat tatcagacct ttacaaaaat
      421 cagataatgt ttaatgaccc cgtgtcgagc tctcgagcca accactgagg atctgtactt
      481 tcagagcgat aacgatggat ccgaaatcgg tactggcttt ccattcgacc cccattatgt
      541 ggaagtcctg ggcgagcgca tgcactacgt cgatgttggt ccgcgcgatg gcacccctgt
      601 gctgttcctg cacggtaacc cgacctcctc ctacgtgtgg cgcaacatca tcccgcatgt
      661 tgcaccgacc catcgctgca ttgctccaga cctgatcggt atgggcaaat ccgacaaacc
      721 agacctgggt tatttcttcg acgaccacgt ccgcttcatg gatgccttca tcgaagccct
      781 gggtctggaa gaggtcgtcc tggtcattca cgactggggc tccgctctgg gtttccactg
      841 ggccaagcgc aatccagagc gcgtcaaagg tattgcattt atggagttca tccgccctat
      901 cccgacctgg gacgaatggc cagaatttgc ccgcgagacc ttccaggcct tccgcaccac
      961 cgacgtcggc cgcaagctga tcatcgatca gaacgttttt atcgagggta cgctgccgat
     1021 gggtgtcgtc cgcccgctga ctgaagtcga gatggaccat taccgcgagc cgttcctgaa
     1081 tcctgttgac cgcgagccac tgtggcgctt cccaaacgag ctgccaatcg ccggtgagcc
     1141 agcgaacatc gtcgcgctgg tcgaagaata catggactgg ctgcaccagt cccctgtccc
     1201 gaagctgctg ttctggggca ccccaggcgt tctgatccca ccggccgaag ccgctcgcct
     1261 ggccaaaagc ctgcctaact gcaaggctgt ggacatcggc ccgggtctga atctgctgca
     1321 agaagacaac ccggacctga tcggcagcga gatcgcgcgc tggctgtcta ctctggagat
     1381 ttccggtcat catcaccatc accactaata gtcaaccgct gagcaataac tagcataacc
     1441 ccttggggcc tctaaacggg tcttgagggg ttttttgctg aaaggaggaa ctatatccgg
     1501 ttcgcttgct gtccataaaa ccgcccagtc tagctatcgc catgtaagcc cactgcaagc
     1561 tacctgcttt ctctttgcgc ttgcgttttc ccttgtccag atagcccagt agctgacatt
     1621 catccggggt cagcaccgtt tctgcggact ggctttctac gtgttccgct tcctttagca
     1681 gcccttgcgc cctgagtgct tgcggcagcg tgagcttcaa aagaattgcc agctggggcg
     1741 ccctctggta aggttgggaa gccctgcaaa gtaaactgga tggctttctt gccgccaagg
     1801 atctgatggc gcaggggatc aagatctgat caagagacag gatgacggtc gtttcgcatg
     1861 cttgaacaag atggattgca cgcaggttct ccggccgctt gggtggagag gctattcggc
     1921 tatgactggg cacaacagac aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg
     1981 caggggcgcc cggttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag
     2041 gacgaggcag cgcggctatc gtggctggcc acgacgggcg ttccttgcgc agctgtgctc
     2101 gacgttgtca ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat
     2161 ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg
     2221 cggctgcata cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc
     2281 gagcgagcac gcactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagag
     2341 catcaggggc tcgcgccagc cgaactgttc gccaggctca aggcgcgtat gccggatggt
     2401 gaggatctcg tcgtgactca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc
     2461 cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata
     2521 gcgttggcta cccgtgatat tgctgaagag cttggcggcg aatgggctga ccgcttcctc
     2581 gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg ccttcttgac
     2641 gagttcttct gagcgggact ctggggttcg aaatgaccga ccaagcgacg cccaaccggt
     2701 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     2761 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     2821 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     2881 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     2941 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     3001 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     3061 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     3121 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     3181 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     3241 gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc tgaagccagt
     3301 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     3361 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatttc aagaagatcc
     3421 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
     3481 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttatagtccg gaaatacagg
     3541 aacgcacgct ggatggccct tcgctgggat ggtgaaacca tgaaaaatgg cagcttcagt
     3601 ggattaagtg ggggtaatgt ggcctgtacc ctctggttgc ataggtattc atacggttaa
     3661 aatttatcag gcgcgattgc ggcagttttt cgggtggttt gttgccattt ttacctgtct
     3721 gctgccgtga tcgcgctgaa cgcgttttag cggtgcgtac aattaaggga ttatggtaaa
     3781 tccacttact gtctgccctc gtagccatcg agataaaccg cagtactccg gccacgatgc
     3841 gtccggcgta gaggatcgag atcttttcag cctgatacag attaaatcag aacgcagaag
     3901 cggtctgata aaacagaatt tgcctggcgg cagtagcgcg gtggtcccac ctgaccccat
     3961 gccgaactca gaagtgaaac gccgtagcgc cgatggtagt gtggggtctc cccatgcgag
     4021 agtagggaac tgccaggcat caaataaaac gaaaggctca gtcgaaagac tgggcctttc
     4081 gttttatctg ttgtttgtcg gtgaacgctc tcctgagtag gacaaatccg ccgggagcgg
     4141 atttgaacgt tgcgaagcaa cggcccggag ggtggcgggc aggacgcccg ccataaactg
     4201 ccaggcatca aattaagcag aaggccatcc tgacggatgg cctttttgcg tttctacaaa
     4261 ctcttttgtt tatttttcta aatacattca aatatgtatc cgctcat
//
