LOCUS       40924_20301        4421 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Flexi(R) vector with a kanamycin resistance marker, for cell-free 
            expression of a protein with a cleavable N-terminal HaloTag(R).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4421)
  AUTHORS   Promega
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 4421)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Subclone a coding sequence between the SgfI/AsiSI and PmeI sites to 
            remove the lethal barnase gene.
FEATURES             Location/Qualifiers
     source          1..4421
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        21..39
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        45..63
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     CDS             80..970
                     /codon_start=1
                     /label=HaloTag(R)
                     /note="modified bacterial dehalogenase that forms covalent
                     bonds with chloroalkane derivatives"
                     /translation="MAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSS
                     YVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVI
                     HDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLII
                     DQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALV
                     EEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDL
                     IGSEIARWLSTLEISG"
     CDS             983..1003
                     /codon_start=1
                     /label=TEV site
                     /note="tobacco etch virus (TEV) protease recognition and 
                     cleavage site"
                     /translation="EDLYFQS"
     CDS             1041..1373
                     /codon_start=1
                     /label=barnase
                     /note="ribonuclease from Bacillus amyloliquefaciens"
                     /translation="MAQVINTFDGVADYLQTYHKLPDNYITKSEAQALGWVASKGNLAD
                     VAPGKSIGGDIFSNREGKLPGKSGRTWREADINYTSGFRNSDRILYSSDWLIYKTTDHY
                     QTFTKIR"
     misc_feature    1386..1441
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     terminator      1544..1591
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             1972..2763
                     /codon_start=1
                     /label=NeoR/KanR
                     /note="aminoglycoside phosphotransferase from Tn5"
                     /translation="MLEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     rep_origin      complement(2938..3526)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     misc_feature    3642..3925
                     /label=cer region
                     /note="ColE1-derived recombination site that helps to
                     maintain plasmids as monomers"
     terminator      4155..4241
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     terminator      4333..4360
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB
                     gene"
ORIGIN
        1 ggatctcgat cccgcgaaat taatacgact cactataggg tacgatttag gtgacactat
       61 agaataagga gaattcatca tggcagaaat cggtactggc tttccattcg acccccatta
      121 tgtggaagtc ctgggcgagc gcatgcacta cgtcgatgtt ggtccgcgcg atggcacccc
      181 tgtgctgttc ctgcacggta acccgacctc ctcctacgtg tggcgcaaca tcatcccgca
      241 tgttgcaccg acccatcgct gcattgctcc agacctgatc ggtatgggca aatccgacaa
      301 accagacctg ggttatttct tcgacgacca cgtccgcttc atggatgcct tcatcgaagc
      361 cctgggtctg gaagaggtcg tcctggtcat tcacgactgg ggctccgctc tgggtttcca
      421 ctgggccaag cgcaatccag agcgcgtcaa aggtattgca tttatggagt tcatccgccc
      481 tatcccgacc tgggacgaat ggccagaatt tgcccgcgag accttccagg ccttccgcac
      541 caccgacgtc ggccgcaagc tgatcatcga tcagaacgtt tttatcgagg gtacgctgcc
      601 gatgggtgtc gtccgcccgc tgactgaagt cgagatggac cattaccgcg agccgttcct
      661 gaatcctgtt gaccgcgagc cactgtggcg cttcccaaac gagctgccaa tcgccggtga
      721 gccagcgaac atcgtcgcgc tggtcgaaga atacatggac tggctgcacc agtcccctgt
      781 cccgaagctg ctgttctggg gcaccccagg cgttctgatc ccaccggccg aagccgctcg
      841 cctggccaaa agcctgccta actgcaaggc tgtggacatc ggcccgggtc tgaatctgct
      901 gcaagaagac aacccggacc tgatcggcag cgagatcgcg cgctggctgt cgacgctcga
      961 gatttccggc gagccaacca ctgaggatct gtactttcag agcgataacg cgatcgccat
     1021 ggaataagta aggaatccac atggcacagg ttatcaacac gtttgacggg gttgcggatt
     1081 atcttcagac atatcataag ctacctgata attacattac aaaatcagaa gcacaagccc
     1141 tcggctgggt ggcatcaaaa gggaaccttg cagacgtcgc tccggggaaa agcatcggcg
     1201 gagacatctt ctcaaacagg gaaggcaaac tcccgggcaa aagcggacga acatggcgtg
     1261 aagcggatat taactataca tcaggcttca gaaattcaga ccggattctt tactcaagcg
     1321 actggctgat ttacaaaaca acggaccatt atcagacctt tacaaaaatc agataatgtt
     1381 taaacgaatt cgggctcggt acccggggat cctctagagt cgacctgcag gcatgcaagc
     1441 tgatccggct gctaacaaag cccgaaagga agctgagttg gctgctgcca ccgctgagca
     1501 ataactagca taaccaaaaa aaaaaaaaaa aaaaaaaaaa aaactagcat aaccccttgg
     1561 ggcctctaaa cgggtcttga ggggtttttt gctgaaagga ggaactatat ccggttcgct
     1621 tgctgtccat aaaaccgccc agtctagcta tcgccatgta agcccactgc aagctacctg
     1681 ctttctcttt gcgcttgcgt tttcccttgt ccagatagcc cagtagctga cattcatccg
     1741 gggtcagcac cgtttctgcg gactggcttt ctacgtgttc cgcttccttt agcagccctt
     1801 gcgccctgag tgcttgcggc agcgtgagct tcaaaagaat tgccagctgg ggcgccctct
     1861 ggtaaggttg ggaagccctg caaagtaaac tggatggctt tcttgccgcc aaggatctga
     1921 tggcgcaggg gatcaagatc tgatcaagag acaggatgac ggtcgtttcg catgcttgaa
     1981 caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt cggctatgac
     2041 tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc agcgcagggg
     2101 cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact gcaggacgag
     2161 gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt
     2221 gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca ggatctcctg
     2281 tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat gcggcggctg
     2341 catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg catcgagcga
     2401 gcacgcactc ggatggaagc cggtcttgtc gatcaggatg atctggacga agagcatcag
     2461 gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc gtatgccgga tggtgaggat
     2521 ctcgtcgtga ctcatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt
     2581 tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga catagcgttg
     2641 gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt
     2701 tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc
     2761 ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc gacgcccaac cggtatcagc
     2821 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
     2881 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     2941 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     3001 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     3061 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     3121 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     3181 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     3241 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     3301 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     3361 ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     3421 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     3481 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tttcaagaag atcctttgat
     3541 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     3601 gagattatca aaaaggatct tcacctagat ccttttatag tccggaaata caggaacgca
     3661 cgctggatgg cccttcgctg ggatggtgaa accatgaaaa atggcagctt cagtggatta
     3721 agtgggggta atgtggcctg taccctctgg ttgcataggt attcatacgg ttaaaattta
     3781 tcaggcgcga ttgcggcagt ttttcgggtg gtttgttgcc atttttacct gtctgctgcc
     3841 gtgatcgcgc tgaacgcgtt ttagcggtgc gtacaattaa gggattatgg taaatccact
     3901 tactgtctgc cctcgtagcc atcgagataa accgcagtac tccggccacg atgcgtccgg
     3961 cgtagaggat cgagatcttt tcagcctgat acagattaaa tcagaacgca gaagcggtct
     4021 gataaaacag aatttgcctg gcggcagtag cgcggtggtc ccacctgacc ccatgccgaa
     4081 ctcagaagtg aaacgccgta gcgccgatgg tagtgtgggg tctccccatg cgagagtagg
     4141 gaactgccag gcatcaaata aaacgaaagg ctcagtcgaa agactgggcc tttcgtttta
     4201 tctgttgttt gtcggtgaac gctctcctga gtaggacaaa tccgccggga gcggatttga
     4261 acgttgcgaa gcaacggccc ggagggtggc gggcaggacg cccgccataa actgccaggc
     4321 atcaaattaa gcagaaggcc atcctgacgg atggcctttt tgcgtttcta caaactcttt
     4381 tgtttatttt tctaaataca ttcaaatatg tatccgctca t
//
