LOCUS       40924_20291        4372 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 N-terminal 
            HaloTag(R).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4372)
  AUTHORS   Promega
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 4372)
  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..4372
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        21..39
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             70..960
                     /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             973..993
                     /codon_start=1
                     /label=TEV site
                     /note="tobacco etch virus (TEV) protease recognition and 
                     cleavage site"
                     /translation="EDLYFQS"
     CDS             1031..1363
                     /codon_start=1
                     /label=barnase
                     /note="ribonuclease from Bacillus amyloliquefaciens"
                     /translation="MAQVINTFDGVADYLQTYHKLPDNYITKSEAQALGWVASKGNLAD
                     VAPGKSIGGDIFSNREGKLPGKSGRTWREADINYTSGFRNSDRILYSSDWLIYKTTDHY
                     QTFTKIR"
     misc_feature    1376..1431
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     terminator      1495..1542
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             1923..2714
                     /codon_start=1
                     /label=NeoR/KanR
                     /note="aminoglycoside phosphotransferase from Tn5"
                     /translation="MLEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     rep_origin      complement(2889..3477)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     misc_feature    3593..3876
                     /label=cer region
                     /note="ColE1-derived recombination site that helps to
                     maintain plasmids as monomers"
     terminator      4106..4192
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     terminator      4284..4311
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB
                     gene"
ORIGIN
        1 ggatctcgat cccgcgaaat taatacgact cactataggg tattttaact ttactaagga
       61 gaattcatca tggcagaaat cggtactggc tttccattcg acccccatta tgtggaagtc
      121 ctgggcgagc gcatgcacta cgtcgatgtt ggtccgcgcg atggcacccc tgtgctgttc
      181 ctgcacggta acccgacctc ctcctacgtg tggcgcaaca tcatcccgca tgttgcaccg
      241 acccatcgct gcattgctcc agacctgatc ggtatgggca aatccgacaa accagacctg
      301 ggttatttct tcgacgacca cgtccgcttc atggatgcct tcatcgaagc cctgggtctg
      361 gaagaggtcg tcctggtcat tcacgactgg ggctccgctc tgggtttcca ctgggccaag
      421 cgcaatccag agcgcgtcaa aggtattgca tttatggagt tcatccgccc tatcccgacc
      481 tgggacgaat ggccagaatt tgcccgcgag accttccagg ccttccgcac caccgacgtc
      541 ggccgcaagc tgatcatcga tcagaacgtt tttatcgagg gtacgctgcc gatgggtgtc
      601 gtccgcccgc tgactgaagt cgagatggac cattaccgcg agccgttcct gaatcctgtt
      661 gaccgcgagc cactgtggcg cttcccaaac gagctgccaa tcgccggtga gccagcgaac
      721 atcgtcgcgc tggtcgaaga atacatggac tggctgcacc agtcccctgt cccgaagctg
      781 ctgttctggg gcaccccagg cgttctgatc ccaccggccg aagccgctcg cctggccaaa
      841 agcctgccta actgcaaggc tgtggacatc ggcccgggtc tgaatctgct gcaagaagac
      901 aacccggacc tgatcggcag cgagatcgcg cgctggctgt cgacgctcga gatttccggc
      961 gagccaacca ctgaggatct gtactttcag agcgataacg cgatcgccat ggaataagta
     1021 aggaatccac atggcacagg ttatcaacac gtttgacggg gttgcggatt atcttcagac
     1081 atatcataag ctacctgata attacattac aaaatcagaa gcacaagccc tcggctgggt
     1141 ggcatcaaaa gggaaccttg cagacgtcgc tccggggaaa agcatcggcg gagacatctt
     1201 ctcaaacagg gaaggcaaac tcccgggcaa aagcggacga acatggcgtg aagcggatat
     1261 taactataca tcaggcttca gaaattcaga ccggattctt tactcaagcg actggctgat
     1321 ttacaaaaca acggaccatt atcagacctt tacaaaaatc agataatgtt taaacgaatt
     1381 cgggctcggt acccggggat cctctagagt cgacctgcag gcatgcaagc tgatccggct
     1441 gctaacaaag cccgaaagga agctgagttg gctgctgcca ccgctgagca ataactagca
     1501 taaccccttg gggcctctaa acgggtcttg aggggttttt tgctgaaagg aggaactata
     1561 tccggttcgc ttgctgtcca taaaaccgcc cagtctagct atcgccatgt aagcccactg
     1621 caagctacct gctttctctt tgcgcttgcg ttttcccttg tccagatagc ccagtagctg
     1681 acattcatcc ggggtcagca ccgtttctgc ggactggctt tctacgtgtt ccgcttcctt
     1741 tagcagccct tgcgccctga gtgcttgcgg cagcgtgagc ttcaaaagaa ttgccagctg
     1801 gggcgccctc tggtaaggtt gggaagccct gcaaagtaaa ctggatggct ttcttgccgc
     1861 caaggatctg atggcgcagg ggatcaagat ctgatcaaga gacaggatga cggtcgtttc
     1921 gcatgcttga acaagatgga ttgcacgcag gttctccggc cgcttgggtg gagaggctat
     1981 tcggctatga ctgggcacaa cagacaatcg gctgctctga tgccgccgtg ttccggctgt
     2041 cagcgcaggg gcgcccggtt ctttttgtca agaccgacct gtccggtgcc ctgaatgaac
     2101 tgcaggacga ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct tgcgcagctg
     2161 tgctcgacgt tgtcactgaa gcgggaaggg actggctgct attgggcgaa gtgccggggc
     2221 aggatctcct gtcatctcac cttgctcctg ccgagaaagt atccatcatg gctgatgcaa
     2281 tgcggcggct gcatacgctt gatccggcta cctgcccatt cgaccaccaa gcgaaacatc
     2341 gcatcgagcg agcacgcact cggatggaag ccggtcttgt cgatcaggat gatctggacg
     2401 aagagcatca ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg cgtatgccgg
     2461 atggtgagga tctcgtcgtg actcatggcg atgcctgctt gccgaatatc atggtggaaa
     2521 atggccgctt ttctggattc atcgactgtg gccggctggg tgtggcggac cgctatcagg
     2581 acatagcgtt ggctacccgt gatattgctg aagagcttgg cggcgaatgg gctgaccgct
     2641 tcctcgtgct ttacggtatc gccgctcccg attcgcagcg catcgccttc tatcgccttc
     2701 ttgacgagtt cttctgagcg ggactctggg gttcgaaatg accgaccaag cgacgcccaa
     2761 ccggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
     2821 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
     2881 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
     2941 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
     3001 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     3061 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc
     3121 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     3181 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     3241 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     3301 tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag
     3361 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     3421 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atttcaagaa
     3481 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
     3541 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttata gtccggaaat
     3601 acaggaacgc acgctggatg gcccttcgct gggatggtga aaccatgaaa aatggcagct
     3661 tcagtggatt aagtgggggt aatgtggcct gtaccctctg gttgcatagg tattcatacg
     3721 gttaaaattt atcaggcgcg attgcggcag tttttcgggt ggtttgttgc catttttacc
     3781 tgtctgctgc cgtgatcgcg ctgaacgcgt tttagcggtg cgtacaatta agggattatg
     3841 gtaaatccac ttactgtctg ccctcgtagc catcgagata aaccgcagta ctccggccac
     3901 gatgcgtccg gcgtagagga tcgagatctt ttcagcctga tacagattaa atcagaacgc
     3961 agaagcggtc tgataaaaca gaatttgcct ggcggcagta gcgcggtggt cccacctgac
     4021 cccatgccga actcagaagt gaaacgccgt agcgccgatg gtagtgtggg gtctccccat
     4081 gcgagagtag ggaactgcca ggcatcaaat aaaacgaaag gctcagtcga aagactgggc
     4141 ctttcgtttt atctgttgtt tgtcggtgaa cgctctcctg agtaggacaa atccgccggg
     4201 agcggatttg aacgttgcga agcaacggcc cggagggtgg cgggcaggac gcccgccata
     4261 aactgccagg catcaaatta agcagaaggc catcctgacg gatggccttt ttgcgtttct
     4321 acaaactctt ttgtttattt ttctaaatac attcaaatat gtatccgctc at
//
