LOCUS       40924_19826        4417 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 C-terminal HaloTag(R).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4417)
  AUTHORS   Promega
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 4417)
  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..4417
                     /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             102..434
                     /codon_start=1
                     /label=barnase
                     /note="ribonuclease from Bacillus amyloliquefaciens"
                     /translation="MAQVINTFDGVADYLQTYHKLPDNYITKSEAQALGWVASKGNLAD
                     VAPGKSIGGDIFSNREGKLPGKSGRTWREADINYTSGFRNSDRILYSSDWLIYKTTDHY
                     QTFTKIR"
     CDS             477..497
                     /codon_start=1
                     /label=TEV site
                     /note="tobacco etch virus (TEV) protease recognition and 
                     cleavage site"
                     /translation="EDLYFQS"
     CDS             510..1397
                     /codon_start=1
                     /label=HaloTag(R)
                     /note="modified bacterial dehalogenase that forms covalent
                     bonds with chloroalkane derivatives"
                     /translation="SEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSY
                     VWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIH
                     DWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLIID
                     QNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVE
                     EYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLI
                     GSEIARWLSTLEISG"
     terminator      1540..1587
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             1968..2759
                     /codon_start=1
                     /label=NeoR/KanR
                     /note="aminoglycoside phosphotransferase from Tn5"
                     /translation="MLEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     rep_origin      complement(2934..3522)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     misc_feature    3638..3921
                     /label=cer region
                     /note="ColE1-derived recombination site that helps to
                     maintain plasmids as monomers"
     terminator      4151..4237
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     terminator      4329..4356
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB
                     gene"
ORIGIN
        1 ggatctcgat cccgcgaaat taatacgact cactataggg tacgatttag gtgacactat
       61 agaataagga gcgatcgcca tggaataagt aaggaatcca catggcacag gttatcaaca
      121 cgtttgacgg ggttgcggat tatcttcaga catatcataa gctacctgat aattacatta
      181 caaaatcaga agcacaagcc ctcggctggg tggcatcaaa agggaacctt gcagacgtcg
      241 ctccggggaa aagcatcggc ggagacatct tctcaaacag ggaaggcaaa ctcccgggca
      301 aaagcggacg aacatggcgt gaagcggata ttaactatac atcaggcttc agaaattcag
      361 accggattct ttactcaagc gactggctga tttacaaaac aacggaccat tatcagacct
      421 ttacaaaaat cagataatgt ttaatgaccc cgtgtcgagc tctcgagcca accactgagg
      481 atctgtactt tcagagcgat aacgatggat ccgaaatcgg tactggcttt ccattcgacc
      541 cccattatgt ggaagtcctg ggcgagcgca tgcactacgt cgatgttggt ccgcgcgatg
      601 gcacccctgt gctgttcctg cacggtaacc cgacctcctc ctacgtgtgg cgcaacatca
      661 tcccgcatgt tgcaccgacc catcgctgca ttgctccaga cctgatcggt atgggcaaat
      721 ccgacaaacc agacctgggt tatttcttcg acgaccacgt ccgcttcatg gatgccttca
      781 tcgaagccct gggtctggaa gaggtcgtcc tggtcattca cgactggggc tccgctctgg
      841 gtttccactg ggccaagcgc aatccagagc gcgtcaaagg tattgcattt atggagttca
      901 tccgccctat cccgacctgg gacgaatggc cagaatttgc ccgcgagacc ttccaggcct
      961 tccgcaccac cgacgtcggc cgcaagctga tcatcgatca gaacgttttt atcgagggta
     1021 cgctgccgat gggtgtcgtc cgcccgctga ctgaagtcga gatggaccat taccgcgagc
     1081 cgttcctgaa tcctgttgac cgcgagccac tgtggcgctt cccaaacgag ctgccaatcg
     1141 ccggtgagcc agcgaacatc gtcgcgctgg tcgaagaata catggactgg ctgcaccagt
     1201 cccctgtccc gaagctgctg ttctggggca ccccaggcgt tctgatccca ccggccgaag
     1261 ccgctcgcct ggccaaaagc ctgcctaact gcaaggctgt ggacatcggc ccgggtctga
     1321 atctgctgca agaagacaac ccggacctga tcggcagcga gatcgcgcgc tggctgtcta
     1381 ctctggagat ttccggttaa tagaattcta gagtcgacct gcaggcatgc aagctgatcc
     1441 ggctgctaac aaagcccgaa aggaagctga gttggctgct gccaccgctg agcaataact
     1501 agcataacca aaaaaaaaaa aaaaaaaaaa aaaaaaaaac tagcataacc ccttggggcc
     1561 tctaaacggg tcttgagggg ttttttgctg aaaggaggaa ctatatccgg ttcgcttgct
     1621 gtccataaaa ccgcccagtc tagctatcgc catgtaagcc cactgcaagc tacctgcttt
     1681 ctctttgcgc ttgcgttttc ccttgtccag atagcccagt agctgacatt catccggggt
     1741 cagcaccgtt tctgcggact ggctttctac gtgttccgct tcctttagca gcccttgcgc
     1801 cctgagtgct tgcggcagcg tgagcttcaa aagaattgcc agctggggcg ccctctggta
     1861 aggttgggaa gccctgcaaa gtaaactgga tggctttctt gccgccaagg atctgatggc
     1921 gcaggggatc aagatctgat caagagacag gatgacggtc gtttcgcatg cttgaacaag
     1981 atggattgca cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg
     2041 cacaacagac aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc
     2101 cggttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag gacgaggcag
     2161 cgcggctatc gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca
     2221 ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat
     2281 ctcaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata
     2341 cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac
     2401 gcactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc
     2461 tcgcgccagc cgaactgttc gccaggctca aggcgcgtat gccggatggt gaggatctcg
     2521 tcgtgactca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg
     2581 gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta
     2641 cccgtgatat tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg
     2701 gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct
     2761 gagcgggact ctggggttcg aaatgaccga ccaagcgacg cccaaccggt atcagctcac
     2821 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     2881 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     2941 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     3001 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     3061 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     3121 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     3181 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     3241 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     3301 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     3361 ggctacacta gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     3421 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     3481 gtttgcaagc agcagattac gcgcagaaaa aaaggatttc aagaagatcc tttgatcttt
     3541 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     3601 ttatcaaaaa ggatcttcac ctagatcctt ttatagtccg gaaatacagg aacgcacgct
     3661 ggatggccct tcgctgggat ggtgaaacca tgaaaaatgg cagcttcagt ggattaagtg
     3721 ggggtaatgt ggcctgtacc ctctggttgc ataggtattc atacggttaa aatttatcag
     3781 gcgcgattgc ggcagttttt cgggtggttt gttgccattt ttacctgtct gctgccgtga
     3841 tcgcgctgaa cgcgttttag cggtgcgtac aattaaggga ttatggtaaa tccacttact
     3901 gtctgccctc gtagccatcg agataaaccg cagtactccg gccacgatgc gtccggcgta
     3961 gaggatcgag atcttttcag cctgatacag attaaatcag aacgcagaag cggtctgata
     4021 aaacagaatt tgcctggcgg cagtagcgcg gtggtcccac ctgaccccat gccgaactca
     4081 gaagtgaaac gccgtagcgc cgatggtagt gtggggtctc cccatgcgag agtagggaac
     4141 tgccaggcat caaataaaac gaaaggctca gtcgaaagac tgggcctttc gttttatctg
     4201 ttgtttgtcg gtgaacgctc tcctgagtag gacaaatccg ccgggagcgg atttgaacgt
     4261 tgcgaagcaa cggcccggag ggtggcgggc aggacgcccg ccataaactg ccaggcatca
     4321 aattaagcag aaggccatcc tgacggatgg cctttttgcg tttctacaaa ctcttttgtt
     4381 tatttttcta aatacattca aatatgtatc cgctcat
//
