LOCUS       Exported                4417 bp ds-DNA    circular SYN 30-6-2015
DEFINITION  Flexi(R) vector with a kanamycin resistance marker, for cell-free 
            expression of a protein with a cleavable C-terminal HaloTag(R).
ACCESSION   EU545997
VERSION     .
KEYWORDS    pFC20K HaloTag T7 SP6
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4417)
  AUTHORS   Promega
  TITLE     Direct Submission
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
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        21..39
                     /note="T7 promoter"
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        45..63
                     /note="SP6 promoter"
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     CDS             102..437
                     /codon_start=1
                     /product="ribonuclease from Bacillus amyloliquefaciens"
                     /note="barnase"
                     /note="The barnase gene is lethal in standard bacterial 
                     transformation strains."
                     /protein_id="
                     "
                     /translation="MAQVINTFDGVADYLQTYHKLPDNYITKSEAQALGWVASKGNLAD
                     VAPGKSIGGDIFSNREGKLPGKSGRTWREADINYTSGFRNSDRILYSSDWLIYKTTDHY
                     QTFTKIR"
     CDS             477..497
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /note="TEV site"
                     /note="TEV protease recognition site"
                     /translation="EDLYFQS"
     CDS             513..1394
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /note="HaloTag(R)"
                     /translation="EIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYV
                     WRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHD
                     WGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLIIDQ
                     NVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEE
                     YMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIG
                     SEIARWLSTLEIS"
     terminator      1540..1587
                     /note="T7 terminator"
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             1968..2762
                     /codon_start=1
                     /gene="aph(3)-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /note="NeoR/KanR"
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MLEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     rep_origin      complement(2934..3522)
                     /direction=LEFT
                     /note="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     misc_feature    3638..3921
                     /note="cer region"
                     /note="ColE1-derived recombination site that helps to 
                     maintain plasmids as monomers"
     terminator      4151..4237
                     /gene="Escherichia coli rrnB"
                     /note="rrnB T1 terminator"
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4329..4356
                     /note="rrnB T2 terminator"
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"