LOCUS       Exported                4422 bp DNA     circular SYN 20-JUL-2025
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 4422)
  AUTHORS   Promega
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 4422)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 4422)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     SGRef: number: 2; 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..4422
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     source          1544
                     /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      1545..1592
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             1973..2764
                     /codon_start=1
                     /label=NeoR/KanR
                     /note="aminoglycoside phosphotransferase from Tn5"
                     /translation="MLEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     rep_origin      complement(2939..3527)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     misc_feature    3643..3926
                     /label=cer region
                     /note="ColE1-derived recombination site that helps to
                     maintain plasmids as monomers"
     terminator      4156..4242
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     terminator      4334..4361
                     /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 aaaactagca taaccccttg
     1561 gggcctctaa acgggtcttg aggggttttt tgctgaaagg aggaactata tccggttcgc
     1621 ttgctgtcca taaaaccgcc cagtctagct atcgccatgt aagcccactg caagctacct
     1681 gctttctctt tgcgcttgcg ttttcccttg tccagatagc ccagtagctg acattcatcc
     1741 ggggtcagca ccgtttctgc ggactggctt tctacgtgtt ccgcttcctt tagcagccct
     1801 tgcgccctga gtgcttgcgg cagcgtgagc ttcaaaagaa ttgccagctg gggcgccctc
     1861 tggtaaggtt gggaagccct gcaaagtaaa ctggatggct ttcttgccgc caaggatctg
     1921 atggcgcagg ggatcaagat ctgatcaaga gacaggatga cggtcgtttc gcatgcttga
     1981 acaagatgga ttgcacgcag gttctccggc cgcttgggtg gagaggctat tcggctatga
     2041 ctgggcacaa cagacaatcg gctgctctga tgccgccgtg ttccggctgt cagcgcaggg
     2101 gcgcccggtt ctttttgtca agaccgacct gtccggtgcc ctgaatgaac tgcaggacga
     2161 ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct tgcgcagctg tgctcgacgt
     2221 tgtcactgaa gcgggaaggg actggctgct attgggcgaa gtgccggggc aggatctcct
     2281 gtcatctcac cttgctcctg ccgagaaagt atccatcatg gctgatgcaa tgcggcggct
     2341 gcatacgctt gatccggcta cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg
     2401 agcacgcact cggatggaag ccggtcttgt cgatcaggat gatctggacg aagagcatca
     2461 ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg cgtatgccgg atggtgagga
     2521 tctcgtcgtg actcatggcg atgcctgctt gccgaatatc atggtggaaa atggccgctt
     2581 ttctggattc atcgactgtg gccggctggg tgtggcggac cgctatcagg acatagcgtt
     2641 ggctacccgt gatattgctg aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct
     2701 ttacggtatc gccgctcccg attcgcagcg catcgccttc tatcgccttc ttgacgagtt
     2761 cttctgagcg ggactctggg gttcgaaatg accgaccaag cgacgcccaa ccggtatcag
     2821 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     2881 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     2941 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     3001 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     3061 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     3121 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     3181 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     3241 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     3301 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     3361 actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag ccagttacct
     3421 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     3481 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atttcaagaa gatcctttga
     3541 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     3601 tgagattatc aaaaaggatc ttcacctaga tccttttata gtccggaaat acaggaacgc
     3661 acgctggatg gcccttcgct gggatggtga aaccatgaaa aatggcagct tcagtggatt
     3721 aagtgggggt aatgtggcct gtaccctctg gttgcatagg tattcatacg gttaaaattt
     3781 atcaggcgcg attgcggcag tttttcgggt ggtttgttgc catttttacc tgtctgctgc
     3841 cgtgatcgcg ctgaacgcgt tttagcggtg cgtacaatta agggattatg gtaaatccac
     3901 ttactgtctg ccctcgtagc catcgagata aaccgcagta ctccggccac gatgcgtccg
     3961 gcgtagagga tcgagatctt ttcagcctga tacagattaa atcagaacgc agaagcggtc
     4021 tgataaaaca gaatttgcct ggcggcagta gcgcggtggt cccacctgac cccatgccga
     4081 actcagaagt gaaacgccgt agcgccgatg gtagtgtggg gtctccccat gcgagagtag
     4141 ggaactgcca ggcatcaaat aaaacgaaag gctcagtcga aagactgggc ctttcgtttt
     4201 atctgttgtt tgtcggtgaa cgctctcctg agtaggacaa atccgccggg agcggatttg
     4261 aacgttgcga agcaacggcc cggagggtgg cgggcaggac gcccgccata aactgccagg
     4321 catcaaatta agcagaaggc catcctgacg gatggccttt ttgcgtttct acaaactctt
     4381 ttgtttattt ttctaaatac attcaaatat gtatccgctc at
//
