LOCUS       40924_20311        5059 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Flexi(R) vector with a kanamycin resistance marker, for mammalian 
            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 5059)
  AUTHORS   Promega
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 5059)
  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..5059
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        139..517
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        518..721
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
     intron          857..989
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     promoter        1033..1051
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1067..1957
                     /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             1970..1990
                     /codon_start=1
                     /label=TEV site
                     /note="tobacco etch virus (TEV) protease recognition and 
                     cleavage site"
                     /translation="EDLYFQS"
     CDS             2028..2360
                     /codon_start=1
                     /label=barnase
                     /note="ribonuclease from Bacillus amyloliquefaciens"
                     /translation="MAQVINTFDGVADYLQTYHKLPDNYITKSEAQALGWVASKGNLAD
                     VAPGKSIGGDIFSNREGKLPGKSGRTWREADINYTSGFRNSDRILYSSDWLIYKTTDHY
                     QTFTKIR"
     misc_feature    2373..2428
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     polyA_signal    complement(2533..2654)
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             3053..3844
                     /codon_start=1
                     /label=NeoR/KanR
                     /note="aminoglycoside phosphotransferase from Tn5"
                     /translation="MLEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     rep_origin      complement(4019..4607)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     misc_feature    4723..5006
                     /label=cer region
                     /note="ColE1-derived recombination site that helps to
                     maintain plasmids as monomers"
ORIGIN
        1 tcaatattgg ccattagcca tattattcat tggttatata gcataaatca atattggcta
       61 ttggccattg catacgttgt atctatatca taatatgtac atttatattg gctcatgtcc
      121 aatatgaccg ccatgttggc attgattatt gactagttat taatagtaat caattacggg
      181 gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc
      241 gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat
      301 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc
      361 ccacttggca gtacatcaag tgtatcatat gccaagtccg ccccctattg acgtcaatga
      421 cggtaaatgg cccgcctggc attatgccca gtacatgacc ttacgggact ttcctacttg
      481 gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacac
      541 caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt
      601 caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaataaccc
      661 cgccccgttg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc
      721 tggtttagtg aaccgtcaga tcactagaag ctttattgcg gtagtttatc acagttaaat
      781 tgctaacgca gtcagtgctt ctgacacaac agtctcgaac ttaagctgca gaagttggtc
      841 gtgaggcact gggcaggtaa gtatcaaggt tacaagacag gtttaaggag accaatagaa
      901 actgggcttg tcgagacaga gaagactctt gcgtttctga taggcaccta ttggtcttac
      961 tgacatccac tttgcctttc tctccacagg tgtccactcc cagttcaatt acagctctta
     1021 aggctagagt attaatacga ctcactatag ggctagcaaa gccaccatgg cagaaatcgg
     1081 tactggcttt ccattcgacc cccattatgt ggaagtcctg ggcgagcgca tgcactacgt
     1141 cgatgttggt ccgcgcgatg gcacccctgt gctgttcctg cacggtaacc cgacctcctc
     1201 ctacgtgtgg cgcaacatca tcccgcatgt tgcaccgacc catcgctgca ttgctccaga
     1261 cctgatcggt atgggcaaat ccgacaaacc agacctgggt tatttcttcg acgaccacgt
     1321 ccgcttcatg gatgccttca tcgaagccct gggtctggaa gaggtcgtcc tggtcattca
     1381 cgactggggc tccgctctgg gtttccactg ggccaagcgc aatccagagc gcgtcaaagg
     1441 tattgcattt atggagttca tccgccctat cccgacctgg gacgaatggc cagaatttgc
     1501 ccgcgagacc ttccaggcct tccgcaccac cgacgtcggc cgcaagctga tcatcgatca
     1561 gaacgttttt atcgagggta cgctgccgat gggtgtcgtc cgcccgctga ctgaagtcga
     1621 gatggaccat taccgcgagc cgttcctgaa tcctgttgac cgcgagccac tgtggcgctt
     1681 cccaaacgag ctgccaatcg ccggtgagcc agcgaacatc gtcgcgctgg tcgaagaata
     1741 catggactgg ctgcaccagt cccctgtccc gaagctgctg ttctggggca ccccaggcgt
     1801 tctgatccca ccggccgaag ccgctcgcct ggccaaaagc ctgcctaact gcaaggctgt
     1861 ggacatcggc ccgggtctga atctgctgca agaagacaac ccggacctga tcggcagcga
     1921 gatcgcgcgc tggctgtcga cgctcgagat ttccggcgag ccaaccactg aggatctgta
     1981 ctttcagagc gataacgcga tcgccatgga ataagtaagg aatccacatg gcacaggtta
     2041 tcaacacgtt tgacggggtt gcggattatc ttcagacata tcataagcta cctgataatt
     2101 acattacaaa atcagaagca caagccctcg gctgggtggc atcaaaaggg aaccttgcag
     2161 acgtcgctcc ggggaaaagc atcggcggag acatcttctc aaacagggaa ggcaaactcc
     2221 cgggcaaaag cggacgaaca tggcgtgaag cggatattaa ctatacatca ggcttcagaa
     2281 attcagaccg gattctttac tcaagcgact ggctgattta caaaacaacg gaccattatc
     2341 agacctttac aaaaatcaga taatgtttaa acgaattcgg gctcggtacc cggggatcct
     2401 ctagagtcga cctgcaggca tgcaagctga tccggctgct aacaaagccc gaaaggaagc
     2461 tgagttggct gctgccaccg ctgagcaata actagcataa ccccttgggg cggccgcttc
     2521 gagcagacat gataagatac attgatgagt ttggacaaac cacaactaga atgcagtgaa
     2581 aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt atttgtaacc attataagct
     2641 gcaataaaca agttaacaac aacaattgca ttcattttat gtttcaggtt cagggggaga
     2701 tgtgggaggt ttttttaagc aagtaaaacc tctacaaatg tggtaaaatc gaattctaat
     2761 ggatcctctt tgcgcttgcg ttttcccttg tccagatagc ccagtagctg acattcatcc
     2821 ggggtcagca ccgtttctgc ggactggctt tctacgtgtt ccgcttcctt tagcagccct
     2881 tgcgccctga gtgcttgcgg cagcgtgagc ttcaaaagaa ttgccagctg gggcgccctc
     2941 tggtaaggtt gggaagccct gcaaagtaaa ctggatggct ttcttgccgc caaggatctg
     3001 atggcgcagg ggatcaagat ctgatcaaga gacaggatga cggtcgtttc gcatgcttga
     3061 acaagatgga ttgcacgcag gttctccggc cgcttgggtg gagaggctat tcggctatga
     3121 ctgggcacaa cagacaatcg gctgctctga tgccgccgtg ttccggctgt cagcgcaggg
     3181 gcgcccggtt ctttttgtca agaccgacct gtccggtgcc ctgaatgaac tgcaggacga
     3241 ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct tgcgcagctg tgctcgacgt
     3301 tgtcactgaa gcgggaaggg actggctgct attgggcgaa gtgccggggc aggatctcct
     3361 gtcatctcac cttgctcctg ccgagaaagt atccatcatg gctgatgcaa tgcggcggct
     3421 gcatacgctt gatccggcta cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg
     3481 agcacgcact cggatggaag ccggtcttgt cgatcaggat gatctggacg aagagcatca
     3541 ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg cgtatgccgg atggtgagga
     3601 tctcgtcgtg actcatggcg atgcctgctt gccgaatatc atggtggaaa atggccgctt
     3661 ttctggattc atcgactgtg gccggctggg tgtggcggac cgctatcagg acatagcgtt
     3721 ggctacccgt gatattgctg aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct
     3781 ttacggtatc gccgctcccg attcgcagcg catcgccttc tatcgccttc ttgacgagtt
     3841 cttctgagcg ggactctggg gttcgaaatg accgaccaag cgacgcccaa ccggtatcag
     3901 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     3961 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     4021 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     4081 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     4141 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     4201 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     4261 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     4321 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     4381 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     4441 actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag ccagttacct
     4501 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     4561 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atttcaagaa gatcctttga
     4621 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     4681 tgagattatc aaaaaggatc ttcacctaga tccttttata gtccggaaat acaggaacgc
     4741 acgctggatg gcccttcgct gggatggtga aaccatgaaa aatggcagct tcagtggatt
     4801 aagtgggggt aatgtggcct gtaccctctg gttgcatagg tattcatacg gttaaaattt
     4861 atcaggcgcg attgcggcag tttttcgggt ggtttgttgc catttttacc tgtctgctgc
     4921 cgtgatcgcg ctgaacgcgt tttagcggtg cgtacaatta agggattatg gtaaatccac
     4981 ttactgtctg ccctcgtagc catcgagata aaccgcagta ctccggccac gatgcgtccg
     5041 gcgtagagga tcgagatct
//
