LOCUS       40924_19831        6524 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Flexi(R) vector with ampicillin and G418 resistance markers, for 
            mammalian 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 6524)
  AUTHORS   Promega
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 6524)
  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..6524
                     /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             1087..1419
                     /codon_start=1
                     /label=barnase
                     /note="ribonuclease from Bacillus amyloliquefaciens"
                     /translation="MAQVINTFDGVADYLQTYHKLPDNYITKSEAQALGWVASKGNLAD
                     VAPGKSIGGDIFSNREGKLPGKSGRTWREADINYTSGFRNSDRILYSSDWLIYKTTDHY
                     QTFTKIR"
     CDS             1462..1482
                     /codon_start=1
                     /label=TEV site
                     /note="tobacco etch virus (TEV) protease recognition and 
                     cleavage site"
                     /translation="EDLYFQS"
     CDS             1495..2382
                     /codon_start=1
                     /label=HaloTag(R)
                     /note="modified bacterial dehalogenase that forms covalent
                     bonds with chloroalkane derivatives"
                     /translation="SEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSY
                     VWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIH
                     DWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLIID
                     QNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVE
                     EYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLI
                     GSEIARWLSTLEISG"
     polyA_signal    complement(2525..2646)
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     promoter        2891..3248
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     CDS             3299..4090
                     /codon_start=1
                     /label=NeoR/KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     polyA_signal    4157..4205
                     /label=poly(A) signal
                     /note="synthetic polyadenylation signal"
     promoter        4361..4465
                     /label=AmpR promoter
     CDS             4466..5323
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      complement(5484..6072)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     misc_feature    6188..6471
                     /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 ggctagcgat cgccatggaa taagtaagga
     1081 atccacatgg cacaggttat caacacgttt gacggggttg cggattatct tcagacatat
     1141 cataagctac ctgataatta cattacaaaa tcagaagcac aagccctcgg ctgggtggca
     1201 tcaaaaggga accttgcaga cgtcgctccg gggaaaagca tcggcggaga catcttctca
     1261 aacagggaag gcaaactccc gggcaaaagc ggacgaacat ggcgtgaagc ggatattaac
     1321 tatacatcag gcttcagaaa ttcagaccgg attctttact caagcgactg gctgatttac
     1381 aaaacaacgg accattatca gacctttaca aaaatcagat aatgtttaat gaccccgtgt
     1441 cgagctctcg agccaaccac tgaggatctg tactttcaga gcgataacga tggatccgaa
     1501 atcggtactg gctttccatt cgacccccat tatgtggaag tcctgggcga gcgcatgcac
     1561 tacgtcgatg ttggtccgcg cgatggcacc cctgtgctgt tcctgcacgg taacccgacc
     1621 tcctcctacg tgtggcgcaa catcatcccg catgttgcac cgacccatcg ctgcattgct
     1681 ccagacctga tcggtatggg caaatccgac aaaccagacc tgggttattt cttcgacgac
     1741 cacgtccgct tcatggatgc cttcatcgaa gccctgggtc tggaagaggt cgtcctggtc
     1801 attcacgact ggggctccgc tctgggtttc cactgggcca agcgcaatcc agagcgcgtc
     1861 aaaggtattg catttatgga gttcatccgc cctatcccga cctgggacga atggccagaa
     1921 tttgcccgcg agaccttcca ggccttccgc accaccgacg tcggccgcaa gctgatcatc
     1981 gatcagaacg tttttatcga gggtacgctg ccgatgggtg tcgtccgccc gctgactgaa
     2041 gtcgagatgg accattaccg cgagccgttc ctgaatcctg ttgaccgcga gccactgtgg
     2101 cgcttcccaa acgagctgcc aatcgccggt gagccagcga acatcgtcgc gctggtcgaa
     2161 gaatacatgg actggctgca ccagtcccct gtcccgaagc tgctgttctg gggcacccca
     2221 ggcgttctga tcccaccggc cgaagccgct cgcctggcca aaagcctgcc taactgcaag
     2281 gctgtggaca tcggcccggg tctgaatctg ctgcaagaag acaacccgga cctgatcggc
     2341 agcgagatcg cgcgctggct gtctactctg gagatttccg gttaatagaa ttctagagtc
     2401 gacctgcagg catgcaagct gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg
     2461 ctgctgccac cgctgagcaa taactagcat aaccccttgg ggcggccgct tcgagcagac
     2521 atgataagat acattgatga gtttggacaa accacaacta gaatgcagtg aaaaaaatgc
     2581 tttatttgtg aaatttgtga tgctattgct ttatttgtaa ccattataag ctgcaataaa
     2641 caagttaaca acaacaattg cattcatttt atgtttcagg ttcaggggga gatgtgggag
     2701 gtttttttaa gcaagtaaaa cctctacaaa tgtggtaaaa tcgaatttta acaaaatatt
     2761 aacgcttaca atttcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac
     2821 cgcatacgcg gatctgcgca gcaccatggc ctgaaataac ctctgaaaga ggaacttggt
     2881 taggtacctt ctgaggcgga aagaaccagc tgtggaatgt gtgtcagtta gggtgtggaa
     2941 agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa
     3001 ccaggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca
     3061 attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca
     3121 gttccgccca ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg
     3181 ccgcctcggc ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct
     3241 tttgcaaaaa gcttgattct tctgacacaa cagtctcgaa cttaaggcta gagccaccat
     3301 gattgaacaa gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg
     3361 ctatgactgg gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc
     3421 gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca
     3481 ggacgaggca gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct
     3541 cgacgttgtc actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga
     3601 tctcctgtca tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg
     3661 gcggctgcat acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat
     3721 cgagcgagca cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga
     3781 gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc aaggcgcgca tgcccgacgg
     3841 cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg
     3901 ccgcttttct ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat
     3961 agcgttggct acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct
     4021 cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga
     4081 cgagttcttc tgagcgggac tctggggttc gaaatgaccg accaagcgac gcccaacctg
     4141 ccatcacgat ggccgcaata aaatatcttt attttcatta catctgtgtg ttggtttttt
     4201 gtgtgaatcg atagcgataa ggatcctctt tgcgcttgcg ttttcccttg tccagatagc
     4261 ccagtagctg acattcatcc ggggtcagca ccgtttctgc ggactggctt tctacgtaat
     4321 ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt
     4381 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct
     4441 tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc
     4501 cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa
     4561 agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg
     4621 taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca ctttcaaagt
     4681 tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg
     4741 catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac
     4801 ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc
     4861 ggccaactta cttctgacaa ctatcggagg accgaaggag ctaaccgctt ttttgcacaa
     4921 catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc
     4981 aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt
     5041 aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga
     5101 taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa
     5161 atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa
     5221 gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa
     5281 tagacagatc gctgagatag gtgcctcact gattaagcat tggtaattcg aaatgaccga
     5341 ccaagcgacg cccaaccggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
     5401 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     5461 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     5521 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     5581 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     5641 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     5701 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     5761 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     5821 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     5881 acagagttct tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc
     5941 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     6001 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     6061 aaaggatttc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     6121 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
     6181 ttatagtccg gaaatacagg aacgcacgct ggatggccct tcgctgggat ggtgaaacca
     6241 tgaaaaatgg cagcttcagt ggattaagtg ggggtaatgt ggcctgtacc ctctggttgc
     6301 ataggtattc atacggttaa aatttatcag gcgcgattgc ggcagttttt cgggtggttt
     6361 gttgccattt ttacctgtct gctgccgtga tcgcgctgaa cgcgttttag cggtgcgtac
     6421 aattaaggga ttatggtaaa tccacttact gtctgccctc gtagccatcg agataaaccg
     6481 cagtactccg gccacgatgc gtccggcgta gaggatcgag atct
//
