LOCUS       40924_20331        4135 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Flexi(R) vector with a kanamycin resistance marker, for mammalian 
            expression from a moderately compromised CMV promoter of a protein 
            with an N-terminal HaloTag(R).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4135)
  AUTHORS   Promega
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 4135)
  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..4135
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..52
                     /label=CMVd2 promoter
                     /note="truncated human cytomegalovirus (CMV) immediate
                     early promoter that yields moderately reduced expression 
                     relative to the full-length promoter"
     promoter        84..102
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        108..126
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     CDS             143..1033
                     /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             1046..1066
                     /codon_start=1
                     /label=TEV site
                     /note="tobacco etch virus (TEV) protease recognition and 
                     cleavage site"
                     /translation="EDLYFQS"
     CDS             1104..1436
                     /codon_start=1
                     /label=barnase
                     /note="ribonuclease from Bacillus amyloliquefaciens"
                     /translation="MAQVINTFDGVADYLQTYHKLPDNYITKSEAQALGWVASKGNLAD
                     VAPGKSIGGDIFSNREGKLPGKSGRTWREADINYTSGFRNSDRILYSSDWLIYKTTDHY
                     QTFTKIR"
     misc_feature    1449..1504
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     polyA_signal    complement(1609..1730)
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             2129..2920
                     /codon_start=1
                     /label=NeoR/KanR
                     /note="aminoglycoside phosphotransferase from Tn5"
                     /translation="MLEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     rep_origin      complement(3095..3683)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     misc_feature    3799..4082
                     /label=cer region
                     /note="ColE1-derived recombination site that helps to
                     maintain plasmids as monomers"
ORIGIN
        1 gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat ataagcagag ctggtttagt
       61 gaaccgtcag atcactagaa gcttaatacg actcactata gggtacgatt taggtgacac
      121 tatagaataa ggagaattca tcatggcaga aatcggtact ggctttccat tcgaccccca
      181 ttatgtggaa gtcctgggcg agcgcatgca ctacgtcgat gttggtccgc gcgatggcac
      241 ccctgtgctg ttcctgcacg gtaacccgac ctcctcctac gtgtggcgca acatcatccc
      301 gcatgttgca ccgacccatc gctgcattgc tccagacctg atcggtatgg gcaaatccga
      361 caaaccagac ctgggttatt tcttcgacga ccacgtccgc ttcatggatg ccttcatcga
      421 agccctgggt ctggaagagg tcgtcctggt cattcacgac tggggctccg ctctgggttt
      481 ccactgggcc aagcgcaatc cagagcgcgt caaaggtatt gcatttatgg agttcatccg
      541 ccctatcccg acctgggacg aatggccaga atttgcccgc gagaccttcc aggccttccg
      601 caccaccgac gtcggccgca agctgatcat cgatcagaac gtttttatcg agggtacgct
      661 gccgatgggt gtcgtccgcc cgctgactga agtcgagatg gaccattacc gcgagccgtt
      721 cctgaatcct gttgaccgcg agccactgtg gcgcttccca aacgagctgc caatcgccgg
      781 tgagccagcg aacatcgtcg cgctggtcga agaatacatg gactggctgc accagtcccc
      841 tgtcccgaag ctgctgttct ggggcacccc aggcgttctg atcccaccgg ccgaagccgc
      901 tcgcctggcc aaaagcctgc ctaactgcaa ggctgtggac atcggcccgg gtctgaatct
      961 gctgcaagaa gacaacccgg acctgatcgg cagcgagatc gcgcgctggc tgtcgacgct
     1021 cgagatttcc ggcgagccaa ccactgagga tctgtacttt cagagcgata acgcgatcgc
     1081 catggaataa gtaaggaatc cacatggcac aggttatcaa cacgtttgac ggggttgcgg
     1141 attatcttca gacatatcat aagctacctg ataattacat tacaaaatca gaagcacaag
     1201 ccctcggctg ggtggcatca aaagggaacc ttgcagacgt cgctccgggg aaaagcatcg
     1261 gcggagacat cttctcaaac agggaaggca aactcccggg caaaagcgga cgaacatggc
     1321 gtgaagcgga tattaactat acatcaggct tcagaaattc agaccggatt ctttactcaa
     1381 gcgactggct gatttacaaa acaacggacc attatcagac ctttacaaaa atcagataat
     1441 gtttaaacga attcgggctc ggtacccggg gatcctctag agtcgacctg caggcatgca
     1501 agctgatccg gctgctaaca aagcccgaaa ggaagctgag ttggctgctg ccaccgctga
     1561 gcaataacta gcataacccc ttggggcggc cgcttcgagc agacatgata agatacattg
     1621 atgagtttgg acaaaccaca actagaatgc agtgaaaaaa atgctttatt tgtgaaattt
     1681 gtgatgctat tgctttattt gtaaccatta taagctgcaa taaacaagtt aacaacaaca
     1741 attgcattca ttttatgttt caggttcagg gggagatgtg ggaggttttt ttaagcaagt
     1801 aaaacctcta caaatgtggt aaaatcgaat tctaatggat cctctttgcg cttgcgtttt
     1861 cccttgtcca gatagcccag tagctgacat tcatccgggg tcagcaccgt ttctgcggac
     1921 tggctttcta cgtgttccgc ttcctttagc agcccttgcg ccctgagtgc ttgcggcagc
     1981 gtgagcttca aaagaattgc cagctggggc gccctctggt aaggttggga agccctgcaa
     2041 agtaaactgg atggctttct tgccgccaag gatctgatgg cgcaggggat caagatctga
     2101 tcaagagaca ggatgacggt cgtttcgcat gcttgaacaa gatggattgc acgcaggttc
     2161 tccggccgct tgggtggaga ggctattcgg ctatgactgg gcacaacaga caatcggctg
     2221 ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac
     2281 cgacctgtcc ggtgccctga atgaactgca ggacgaggca gcgcggctat cgtggctggc
     2341 cacgacgggc gttccttgcg cagctgtgct cgacgttgtc actgaagcgg gaagggactg
     2401 gctgctattg ggcgaagtgc cggggcagga tctcctgtca tctcaccttg ctcctgccga
     2461 gaaagtatcc atcatggctg atgcaatgcg gcggctgcat acgcttgatc cggctacctg
     2521 cccattcgac caccaagcga aacatcgcat cgagcgagca cgcactcgga tggaagccgg
     2581 tcttgtcgat caggatgatc tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt
     2641 cgccaggctc aaggcgcgta tgccggatgg tgaggatctc gtcgtgactc atggcgatgc
     2701 ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct ggattcatcg actgtggccg
     2761 gctgggtgtg gcggaccgct atcaggacat agcgttggct acccgtgata ttgctgaaga
     2821 gcttggcggc gaatgggctg accgcttcct cgtgctttac ggtatcgccg ctcccgattc
     2881 gcagcgcatc gccttctatc gccttcttga cgagttcttc tgagcgggac tctggggttc
     2941 gaaatgaccg accaagcgac gcccaaccgg tatcagctca ctcaaaggcg gtaatacggt
     3001 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     3061 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     3121 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     3181 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     3241 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     3301 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     3361 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     3421 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     3481 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag
     3541 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     3601 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     3661 cgcgcagaaa aaaaggattt caagaagatc ctttgatctt ttctacgggg tctgacgctc
     3721 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     3781 cctagatcct tttatagtcc ggaaatacag gaacgcacgc tggatggccc ttcgctggga
     3841 tggtgaaacc atgaaaaatg gcagcttcag tggattaagt gggggtaatg tggcctgtac
     3901 cctctggttg cataggtatt catacggtta aaatttatca ggcgcgattg cggcagtttt
     3961 tcgggtggtt tgttgccatt tttacctgtc tgctgccgtg atcgcgctga acgcgtttta
     4021 gcggtgcgta caattaaggg attatggtaa atccacttac tgtctgccct cgtagccatc
     4081 gagataaacc gcagtactcc ggccacgatg cgtccggcgt agaggatcga gatct
//
