LOCUS       40924_20286        4377 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Flexi(R) vector with an ampicillin resistance marker, for bacterial 
            or 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 4377)
  AUTHORS   Promega
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 4377)
  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..4377
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        21..39
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             70..960
                     /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             973..993
                     /codon_start=1
                     /label=TEV site
                     /note="tobacco etch virus (TEV) protease recognition and 
                     cleavage site"
                     /translation="EDLYFQS"
     CDS             1031..1363
                     /codon_start=1
                     /label=barnase
                     /note="ribonuclease from Bacillus amyloliquefaciens"
                     /translation="MAQVINTFDGVADYLQTYHKLPDNYITKSEAQALGWVASKGNLAD
                     VAPGKSIGGDIFSNREGKLPGKSGRTWREADINYTSGFRNSDRILYSSDWLIYKTTDHY
                     QTFTKIR"
     misc_feature    1376..1431
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     terminator      1495..1542
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        1771..1875
                     /label=AmpR promoter
     CDS             1876..2733
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      complement(2894..3482)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     misc_feature    3598..3881
                     /label=cer region
                     /note="ColE1-derived recombination site that helps to
                     maintain plasmids as monomers"
     terminator      4111..4197
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     terminator      4289..4316
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB
                     gene"
ORIGIN
        1 ggatctcgat cccgcgaaat taatacgact cactataggg tattttaact ttactaagga
       61 gaattcatca tggcagaaat cggtactggc tttccattcg acccccatta tgtggaagtc
      121 ctgggcgagc gcatgcacta cgtcgatgtt ggtccgcgcg atggcacccc tgtgctgttc
      181 ctgcacggta acccgacctc ctcctacgtg tggcgcaaca tcatcccgca tgttgcaccg
      241 acccatcgct gcattgctcc agacctgatc ggtatgggca aatccgacaa accagacctg
      301 ggttatttct tcgacgacca cgtccgcttc atggatgcct tcatcgaagc cctgggtctg
      361 gaagaggtcg tcctggtcat tcacgactgg ggctccgctc tgggtttcca ctgggccaag
      421 cgcaatccag agcgcgtcaa aggtattgca tttatggagt tcatccgccc tatcccgacc
      481 tgggacgaat ggccagaatt tgcccgcgag accttccagg ccttccgcac caccgacgtc
      541 ggccgcaagc tgatcatcga tcagaacgtt tttatcgagg gtacgctgcc gatgggtgtc
      601 gtccgcccgc tgactgaagt cgagatggac cattaccgcg agccgttcct gaatcctgtt
      661 gaccgcgagc cactgtggcg cttcccaaac gagctgccaa tcgccggtga gccagcgaac
      721 atcgtcgcgc tggtcgaaga atacatggac tggctgcacc agtcccctgt cccgaagctg
      781 ctgttctggg gcaccccagg cgttctgatc ccaccggccg aagccgctcg cctggccaaa
      841 agcctgccta actgcaaggc tgtggacatc ggcccgggtc tgaatctgct gcaagaagac
      901 aacccggacc tgatcggcag cgagatcgcg cgctggctgt cgacgctcga gatttccggc
      961 gagccaacca ctgaggatct gtactttcag agcgataacg cgatcgccat ggaataagta
     1021 aggaatccac atggcacagg ttatcaacac gtttgacggg gttgcggatt atcttcagac
     1081 atatcataag ctacctgata attacattac aaaatcagaa gcacaagccc tcggctgggt
     1141 ggcatcaaaa gggaaccttg cagacgtcgc tccggggaaa agcatcggcg gagacatctt
     1201 ctcaaacagg gaaggcaaac tcccgggcaa aagcggacga acatggcgtg aagcggatat
     1261 taactataca tcaggcttca gaaattcaga ccggattctt tactcaagcg actggctgat
     1321 ttacaaaaca acggaccatt atcagacctt tacaaaaatc agataatgtt taaacgaatt
     1381 cgggctcggt acccggggat cctctagagt cgacctgcag gcatgcaagc tgatccggct
     1441 gctaacaaag cccgaaagga agctgagttg gctgctgcca ccgctgagca ataactagca
     1501 taaccccttg gggcctctaa acgggtcttg aggggttttt tgctgaaagg aggaactata
     1561 tccggttcgc ttgctgtcca taaaaccgcc cagtctagct atcgccatgt aagcccactg
     1621 caagctacct gctttctctt tgcgcttgcg ttttcccttg tccagatagc ccagtagctg
     1681 acattcatcc ggggtcagca ccgtttctgc ggactggctt tctacgtaat ggtttcttag
     1741 acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
     1801 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat
     1861 tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg
     1921 gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa
     1981 gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt
     2041 gagagttttc gccccgaaga acgttttcca atgatgagca ctttcaaagt tctgctatgt
     2101 ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat
     2161 tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg
     2221 acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta
     2281 cttctgacaa ctatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat
     2341 catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag
     2401 cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa
     2461 ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca
     2521 ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc
     2581 ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt
     2641 atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc
     2701 gctgagatag gtgcctcact gattaagcat tggtaattcg aaatgaccga ccaagcgacg
     2761 cccaaccggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
     2821 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     2881 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     2941 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     3001 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     3061 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     3121 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     3181 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     3241 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     3301 tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc
     3361 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     3421 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatttc
     3481 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     3541 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttatagtccg
     3601 gaaatacagg aacgcacgct ggatggccct tcgctgggat ggtgaaacca tgaaaaatgg
     3661 cagcttcagt ggattaagtg ggggtaatgt ggcctgtacc ctctggttgc ataggtattc
     3721 atacggttaa aatttatcag gcgcgattgc ggcagttttt cgggtggttt gttgccattt
     3781 ttacctgtct gctgccgtga tcgcgctgaa cgcgttttag cggtgcgtac aattaaggga
     3841 ttatggtaaa tccacttact gtctgccctc gtagccatcg agataaaccg cagtactccg
     3901 gccacgatgc gtccggcgta gaggatcgag atcttttcag cctgatacag attaaatcag
     3961 aacgcagaag cggtctgata aaacagaatt tgcctggcgg cagtagcgcg gtggtcccac
     4021 ctgaccccat gccgaactca gaagtgaaac gccgtagcgc cgatggtagt gtggggtctc
     4081 cccatgcgag agtagggaac tgccaggcat caaataaaac gaaaggctca gtcgaaagac
     4141 tgggcctttc gttttatctg ttgtttgtcg gtgaacgctc tcctgagtag gacaaatccg
     4201 ccgggagcgg atttgaacgt tgcgaagcaa cggcccggag ggtggcgggc aggacgcccg
     4261 ccataaactg ccaggcatca aattaagcag aaggccatcc tgacggatgg cctttttgcg
     4321 tttctacaaa ctcttttgtt tatttttcta aatacattca aatatgtatc cgctcat
//
