LOCUS       40924_20296        4428 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Flexi(R) vector with an ampicillin 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 4428)
  AUTHORS   Promega
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 4428)
  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..4428
                     /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      1546..1593
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        1822..1926
                     /label=AmpR promoter
     CDS             1927..2784
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      complement(2945..3533)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     misc_feature    3649..3932
                     /label=cer region
                     /note="ColE1-derived recombination site that helps to
                     maintain plasmids as monomers"
     terminator      4162..4248
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     terminator      4340..4367
                     /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 aaaaactagc ataacccctt
     1561 ggggcctcta aacgggtctt gaggggtttt ttgctgaaag gaggaactat atccggttcg
     1621 cttgctgtcc ataaaaccgc ccagtctagc tatcgccatg taagcccact gcaagctacc
     1681 tgctttctct ttgcgcttgc gttttccctt gtccagatag cccagtagct gacattcatc
     1741 cggggtcagc accgtttctg cggactggct ttctacgtaa tggtttctta gacgtcaggt
     1801 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca
     1861 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg
     1921 aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc
     1981 cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg
     2041 ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt
     2101 cgccccgaag aacgttttcc aatgatgagc actttcaaag ttctgctatg tggcgcggta
     2161 ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat
     2221 gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga
     2281 gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca
     2341 actatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact
     2401 cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc
     2461 acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact
     2521 ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt
     2581 ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt
     2641 gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt
     2701 atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata
     2761 ggtgcctcac tgattaagca ttggtaattc gaaatgaccg accaagcgac gcccaaccgg
     2821 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
     2881 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     2941 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     3001 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
     3061 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     3121 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     3181 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     3241 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     3301 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     3361 ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag
     3421 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     3481 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggattt caagaagatc
     3541 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     3601 tggtcatgag attatcaaaa aggatcttca cctagatcct tttatagtcc ggaaatacag
     3661 gaacgcacgc tggatggccc ttcgctggga tggtgaaacc atgaaaaatg gcagcttcag
     3721 tggattaagt gggggtaatg tggcctgtac cctctggttg cataggtatt catacggtta
     3781 aaatttatca ggcgcgattg cggcagtttt tcgggtggtt tgttgccatt tttacctgtc
     3841 tgctgccgtg atcgcgctga acgcgtttta gcggtgcgta caattaaggg attatggtaa
     3901 atccacttac tgtctgccct cgtagccatc gagataaacc gcagtactcc ggccacgatg
     3961 cgtccggcgt agaggatcga gatcttttca gcctgataca gattaaatca gaacgcagaa
     4021 gcggtctgat aaaacagaat ttgcctggcg gcagtagcgc ggtggtccca cctgacccca
     4081 tgccgaactc agaagtgaaa cgccgtagcg ccgatggtag tgtggggtct ccccatgcga
     4141 gagtagggaa ctgccaggca tcaaataaaa cgaaaggctc agtcgaaaga ctgggccttt
     4201 cgttttatct gttgtttgtc ggtgaacgct ctcctgagta ggacaaatcc gccgggagcg
     4261 gatttgaacg ttgcgaagca acggcccgga gggtggcggg caggacgccc gccataaact
     4321 gccaggcatc aaattaagca gaaggccatc ctgacggatg gcctttttgc gtttctacaa
     4381 actcttttgt ttatttttct aaatacattc aaatatgtat ccgctcat
//
