LOCUS       40924_19821        4422 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 C-terminal HaloTag(R).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4422)
  AUTHORS   Promega
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 4422)
  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..4422
                     /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             102..434
                     /codon_start=1
                     /label=barnase
                     /note="ribonuclease from Bacillus amyloliquefaciens"
                     /translation="MAQVINTFDGVADYLQTYHKLPDNYITKSEAQALGWVASKGNLAD
                     VAPGKSIGGDIFSNREGKLPGKSGRTWREADINYTSGFRNSDRILYSSDWLIYKTTDHY
                     QTFTKIR"
     CDS             477..497
                     /codon_start=1
                     /label=TEV site
                     /note="tobacco etch virus (TEV) protease recognition and 
                     cleavage site"
                     /translation="EDLYFQS"
     CDS             510..1397
                     /codon_start=1
                     /label=HaloTag(R)
                     /note="modified bacterial dehalogenase that forms covalent
                     bonds with chloroalkane derivatives"
                     /translation="SEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSY
                     VWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIH
                     DWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLIID
                     QNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVE
                     EYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLI
                     GSEIARWLSTLEISG"
     terminator      1540..1587
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        1816..1920
                     /label=AmpR promoter
     CDS             1921..2778
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      complement(2939..3527)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     misc_feature    3643..3926
                     /label=cer region
                     /note="ColE1-derived recombination site that helps to
                     maintain plasmids as monomers"
     terminator      4156..4242
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     terminator      4334..4361
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB
                     gene"
ORIGIN
        1 ggatctcgat cccgcgaaat taatacgact cactataggg tacgatttag gtgacactat
       61 agaataagga gcgatcgcca tggaataagt aaggaatcca catggcacag gttatcaaca
      121 cgtttgacgg ggttgcggat tatcttcaga catatcataa gctacctgat aattacatta
      181 caaaatcaga agcacaagcc ctcggctggg tggcatcaaa agggaacctt gcagacgtcg
      241 ctccggggaa aagcatcggc ggagacatct tctcaaacag ggaaggcaaa ctcccgggca
      301 aaagcggacg aacatggcgt gaagcggata ttaactatac atcaggcttc agaaattcag
      361 accggattct ttactcaagc gactggctga tttacaaaac aacggaccat tatcagacct
      421 ttacaaaaat cagataatgt ttaatgaccc cgtgtcgagc tctcgagcca accactgagg
      481 atctgtactt tcagagcgat aacgatggat ccgaaatcgg tactggcttt ccattcgacc
      541 cccattatgt ggaagtcctg ggcgagcgca tgcactacgt cgatgttggt ccgcgcgatg
      601 gcacccctgt gctgttcctg cacggtaacc cgacctcctc ctacgtgtgg cgcaacatca
      661 tcccgcatgt tgcaccgacc catcgctgca ttgctccaga cctgatcggt atgggcaaat
      721 ccgacaaacc agacctgggt tatttcttcg acgaccacgt ccgcttcatg gatgccttca
      781 tcgaagccct gggtctggaa gaggtcgtcc tggtcattca cgactggggc tccgctctgg
      841 gtttccactg ggccaagcgc aatccagagc gcgtcaaagg tattgcattt atggagttca
      901 tccgccctat cccgacctgg gacgaatggc cagaatttgc ccgcgagacc ttccaggcct
      961 tccgcaccac cgacgtcggc cgcaagctga tcatcgatca gaacgttttt atcgagggta
     1021 cgctgccgat gggtgtcgtc cgcccgctga ctgaagtcga gatggaccat taccgcgagc
     1081 cgttcctgaa tcctgttgac cgcgagccac tgtggcgctt cccaaacgag ctgccaatcg
     1141 ccggtgagcc agcgaacatc gtcgcgctgg tcgaagaata catggactgg ctgcaccagt
     1201 cccctgtccc gaagctgctg ttctggggca ccccaggcgt tctgatccca ccggccgaag
     1261 ccgctcgcct ggccaaaagc ctgcctaact gcaaggctgt ggacatcggc ccgggtctga
     1321 atctgctgca agaagacaac ccggacctga tcggcagcga gatcgcgcgc tggctgtcta
     1381 ctctggagat ttccggttaa tagaattcta gagtcgacct gcaggcatgc aagctgatcc
     1441 ggctgctaac aaagcccgaa aggaagctga gttggctgct gccaccgctg agcaataact
     1501 agcataacca aaaaaaaaaa aaaaaaaaaa aaaaaaaaac tagcataacc ccttggggcc
     1561 tctaaacggg tcttgagggg ttttttgctg aaaggaggaa ctatatccgg ttcgcttgct
     1621 gtccataaaa ccgcccagtc tagctatcgc catgtaagcc cactgcaagc tacctgcttt
     1681 ctctttgcgc ttgcgttttc ccttgtccag atagcccagt agctgacatt catccggggt
     1741 cagcaccgtt tctgcggact ggctttctac gtaatggttt cttagacgtc aggtggcact
     1801 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
     1861 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt
     1921 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct
     1981 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca
     2041 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc
     2101 gaagaacgtt ttccaatgat gagcactttc aaagttctgc tatgtggcgc ggtattatcc
     2161 cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg
     2221 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta
     2281 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaactatc
     2341 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt
     2401 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg
     2461 cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct
     2521 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc
     2581 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct
     2641 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac
     2701 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc
     2761 tcactgatta agcattggta attcgaaatg accgaccaag cgacgcccaa ccggtatcag
     2821 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     2881 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     2941 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     3001 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     3061 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     3121 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     3181 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     3241 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     3301 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     3361 actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag ccagttacct
     3421 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     3481 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atttcaagaa gatcctttga
     3541 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     3601 tgagattatc aaaaaggatc ttcacctaga tccttttata gtccggaaat acaggaacgc
     3661 acgctggatg gcccttcgct gggatggtga aaccatgaaa aatggcagct tcagtggatt
     3721 aagtgggggt aatgtggcct gtaccctctg gttgcatagg tattcatacg gttaaaattt
     3781 atcaggcgcg attgcggcag tttttcgggt ggtttgttgc catttttacc tgtctgctgc
     3841 cgtgatcgcg ctgaacgcgt tttagcggtg cgtacaatta agggattatg gtaaatccac
     3901 ttactgtctg ccctcgtagc catcgagata aaccgcagta ctccggccac gatgcgtccg
     3961 gcgtagagga tcgagatctt ttcagcctga tacagattaa atcagaacgc agaagcggtc
     4021 tgataaaaca gaatttgcct ggcggcagta gcgcggtggt cccacctgac cccatgccga
     4081 actcagaagt gaaacgccgt agcgccgatg gtagtgtggg gtctccccat gcgagagtag
     4141 ggaactgcca ggcatcaaat aaaacgaaag gctcagtcga aagactgggc ctttcgtttt
     4201 atctgttgtt tgtcggtgaa cgctctcctg agtaggacaa atccgccggg agcggatttg
     4261 aacgttgcga agcaacggcc cggagggtgg cgggcaggac gcccgccata aactgccagg
     4321 catcaaatta agcagaaggc catcctgacg gatggccttt ttgcgtttct acaaactctt
     4381 ttgtttattt ttctaaatac attcaaatat gtatccgctc at
//
