LOCUS       pET-58-DEST.        8395 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Gateway(R) Nova bacterial destination vector for expressing proteins
            tagged at the N-terminus with NusA and at the C-terminus with S-Tag.
ACCESSION   .
VERSION     .
KEYWORDS    pET-58-DEST
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8395)
  AUTHORS   Novagen (EMD Millipore)
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 8395)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..8395
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      12..467
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     CDS             complement(585..1442)
                     /label=AmpR
                     /note="beta-lactamase"
     promoter        complement(1443..1535)
                     /label=AmpR promoter
     rep_origin      1616..2204
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     misc_feature    complement(2390..2532)
                     /label=bom
                     /note="basis of mobility region from pBR322"
     CDS             complement(2637..2825)
                     /label=rop
                     /note="Rop protein, which maintains plasmids at low copy
                     number"
     protein_bind    complement(3363..3384)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             complement(3400..4479)
                     /label=lacI
                     /note="lac repressor"
     promoter        complement(4480..4557)
                     /label=lacI promoter
     promoter        4870..4888
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4889..4913
                     /label=lac operator
                     /note="The lac repressor binds to the lac operator to
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     RBS             4928..4950
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             4957..4959
                     /codon_start=1
                     /product="start codon"
                     /label=start codon
                     /note="ATG"
                     /translation="M"
     CDS             4969..4986
                     /label=6xHis
                     /note="6xHis affinity tag"
     CDS             5002..6483
                     /label=NusA
                     /note="transcription elongation protein (N utilization
                     substance protein A)"
     CDS             6493..6510
                     /label=thrombin site
                     /note="thrombin recognition and cleavage site"
     protein_bind    6514..6638
                     /label=attR1
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        6663..6693
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an 'up' mutation"
     CDS             6747..7403
                     /label=CmR
                     /note="chloramphenicol acetyltransferase"
     CDS             7748..8050
                     /label=ccdB
                     /note="CcdB, a bacterial toxin that poisons DNA gyrase"
     protein_bind    complement(8094..8218)
                     /label=attR2
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             8223..8267
                     /label=S-Tag
                     /note="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
     terminator      8323..8370
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
       61 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
      121 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
      181 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
      241 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
      301 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
      361 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
      421 acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttctg gcggcacgat
      481 ggcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
      541 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
      601 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
      661 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
      721 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
      781 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
      841 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
      901 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
      961 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     1021 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     1081 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     1141 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     1201 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     1261 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     1321 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     1381 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     1441 atactcttcc tttttcaatc atgattgaag catttatcag ggttattgtc tcatgagcgg
     1501 atacatattt gaatgtattt agaaaaataa acaaataggt catgaccaaa atcccttaac
     1561 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     1621 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     1681 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     1741 gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga
     1801 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     1861 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     1921 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     1981 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     2041 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     2101 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     2161 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     2221 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
     2281 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca
     2341 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt
     2401 attttctcct tacgcatctg tgcggtattt cacaccgcat atatggtgca ctctcagtac
     2461 aatctgctct gatgccgcat agttaagcca gtatacactc cgctatcgct acgtgactgg
     2521 gtcatggctg cgccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg
     2581 ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg
     2641 ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg
     2701 tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca gctcgttgag tttctccaga
     2761 agcgttaatg tctggcttct gataaagcgg gccatgttaa gggcggtttt ttcctgtttg
     2821 gtcactgatg cctccgtgta agggggattt ctgttcatgg gggtaatgat accgatgaaa
     2881 cgagagagga tgctcacgat acgggttact gatgatgaac atgcccggtt actggaacgt
     2941 tgtgagggta aacaactggc ggtatggatg cggcgggacc agagaaaaat cactcagggt
     3001 caatgccagc gcttcgttaa tacagatgta ggtgttccac agggtagcca gcagcatcct
     3061 gcgatgcaga tccggaacat aatggtgcag ggcgctgact tccgcgtttc cagactttac
     3121 gaaacacgga aaccgaagac cattcatgtt gttgctcagg tcgcagacgt tttgcagcag
     3181 cagtcgcttc acgttcgctc gcgtatcggt gattcattct gctaaccagt aaggcaaccc
     3241 cgccagccta gccgggtcct caacgacagg agcacgatca tgctagtcat gccccgcgcc
     3301 caccggaagg agctgactgg gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc
     3361 taatgagtga gctaacttac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     3421 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     3481 attgggcgcc agggtggttt ttcttttcac cagtgagacg ggcaacagct gattgccctt
     3541 caccgcctgg ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg
     3601 aaaatcctgt ttgatggtgg ttaacggcgg gatataacat gagctgtctt cggtatcgtc
     3661 gtatcccact accgagatgt ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat
     3721 tgcgcccagc gccatctgat cgttggcaac cagcatcgca gtgggaacga tgccctcatt
     3781 cagcatttgc atggtttgtt gaaaaccgga catggcactc cagtcgcctt cccgttccgc
     3841 tatcggctga atttgattgc gagtgagata tttatgccag ccagccagac gcagacgcgc
     3901 cgagacagaa cttaatgggc ccgctaacag cgcgatttgc tggtgaccca atgcgaccag
     3961 atgctccacg cccagtcgcg taccgtcttc atgggagaaa ataatactgt tgatgggtgt
     4021 ctggtcagag acatcaagaa ataacgccgg aacattagtg caggcagctt ccacagcaat
     4081 ggcatcctgg tcatccagcg gatagttaat gatcagccca ctgacgcgtt gcgcgagaag
     4141 attgtgcacc gccgctttac aggcttcgac gccgcttcgt tctaccatcg acaccaccac
     4201 gctggcaccc agttgatcgg cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg
     4261 cagggccaga ctggaggtgg caacgccaat cagcaacgac tgtttgcccg ccagttgttg
     4321 tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc gcttccactt tttcccgcgt
     4381 tttcgcagaa acgtggctgg cctggttcac cacgcgggaa acggtctgat aagagacacc
     4441 ggcatactct gcgacatcgt ataacgttac tggtttcaca ttcaccaccc tgaattgact
     4501 ctcttccggg cgctatcatg ccataccgcg aaaggttttg cgccattcga tggtgtccgg
     4561 gatctcgacg ctctccctta tgcgactcct gcattaggaa gcagcccagt agtaggttga
     4621 ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc
     4681 ccccggccac ggggcctgcc accataccca cgccgaaaca agcgctcatg agcccgaagt
     4741 ggcgagcccg atcttcccca tcggtgatgt cggcgatata ggcgccagca accgcacctg
     4801 tggcgccggt gatgccggcc acgatgcgtc cggcgtagag gatcgagatc gatctcgatc
     4861 ccgcgaaatt aatacgactc actatagggg aattgtgagc ggataacaat tcccctctag
     4921 aaataatttt gtttaacttt aagaaggaga tataccatgg gcagcagcca tcatcatcat
     4981 catcacagcc atatggctag caacaaagaa attttggctg tagttgaagc cgtatccaat
     5041 gaaaaggcgc tacctcgcga gaagattttc gaagcattgg aaagcgcgct ggcgacagca
     5101 acaaagaaaa aatatgaaca agagatcgac gtccgcgtac agatcgatcg caaaagcggt
     5161 gattttgaca ctttccgtcg ctggttagtt gttgatgaag tcacccagcc gaccaaggaa
     5221 atcacccttg aagccgcacg ttatgaagat gaaagcctga acctgggcga ttacgttgaa
     5281 gatcagattg agtctgttac ctttgaccgt atcactaccc agacggcaaa acaggttatc
     5341 gtgcagaaag tgcgtgaagc cgaacgtgcg atggtggttg atcagttccg tgaacacgaa
     5401 ggtgaaatca tcaccggcgt ggtgaaaaaa gtaaaccgcg acaacatctc tctggatctg
     5461 ggcaacaacg ctgaagccgt gatcctgcgc gaagatatgc tgccgcgtga aaacttccgc
     5521 cctggcgacc gcgttcgtgg cgtgctctat tccgttcgcc cggaagcgcg tggcgcgcaa
     5581 ctgttcgtca ctcgttccaa gccggaaatg ctgatcgaac tgttccgtat tgaagtgcca
     5641 gaaatcggcg aagaagtgat tgaaattaaa gcagcggctc gcgatccggg ttctcgtgcg
     5701 aaaatcgcgg tgaaaaccaa cgataaacgt atcgatccgg taggtgcttg cgtaggtatg
     5761 cgtggcgcgc gtgttcaggc ggtgtctact gaactgggtg gcgagcgtat cgatatcgtc
     5821 ctgtgggatg ataacccggc gcagttcgtg attaacgcaa tggcaccggc agacgttgct
     5881 tctatcgtgg tggatgaaga taaacacacc atggacatcg ccgttgaagc cggtaatctg
     5941 gcgcaggcga ttggccgtaa cggtcagaac gtgcgtctgg cttcgcaact gagcggttgg
     6001 gaactcaacg tgatgaccgt tgacgacctg caagctaagc atcaggcgga agcgcacgca
     6061 gcgatcgaca ccttcaccaa atatctcgac atcgacgaag acttcgcgac tgttctggta
     6121 gaagaaggct tctcgacgct ggaagaattg gcctatgtgc cgatgaaaga gctgttggaa
     6181 atcgaaggcc ttgatgagcc gaccgttgaa gcactgcgcg agcgtgctaa aaatgcactg
     6241 gccaccattg cacaggccca ggaagaaagc ctcggtgata acaaaccggc tgacgatctg
     6301 ctgaaccttg aaggggtaga tcgtgatttg gcattcaaac tggccgcccg tggcgtttgt
     6361 acgctggaag atctcgccga acagggcatt gatgatctgg ctgatatcga agggttgacc
     6421 gacgaaaaag ccggagcact gattatggct gcccgtaata tttgctggtt cggtgacgaa
     6481 gcgtctggag ctcttgttcc acgtggttct gtcacaagtt tgtacaaaaa agctgaacga
     6541 gaaacgtaaa atgatataaa tatcaatata ttaaattaga ttttgcataa aaaacagact
     6601 acataatact gtaaaacaca acatatccag tcactatggc ggccgcatta ggcaccccag
     6661 gctttacact ttatgcttcc ggctcgtata atgtgtggat tttgagttag gatccgtcga
     6721 gattttcagg agctaaggaa gctaaaatgg agaaaaaaat cactggatat accaccgttg
     6781 atatatccca atggcatcgt aaagaacatt ttgaggcatt tcagtcagtt gctcaatgta
     6841 cctataacca gaccgttcag ctggatatta cggccttttt aaagaccgta aagaaaaata
     6901 agcacaagtt ttatccggcc tttattcaca ttcttgcccg cctgatgaat gctcatccgg
     6961 aattccgtat ggcaatgaaa gacggtgagc tggtgatatg ggatagtgtt cacccttgtt
     7021 acaccgtttt ccatgagcaa actgaaacgt tttcatcgct ctggagtgaa taccacgacg
     7081 atttccggca gtttctacac atatattcgc aagatgtggc gtgttacggt gaaaacctgg
     7141 cctatttccc taaagggttt attgagaata tgtttttcgt ctcagccaat ccctgggtga
     7201 gtttcaccag ttttgattta aacgtggcca atatggacaa cttcttcgcc cccgttttca
     7261 ctatgggcaa atattatacg caaggcgaca aggtgctgat gccgctggcg attcaggttc
     7321 atcatgccgt ttgtgatggc ttccatgtcg gcagaatgct taatgaatta caacagtact
     7381 gcgatgagtg gcagggcggg gcgtaaacgc gtggatccgg cttactaaaa gccagataac
     7441 agtatgcgta tttgcgcgct gatttttgcg gtataagaat atatactgat atgtataccc
     7501 gaagtatgtc aaaaagaggt atgctatgaa gcagcgtatt acagtgacag ttgacagcga
     7561 cagctatcag ttgctcaagg catatatgat gtcaatatct ccggtctggt aagcacaacc
     7621 atgcagaatg aagcccgtcg tctgcgtgcc gaacgctgga aagcggaaaa tcaggaaggg
     7681 atggctgagg tcgcccggtt tattgaaatg aacggctctt ttgctgacga gaacaggggc
     7741 tggtgaaatg cagtttaagg tttacaccta taaaagagag agccgttatc gtctgtttgt
     7801 ggatgtacag agtgatatta ttgacacgcc cgggcgacgg atggtgatcc ccctggccag
     7861 tgcacgtctg ctgtcagata aagtctcccg tgaactttac ccggtggtgc atatcgggga
     7921 tgaaagctgg cgcatgatga ccaccgatat ggccagtgtg ccggtttccg ttatcgggga
     7981 agaagtggct gatctcagcc accgcgaaaa tgacatcaaa aacgccatta acctgatgtt
     8041 ctggggaata taaatgtcag gctcccttat acacagccag tctgcaggtc gaccatagtg
     8101 actggatatg ttgtgtttta cagtattatg tagtctgttt tttatgcaaa atctaattta
     8161 atatattgat atttatatca ttttacgttt ctcgttcagc tttcttgtac aaagtggttg
     8221 gtaaagaaac cgctgctgcg aaatttgaac gccagcacat ggactcgtct actagcgcag
     8281 cttaagtgat taacctaggc tgctgccacc gctgagcaat aactagcata accccttggg
     8341 gcctctaaac gggtcttgag gggttttttg ctgaaaggag gaactatatc cggat
//
