LOCUS       40924_30206        6453 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Bacterial vector with an MBP-TVMV-10xHis-TEV leader for 
            high-throughput purification of recombinant proteins.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6453)
  AUTHORS   Donnelly MI, Zhou M, Millard CS, Clancy S, Stols L, Eschenfeldt WH, 
            Collart FR, Joachimiak A.
  TITLE     An expression vector tailored for large-scale, high-throughput 
            purification of recombinant proteins.
  JOURNAL   Protein Expr. Purif. 2006;47:446-54.
  PUBMED    16497515
REFERENCE   2  (bases 1 to 6453)
  AUTHORS   Midwest Center for Structural Genomics
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 6453)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Protein 
            Expr. Purif."; date: "2006"; volume: "47"; pages: "446-54"
COMMENT     SGRef: number: 2; type: "Journal Article"
COMMENT     For ligation-independent cloning (LIC), linearize with SspI and 
            treat with T4 DNA polymerase plus dGTP.
FEATURES             Location/Qualifiers
     source          1..6453
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(26..73)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(140..157)
                     /codon_start=1
                     /label=6xHis
                     /note="6xHis affinity tag"
                     /translation="HHHHHH"
     CDS             complement(223..243)
                     /codon_start=1
                     /label=TEV site
                     /note="tobacco etch virus (TEV) protease recognition and 
                     cleavage site"
                     /translation="ENLYFQS"
     CDS             complement(271..297)
                     /codon_start=1
                     /label=9xHis
                     /note="9xHis affinity tag"
                     /translation="HHHHHHHHH"
     CDS             complement(298..318)
                     /codon_start=1
                     /label=TVMV site
                     /note="tobacco vein mottling virus (TVMV) NIa protease 
                     recognition and cleavage site"
                     /translation="ETVRFQS"
     protein_bind    complement(321..345)
                     /label=attB1
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             complement(355..1455)
                     /codon_start=1
                     /label=MBP
                     /note="maltose binding protein from E. coli"
                     /translation="MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE
                     EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL
                     IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA
                     ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE
                     TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL
                     ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF
                     WYAVRTAVINAASGRQTVDEALKDAQT"
     RBS             complement(1463..1485)
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    complement(1500..1524)
                     /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)."
     promoter        complement(1525..1543)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        1856..1933
                     /label=lacI promoter
     CDS             1934..3013
                     /codon_start=1
                     /label=lacI
                     /note="lac repressor"
                     /translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     protein_bind    3029..3050
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             3825..4013
                     /codon_start=1
                     /label=rop
                     /note="Rop protein, which maintains plasmids at low copy
                     number"
                     /translation="VTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     misc_feature    4118..4260
                     /label=bom
                     /note="basis of mobility region from pBR322"
     rep_origin      complement(4446..5034)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(5208..6065)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(6066..6169)
                     /label=AmpR promoter
ORIGIN
        1 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa
       61 ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt
      121 tgttagcagc cggatctcag tggtggtggt ggtggtgctc gagtgcggcc gcaagcttgt
      181 cgacggagct cgaattcgga tccgttatcc acttccaata ttggattgga agtacaggtt
      241 ctcggtaccc agatccacgc cagaagaatg gtgatggtgg tgatgatgat ggtggtgaga
      301 ctggaaacgc acggtttccg agcctgcttt tttgtacaaa cttgtgatcg aattagtctg
      361 cgcgtctttc agggcttcat cgacagtctg acgaccgctg gcggcgttga tcaccgcagt
      421 acgcacggca taccagaaag cggacatctg cgggatgttc ggcatgattt cacctttctg
      481 ggcgttttcc atggtggcgg caatacgtgg atctttcgcc aactcttcct cgtaagactt
      541 cagcgctacg gcacccagcg gtttgtcttt attaaccgct tccagacctt catcagtcag
      601 cagatagttt tcgaggaact cttttgccag ctctttgttc ggactggcgg cgttaatacc
      661 tgcgctcagc acgccaacga acggtttgga tggttgaccc ttgaaggtcg gcagtaccgt
      721 tacaccataa ttcactttgc tggtgtcgat gttggaccat gcccacgggc cgttgatggt
      781 catcgctgtt tcgcctttat taaaggcagc ttctgcgatg gagtaatcgg tgtctgcatt
      841 catgtgtttg tttttaatca ggtcaaccag gaaggtcaga cccgctttcg cgccagcgtt
      901 atccacgccc acgtctttaa tgtcgtactt gccgttttca tacttgaacg cataaccccc
      961 gtcagcagca atcagcggcc aggtgaagta cggttcttgc aggttgaaca tcagcgcgct
     1021 cttacctttc gctttcagtt ctttatccag cgccgggatc tcttcccagg tttttggcgg
     1081 gttcggcagc agatctttgt tataaatcag cgataacgct tcaacagcga tcgggtaagc
     1141 aatcagcttg ccgttgtaac gtacggcatc ccaggtaaac ggatacagct tgtcctggaa
     1201 cgctttgtcc ggggtgattt cagccaacag gccagattga gcgtagccac caaagcggtc
     1261 gtgtgcccag aagataatgt cagggccatc gccagttgcc gcaacctgtg ggaatttctc
     1321 ttccagttta tccggatgct caacggtgac tttaattccg gtatctttct cgaatttctt
     1381 accgacttca gcgagaccgt tatagccttt atcgccgtta atccagatta ccagtttacc
     1441 ttcttcgatt ttcatatgta tatctccttc ttaaagttaa acaaaattat ttctagaggg
     1501 gaattgttat ccgctcacaa ttcccctata gtgagtcgta ttaatttcgc gggatcgaga
     1561 tcgatctcga tcctctacgc cggacgcatc gtggccggca tcaccggcgc cacaggtgcg
     1621 gttgctggcg cctatatcgc cgacatcacc gatggggaag atcgggctcg ccacttcggg
     1681 ctcatgagcg cttgtttcgg cgtgggtatg gtggcaggcc ccgtggccgg gggactgttg
     1741 ggcgccatct ccttgcatgc accattcctt gcggcggcgg tgctcaacgg cctcaaccta
     1801 ctactgggct gcttcctaat gcaggagtcg cataagggag agcgtcgaga tcccggacac
     1861 catcgaatgg cgcaaaacct ttcgcggtat ggcatgatag cgcccggaag agagtcaatt
     1921 cagggtggtg aatgtgaaac cagtaacgtt atacgatgtc gcagagtatg ccggtgtctc
     1981 ttatcagacc gtttcccgcg tggtgaacca ggccagccac gtttctgcga aaacgcggga
     2041 aaaagtggaa gcggcgatgg cggagctgaa ttacattccc aaccgcgtgg cacaacaact
     2101 ggcgggcaaa cagtcgttgc tgattggcgt tgccacctcc agtctggccc tgcacgcgcc
     2161 gtcgcaaatt gtcgcggcga ttaaatctcg cgccgatcaa ctgggtgcca gcgtggtggt
     2221 gtcgatggta gaacgaagcg gcgtcgaagc ctgtaaagcg gcggtgcaca atcttctcgc
     2281 gcaacgcgtc agtgggctga tcattaacta tccgctggat gaccaggatg ccattgctgt
     2341 ggaagctgcc tgcactaatg ttccggcgtt atttcttgat gtctctgacc agacacccat
     2401 caacagtatt attttctccc atgaagacgg tacgcgactg ggcgtggagc atctggtcgc
     2461 attgggtcac cagcaaatcg cgctgttagc gggcccatta agttctgtct cggcgcgtct
     2521 gcgtctggct ggctggcata aatatctcac tcgcaatcaa attcagccga tagcggaacg
     2581 ggaaggcgac tggagtgcca tgtccggttt tcaacaaacc atgcaaatgc tgaatgaggg
     2641 catcgttccc actgcgatgc tggttgccaa cgatcagatg gcgctgggcg caatgcgcgc
     2701 cattaccgag tccgggctgc gcgttggtgc ggatatctcg gtagtgggat acgacgatac
     2761 cgaagacagc tcatgttata tcccgccgtt aaccaccatc aaacaggatt ttcgcctgct
     2821 ggggcaaacc agcgtggacc gcttgctgca actctctcag ggccaggcgg tgaagggcaa
     2881 tcagctgttg cccgtctcac tggtgaaaag aaaaaccacc ctggcgccca atacgcaaac
     2941 cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact
     3001 ggaaagcggg cagtgagcgc aacgcaatta atgtaagtta gctcactcat taggcaccgg
     3061 gatctcgacc gatgcccttg agagccttca acccagtcag ctccttccgg tgggcgcggg
     3121 gcatgactat cgtcgccgca cttatgactg tcttctttat catgcaactc gtaggacagg
     3181 tgccggcagc gctctgggtc attttcggcg aggaccgctt tcgctggagc gcgacgatga
     3241 tcggcctgtc gcttgcggta ttcggaatct tgcacgccct cgctcaagcc ttcgtcactg
     3301 gtcccgccac caaacgtttc ggcgagaagc aggccattat cgccggcatg gcggccccac
     3361 gggtgcgcat gatcgtgctc ctgtcgttga ggacccggct aggctggcgg ggttgcctta
     3421 ctggttagca gaatgaatca ccgatacgcg agcgaacgtg aagcgactgc tgctgcaaaa
     3481 cgtctgcgac ctgagcaaca acatgaatgg tcttcggttt ccgtgtttcg taaagtctgg
     3541 aaacgcggaa gtcagcgccc tgcaccatta tgttccggat ctgcatcgca ggatgctgct
     3601 ggctaccctg tggaacacct acatctgtat taacgaagcg ctggcattga ccctgagtga
     3661 tttttctctg gtcccgccgc atccataccg ccagttgttt accctcacaa cgttccagta
     3721 accgggcatg ttcatcatca gtaacccgta tcgtgagcat cctctctcgt ttcatcggta
     3781 tcattacccc catgaacaga aatccccctt acacggaggc atcagtgacc aaacaggaaa
     3841 aaaccgccct taacatggcc cgctttatca gaagccagac attaacgctt ctggagaaac
     3901 tcaacgagct ggacgcggat gaacaggcag acatctgtga atcgcttcac gaccacgctg
     3961 atgagcttta ccgcagctgc ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca
     4021 tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc
     4081 gtcagggcgc gtcagcgggt gttggcgggt gtcggggcgc agccatgacc cagtcacgta
     4141 gcgatagcgg agtgtatact ggcttaacta tgcggcatca gagcagattg tactgagagt
     4201 gcaccatata tgcggtgtga aataccgcac agatgcgtaa ggagaaaata ccgcatcagg
     4261 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
     4321 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     4381 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     4441 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     4501 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     4561 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     4621 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     4681 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     4741 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     4801 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     4861 tggcctaact acggctacac tagaaggaca gtatttggta tctgcgctct gctgaagcca
     4921 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     4981 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     5041 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     5101 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     5161 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     5221 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     5281 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     5341 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     5401 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     5461 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     5521 gcaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     5581 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     5641 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     5701 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     5761 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     5821 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     5881 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     5941 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     6001 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     6061 ctcatactct tcctttttca aattattgaa gcatttatca gggttattgt ctcatgagcg
     6121 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
     6181 gaaaagtgcc acctaaattg taagcgttaa tgtgaaccat caccctaatc aagttttttg
     6241 gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg atttagagct
     6301 tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc
     6361 gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt
     6421 aatgcgccgc tacagggcgc gtcccattcg cca
//
