LOCUS       40924_30086        6441 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Bacterial vector with an MBP-TVMV-6xHis-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 6441)
  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 6441)
  AUTHORS   Midwest Center for Structural Genomics
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 6441)
  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..6441
                     /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(268..285)
                     /codon_start=1
                     /label=6xHis
                     /note="6xHis affinity tag"
                     /translation="HHHHHH"
     CDS             complement(286..306)
                     /codon_start=1
                     /label=TVMV site
                     /note="tobacco vein mottling virus (TVMV) NIa protease 
                     recognition and cleavage site"
                     /translation="ETVRFQS"
     protein_bind    complement(309..333)
                     /label=attB1
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             complement(343..1443)
                     /codon_start=1
                     /label=MBP
                     /note="maltose binding protein from E. coli"
                     /translation="MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE
                     EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL
                     IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA
                     ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE
                     TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL
                     ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF
                     WYAVRTAVINAASGRQTVDEALKDAQT"
     RBS             complement(1451..1473)
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    complement(1488..1512)
                     /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(1513..1531)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        1844..1921
                     /label=lacI promoter
     CDS             1922..3001
                     /codon_start=1
                     /label=lacI
                     /note="lac repressor"
                     /translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     protein_bind    3017..3038
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             3813..4001
                     /codon_start=1
                     /label=rop
                     /note="Rop protein, which maintains plasmids at low copy
                     number"
                     /translation="VTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     misc_feature    4106..4248
                     /label=bom
                     /note="basis of mobility region from pBR322"
     rep_origin      complement(4434..5022)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(5196..6053)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(6054..6157)
                     /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 cagaagagtg atgatgatgg tggtgagact ggaaacgcac
      301 ggtttccgag cctgcttttt tgtacaaact tgtgatcgaa ttagtctgcg cgtctttcag
      361 ggcttcatcg acagtctgac gaccgctggc ggcgttgatc accgcagtac gcacggcata
      421 ccagaaagcg gacatctgcg ggatgttcgg catgatttca cctttctggg cgttttccat
      481 ggtggcggca atacgtggat ctttcgccaa ctcttcctcg taagacttca gcgctacggc
      541 acccagcggt ttgtctttat taaccgcttc cagaccttca tcagtcagca gatagttttc
      601 gaggaactct tttgccagct ctttgttcgg actggcggcg ttaatacctg cgctcagcac
      661 gccaacgaac ggtttggatg gttgaccctt gaaggtcggc agtaccgtta caccataatt
      721 cactttgctg gtgtcgatgt tggaccatgc ccacgggccg ttgatggtca tcgctgtttc
      781 gcctttatta aaggcagctt ctgcgatgga gtaatcggtg tctgcattca tgtgtttgtt
      841 tttaatcagg tcaaccagga aggtcagacc cgctttcgcg ccagcgttat ccacgcccac
      901 gtctttaatg tcgtacttgc cgttttcata cttgaacgca taacccccgt cagcagcaat
      961 cagcggccag gtgaagtacg gttcttgcag gttgaacatc agcgcgctct tacctttcgc
     1021 tttcagttct ttatccagcg ccgggatctc ttcccaggtt tttggcgggt tcggcagcag
     1081 atctttgtta taaatcagcg ataacgcttc aacagcgatc gggtaagcaa tcagcttgcc
     1141 gttgtaacgt acggcatccc aggtaaacgg atacagcttg tcctggaacg ctttgtccgg
     1201 ggtgatttca gccaacaggc cagattgagc gtagccacca aagcggtcgt gtgcccagaa
     1261 gataatgtca gggccatcgc cagttgccgc aacctgtggg aatttctctt ccagtttatc
     1321 cggatgctca acggtgactt taattccggt atctttctcg aatttcttac cgacttcagc
     1381 gagaccgtta tagcctttat cgccgttaat ccagattacc agtttacctt cttcgatttt
     1441 catatgtata tctccttctt aaagttaaac aaaattattt ctagagggga attgttatcc
     1501 gctcacaatt cccctatagt gagtcgtatt aatttcgcgg gatcgagatc gatctcgatc
     1561 ctctacgccg gacgcatcgt ggccggcatc accggcgcca caggtgcggt tgctggcgcc
     1621 tatatcgccg acatcaccga tggggaagat cgggctcgcc acttcgggct catgagcgct
     1681 tgtttcggcg tgggtatggt ggcaggcccc gtggccgggg gactgttggg cgccatctcc
     1741 ttgcatgcac cattccttgc ggcggcggtg ctcaacggcc tcaacctact actgggctgc
     1801 ttcctaatgc aggagtcgca taagggagag cgtcgagatc ccggacacca tcgaatggcg
     1861 caaaaccttt cgcggtatgg catgatagcg cccggaagag agtcaattca gggtggtgaa
     1921 tgtgaaacca gtaacgttat acgatgtcgc agagtatgcc ggtgtctctt atcagaccgt
     1981 ttcccgcgtg gtgaaccagg ccagccacgt ttctgcgaaa acgcgggaaa aagtggaagc
     2041 ggcgatggcg gagctgaatt acattcccaa ccgcgtggca caacaactgg cgggcaaaca
     2101 gtcgttgctg attggcgttg ccacctccag tctggccctg cacgcgccgt cgcaaattgt
     2161 cgcggcgatt aaatctcgcg ccgatcaact gggtgccagc gtggtggtgt cgatggtaga
     2221 acgaagcggc gtcgaagcct gtaaagcggc ggtgcacaat cttctcgcgc aacgcgtcag
     2281 tgggctgatc attaactatc cgctggatga ccaggatgcc attgctgtgg aagctgcctg
     2341 cactaatgtt ccggcgttat ttcttgatgt ctctgaccag acacccatca acagtattat
     2401 tttctcccat gaagacggta cgcgactggg cgtggagcat ctggtcgcat tgggtcacca
     2461 gcaaatcgcg ctgttagcgg gcccattaag ttctgtctcg gcgcgtctgc gtctggctgg
     2521 ctggcataaa tatctcactc gcaatcaaat tcagccgata gcggaacggg aaggcgactg
     2581 gagtgccatg tccggttttc aacaaaccat gcaaatgctg aatgagggca tcgttcccac
     2641 tgcgatgctg gttgccaacg atcagatggc gctgggcgca atgcgcgcca ttaccgagtc
     2701 cgggctgcgc gttggtgcgg atatctcggt agtgggatac gacgataccg aagacagctc
     2761 atgttatatc ccgccgttaa ccaccatcaa acaggatttt cgcctgctgg ggcaaaccag
     2821 cgtggaccgc ttgctgcaac tctctcaggg ccaggcggtg aagggcaatc agctgttgcc
     2881 cgtctcactg gtgaaaagaa aaaccaccct ggcgcccaat acgcaaaccg cctctccccg
     2941 cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca
     3001 gtgagcgcaa cgcaattaat gtaagttagc tcactcatta ggcaccggga tctcgaccga
     3061 tgcccttgag agccttcaac ccagtcagct ccttccggtg ggcgcggggc atgactatcg
     3121 tcgccgcact tatgactgtc ttctttatca tgcaactcgt aggacaggtg ccggcagcgc
     3181 tctgggtcat tttcggcgag gaccgctttc gctggagcgc gacgatgatc ggcctgtcgc
     3241 ttgcggtatt cggaatcttg cacgccctcg ctcaagcctt cgtcactggt cccgccacca
     3301 aacgtttcgg cgagaagcag gccattatcg ccggcatggc ggccccacgg gtgcgcatga
     3361 tcgtgctcct gtcgttgagg acccggctag gctggcgggg ttgccttact ggttagcaga
     3421 atgaatcacc gatacgcgag cgaacgtgaa gcgactgctg ctgcaaaacg tctgcgacct
     3481 gagcaacaac atgaatggtc ttcggtttcc gtgtttcgta aagtctggaa acgcggaagt
     3541 cagcgccctg caccattatg ttccggatct gcatcgcagg atgctgctgg ctaccctgtg
     3601 gaacacctac atctgtatta acgaagcgct ggcattgacc ctgagtgatt tttctctggt
     3661 cccgccgcat ccataccgcc agttgtttac cctcacaacg ttccagtaac cgggcatgtt
     3721 catcatcagt aacccgtatc gtgagcatcc tctctcgttt catcggtatc attaccccca
     3781 tgaacagaaa tcccccttac acggaggcat cagtgaccaa acaggaaaaa accgccctta
     3841 acatggcccg ctttatcaga agccagacat taacgcttct ggagaaactc aacgagctgg
     3901 acgcggatga acaggcagac atctgtgaat cgcttcacga ccacgctgat gagctttacc
     3961 gcagctgcct cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg
     4021 agacggtcac agcttgtctg taagcggatg ccgggagcag acaagcccgt cagggcgcgt
     4081 cagcgggtgt tggcgggtgt cggggcgcag ccatgaccca gtcacgtagc gatagcggag
     4141 tgtatactgg cttaactatg cggcatcaga gcagattgta ctgagagtgc accatatatg
     4201 cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc gcatcaggcg ctcttccgct
     4261 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
     4321 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     4381 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     4441 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     4501 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     4561 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     4621 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     4681 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     4741 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     4801 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     4861 ggctacacta gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     4921 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     4981 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     5041 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     5101 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     5161 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     5221 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     5281 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
     5341 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     5401 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     5461 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctgc aggcatcgtg
     5521 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     5581 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     5641 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     5701 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     5761 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
     5821 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     5881 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     5941 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     6001 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     6061 ctttttcaaa ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     6121 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
     6181 ctaaattgta agcgttaatg tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc
     6241 cgtaaagcac taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag
     6301 ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg
     6361 gcaagtgtag cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta
     6421 cagggcgcgt cccattcgcc a
//
