LOCUS       40924_30091        7468 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Bacterial expression vector with an MBP-TVMV-6xHis-TEV leader and 
            inducible TVMV protease. See also pMCSG19C.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7468)
  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 7468)
  AUTHORS   Midwest Center for Structural Genomics
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 7468)
  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..7468
                     /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"
     terminator      complement(1762..1848)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     CDS             complement(1884..2591)
                     /codon_start=1
                     /label=TVMV protease
                     /note="tobacco vein mottling virus NIa protease
                     (Nallamsetty et al., 2004)"
                     /translation="MSKALLKGVRDFNPISACVCLLENSSDGHSERLFGIGFGPYIIAN
                     QHLFRRNNGELTIKTMHGEFKVKNSTQLQMKPVEGRDIIVIKMAKDFPPFPQKLKFRQP
                     TIKDRVCMVSTNFQQKSVSSLVSESSHIVHKEDTSFWQHWITTKDGQCGSPLVSIIDGN
                     ILGIHSLTHTTNGSNYFVEFPEKFVATYLDAADGWCKNWKFNADKISWGSFTLVEDAPE
                     DDFMAKKTVAAIMD"
     promoter        complement(2615..2688)
                     /label=PLtetO-1 promoter
                     /note="modified phage lambda PL promoter with tet operator
                     sites (Lutz and Bujard, 1997)"
     promoter        2871..2948
                     /label=lacI promoter
     CDS             2949..4028
                     /codon_start=1
                     /label=lacI
                     /note="lac repressor"
                     /translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     protein_bind    4044..4065
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             4840..5028
                     /codon_start=1
                     /label=rop
                     /note="Rop protein, which maintains plasmids at low copy
                     number"
                     /translation="VTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     misc_feature    5133..5275
                     /label=bom
                     /note="basis of mobility region from pBR322"
     rep_origin      complement(5461..6049)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(6223..7080)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(7081..7184)
                     /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 ttgcatgctc tagggcggcg gatttgtcct actcaggaga gcgttcaccg acaaacaaca
     1801 gataaaacga aaggcccagt ctttcgactg agcctttcgt tttatttgat gcctctagca
     1861 cgcgtaccat gggatcttta ttagtccatg atggcggcaa cagttttttt ggccatgaag
     1921 tcatcttccg gcgcatcttc aaccagggta aaggaacccc agctgatttt atccgcgttg
     1981 aatttccagt ttttgcacca accatccgcg gcatctagat aagtcgccac gaatttttcc
     2041 ggaaattcca cgaagtagtt gctaccgttg gtggtatgag tcaggctgtg gatgcccaga
     2101 atgttgccat caatgatgga aactagtggg ctgccacact ggccatcttt agtggtgatc
     2161 cagtgctgcc agaaagaagt gtcttcttta tgcacaatgt gagaggattc agacaccagg
     2221 ctcgagacgc ttttctgctg aaagttggtg gacaccatgc acacacgatc tttgatggtc
     2281 ggctgacgga atttcagttt ctgcgggaac ggcgggaagt ctttagccat tttgataacg
     2341 ataatgtcac ggccttcaac cggtttcatc tgcagctggg tagagttttt gactttgaat
     2401 tcaccatgca tggttttgat ggtcagttcg ccattgttac gacgaaacag atgctggttg
     2461 gcaatgatat acgggccaaa accaatgcca aacagacgtt cactatgacc atccgaggag
     2521 ttttccagca ggcatacgca agcagagatc ggattaaaat cgcgcacgcc cttcagcaaa
     2581 gctttagaca tatgttacct ccttaaatca attcggtcag tgcgtcctgc tgatgtgctc
     2641 agtatctcta tcactgatag ggatgtcaat ctctatcact gatagggact cgaggtgaag
     2701 acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc
     2761 ttagacgtcg catgcaccat tccttgcggc ggcggtgctc aacggcctca acctactact
     2821 gggctgcttc ctaatgcagg agtcgcataa gggagagcgt cgagatcccg gacaccatcg
     2881 aatggcgcaa aacctttcgc ggtatggcat gatagcgccc ggaagagagt caattcaggg
     2941 tggtgaatgt gaaaccagta acgttatacg atgtcgcaga gtatgccggt gtctcttatc
     3001 agaccgtttc ccgcgtggtg aaccaggcca gccacgtttc tgcgaaaacg cgggaaaaag
     3061 tggaagcggc gatggcggag ctgaattaca ttcccaaccg cgtggcacaa caactggcgg
     3121 gcaaacagtc gttgctgatt ggcgttgcca cctccagtct ggccctgcac gcgccgtcgc
     3181 aaattgtcgc ggcgattaaa tctcgcgccg atcaactggg tgccagcgtg gtggtgtcga
     3241 tggtagaacg aagcggcgtc gaagcctgta aagcggcggt gcacaatctt ctcgcgcaac
     3301 gcgtcagtgg gctgatcatt aactatccgc tggatgacca ggatgccatt gctgtggaag
     3361 ctgcctgcac taatgttccg gcgttatttc ttgatgtctc tgaccagaca cccatcaaca
     3421 gtattatttt ctcccatgaa gacggtacgc gactgggcgt ggagcatctg gtcgcattgg
     3481 gtcaccagca aatcgcgctg ttagcgggcc cattaagttc tgtctcggcg cgtctgcgtc
     3541 tggctggctg gcataaatat ctcactcgca atcaaattca gccgatagcg gaacgggaag
     3601 gcgactggag tgccatgtcc ggttttcaac aaaccatgca aatgctgaat gagggcatcg
     3661 ttcccactgc gatgctggtt gccaacgatc agatggcgct gggcgcaatg cgcgccatta
     3721 ccgagtccgg gctgcgcgtt ggtgcggata tctcggtagt gggatacgac gataccgaag
     3781 acagctcatg ttatatcccg ccgttaacca ccatcaaaca ggattttcgc ctgctggggc
     3841 aaaccagcgt ggaccgcttg ctgcaactct ctcagggcca ggcggtgaag ggcaatcagc
     3901 tgttgcccgt ctcactggtg aaaagaaaaa ccaccctggc gcccaatacg caaaccgcct
     3961 ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa
     4021 gcgggcagtg agcgcaacgc aattaatgta agttagctca ctcattaggc accgggatct
     4081 cgaccgatgc ccttgagagc cttcaaccca gtcagctcct tccggtgggc gcggggcatg
     4141 actatcgtcg ccgcacttat gactgtcttc tttatcatgc aactcgtagg acaggtgccg
     4201 gcagcgctct gggtcatttt cggcgaggac cgctttcgct ggagcgcgac gatgatcggc
     4261 ctgtcgcttg cggtattcgg aatcttgcac gccctcgctc aagccttcgt cactggtccc
     4321 gccaccaaac gtttcggcga gaagcaggcc attatcgccg gcatggcggc cccacgggtg
     4381 cgcatgatcg tgctcctgtc gttgaggacc cggctaggct ggcggggttg ccttactggt
     4441 tagcagaatg aatcaccgat acgcgagcga acgtgaagcg actgctgctg caaaacgtct
     4501 gcgacctgag caacaacatg aatggtcttc ggtttccgtg tttcgtaaag tctggaaacg
     4561 cggaagtcag cgccctgcac cattatgttc cggatctgca tcgcaggatg ctgctggcta
     4621 ccctgtggaa cacctacatc tgtattaacg aagcgctggc attgaccctg agtgattttt
     4681 ctctggtccc gccgcatcca taccgccagt tgtttaccct cacaacgttc cagtaaccgg
     4741 gcatgttcat catcagtaac ccgtatcgtg agcatcctct ctcgtttcat cggtatcatt
     4801 acccccatga acagaaatcc cccttacacg gaggcatcag tgaccaaaca ggaaaaaacc
     4861 gcccttaaca tggcccgctt tatcagaagc cagacattaa cgcttctgga gaaactcaac
     4921 gagctggacg cggatgaaca ggcagacatc tgtgaatcgc ttcacgacca cgctgatgag
     4981 ctttaccgca gctgcctcgc gcgtttcggt gatgacggtg aaaacctctg acacatgcag
     5041 ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag
     5101 ggcgcgtcag cgggtgttgg cgggtgtcgg ggcgcagcca tgacccagtc acgtagcgat
     5161 agcggagtgt atactggctt aactatgcgg catcagagca gattgtactg agagtgcacc
     5221 atatatgcgg tgtgaaatac cgcacagatg cgtaaggaga aaataccgca tcaggcgctc
     5281 ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc
     5341 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
     5401 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
     5461 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
     5521 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
     5581 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
     5641 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     5701 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
     5761 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
     5821 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
     5881 taactacggc tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac
     5941 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
     6001 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     6061 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
     6121 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
     6181 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
     6241 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
     6301 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
     6361 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
     6421 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
     6481 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctgcagg
     6541 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
     6601 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
     6661 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
     6721 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
     6781 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg
     6841 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
     6901 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
     6961 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
     7021 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
     7081 actcttcctt tttcaaatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
     7141 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
     7201 gtgccaccta aattgtaagc gttaatgtga accatcaccc taatcaagtt ttttggggtc
     7261 gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg
     7321 gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctag
     7381 ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc
     7441 gccgctacag ggcgcgtccc attcgcca
//
