LOCUS       40924_30176        7445 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Bacterial vector encoding N-terminal MBP-TVMV and C-terminal 
            TEV-6xHis plus TVMV protease. See also pMCSG34.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7445)
  AUTHORS   Eschenfeldt WH, Maltseva N, Stols L, Donnelly MI, Gu M, Nocek B, Tan
            K, Kim Y, Joachimiak A.
  TITLE     Cleavable C-terminal His-tag vectors for structure determination.
  JOURNAL   J. Struct. Funct. Genomics 2010;11:31-9.
  PUBMED    20213425
REFERENCE   2  (bases 1 to 7445)
  AUTHORS   Midwest Center for Structural Genomics
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 7445)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "J. Struct. 
            Funct. Genomics"; date: "2010"; volume: "11"; pages: "31-9"
COMMENT     SGRef: number: 2; type: "Journal Article"
COMMENT     For ligation-independent cloning (LIC), linearize with SmaI and 
            treat with T4 DNA polymerase plus dATP.
FEATURES             Location/Qualifiers
     source          1..7445
                     /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(208..225)
                     /codon_start=1
                     /label=6xHis
                     /note="6xHis affinity tag"
                     /translation="HHHHHH"
     CDS             complement(232..252)
                     /codon_start=1
                     /label=TEV site
                     /note="tobacco etch virus (TEV) protease recognition and 
                     cleavage site"
                     /translation="ENLYFQS"
     CDS             complement(263..283)
                     /codon_start=1
                     /label=TVMV site
                     /note="tobacco vein mottling virus (TVMV) NIa protease 
                     recognition and cleavage site"
                     /translation="ETVRFQS"
     protein_bind    complement(286..310)
                     /label=attB1
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             complement(320..1420)
                     /codon_start=1
                     /label=MBP
                     /note="maltose binding protein from E. coli"
                     /translation="MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE
                     EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL
                     IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA
                     ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE
                     TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL
                     ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF
                     WYAVRTAVINAASGRQTVDEALKDAQT"
     RBS             complement(1428..1450)
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    complement(1465..1489)
                     /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(1490..1508)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     terminator      complement(1739..1825)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     CDS             complement(1861..2568)
                     /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(2592..2665)
                     /label=PLtetO-1 promoter
                     /note="modified phage lambda PL promoter with tet operator
                     sites (Lutz and Bujard, 1997)"
     promoter        2848..2925
                     /label=lacI promoter
     CDS             2926..4005
                     /codon_start=1
                     /label=lacI
                     /note="lac repressor"
                     /translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     protein_bind    4021..4042
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             4817..5005
                     /codon_start=1
                     /label=rop
                     /note="Rop protein, which maintains plasmids at low copy
                     number"
                     /translation="VTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     misc_feature    5110..5252
                     /label=bom
                     /note="basis of mobility region from pBR322"
     rep_origin      complement(5438..6026)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(6200..7057)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(7058..7161)
                     /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 tccgttaatg atgatgatgg tggtgcccgg cggattggaa
      241 gtacaggttc tccccgggag agactctgga aacgcacggt ttccgagcct gcttttttgt
      301 acaaacttgt gatcgaatta gtctgcgcgt ctttcagggc ttcatcgaca gtctgacgac
      361 cgctggcggc gttgatcacc gcagtacgca cggcatacca gaaagcggac atctgcggga
      421 tgttcggcat gatttcacct ttctgggcgt tttccatggt ggcggcaata cgtggatctt
      481 tcgccaactc ttcctcgtaa gacttcagcg ctacggcacc cagcggtttg tctttattaa
      541 ccgcttccag accttcatca gtcagcagat agttttcgag gaactctttt gccagctctt
      601 tgttcggact ggcggcgtta atacctgcgc tcagcacgcc aacgaacggt ttggatggtt
      661 gacccttgaa ggtcggcagt accgttacac cataattcac tttgctggtg tcgatgttgg
      721 accatgccca cgggccgttg atggtcatcg ctgtttcgcc tttattaaag gcagcttctg
      781 cgatggagta atcggtgtct gcattcatgt gtttgttttt aatcaggtca accaggaagg
      841 tcagacccgc tttcgcgcca gcgttatcca cgcccacgtc tttaatgtcg tacttgccgt
      901 tttcatactt gaacgcataa cccccgtcag cagcaatcag cggccaggtg aagtacggtt
      961 cttgcaggtt gaacatcagc gcgctcttac ctttcgcttt cagttcttta tccagcgccg
     1021 ggatctcttc ccaggttttt ggcgggttcg gcagcagatc tttgttataa atcagcgata
     1081 acgcttcaac agcgatcggg taagcaatca gcttgccgtt gtaacgtacg gcatcccagg
     1141 taaacggata cagcttgtcc tggaacgctt tgtccggggt gatttcagcc aacaggccag
     1201 attgagcgta gccaccaaag cggtcgtgtg cccagaagat aatgtcaggg ccatcgccag
     1261 ttgccgcaac ctgtgggaat ttctcttcca gtttatccgg atgctcaacg gtgactttaa
     1321 ttccggtatc tttctcgaat ttcttaccga cttcagcgag accgttatag cctttatcgc
     1381 cgttaatcca gattaccagt ttaccttctt cgattttcat atgtatatct ccttcttaaa
     1441 gttaaacaaa attatttcta gaggggaatt gttatccgct cacaattccc ctatagtgag
     1501 tcgtattaat ttcgcgggat cgagatcgat ctcgatcctc tacgccggac gcatcgtggc
     1561 cggcatcacc ggcgccacag gtgcggttgc tggcgcctat atcgccgaca tcaccgatgg
     1621 ggaagatcgg gctcgccact tcgggctcat gagcgcttgt ttcggcgtgg gtatggtggc
     1681 aggccccgtg gccgggggac tgttgggcgc catctccttg catgctctag ggcggcggat
     1741 ttgtcctact caggagagcg ttcaccgaca aacaacagat aaaacgaaag gcccagtctt
     1801 tcgactgagc ctttcgtttt atttgatgcc tctagcacgc gtaccatggg atctttatta
     1861 gtccatgatg gcggcaacag tttttttggc catgaagtca tcttccggcg catcttcaac
     1921 cagggtaaag gaaccccagc tgattttatc cgcgttgaat ttccagtttt tgcaccaacc
     1981 atccgcggca tctagataag tcgccacgaa tttttccgga aattccacga agtagttgct
     2041 accgttggtg gtatgagtca ggctgtggat gcccagaatg ttgccatcaa tgatggaaac
     2101 tagtgggctg ccacactggc catctttagt ggtgatccag tgctgccaga aagaagtgtc
     2161 ttctttatgc acaatgtgag aggattcaga caccaggctc gagacgcttt tctgctgaaa
     2221 gttggtggac accatgcaca cacgatcttt gatggtcggc tgacggaatt tcagtttctg
     2281 cgggaacggc gggaagtctt tagccatttt gataacgata atgtcacggc cttcaaccgg
     2341 tttcatctgc agctgggtag agtttttgac tttgaattca ccatgcatgg ttttgatggt
     2401 cagttcgcca ttgttacgac gaaacagatg ctggttggca atgatatacg ggccaaaacc
     2461 aatgccaaac agacgttcac tatgaccatc cgaggagttt tccagcaggc atacgcaagc
     2521 agagatcgga ttaaaatcgc gcacgccctt cagcaaagct ttagacatat gttacctcct
     2581 taaatcaatt cggtcagtgc gtcctgctga tgtgctcagt atctctatca ctgataggga
     2641 tgtcaatctc tatcactgat agggactcga ggtgaagacg aaagggcctc gtgatacgcc
     2701 tatttttata ggttaatgtc atgataataa tggtttctta gacgtcgcat gcaccattcc
     2761 ttgcggcggc ggtgctcaac ggcctcaacc tactactggg ctgcttccta atgcaggagt
     2821 cgcataaggg agagcgtcga gatcccggac accatcgaat ggcgcaaaac ctttcgcggt
     2881 atggcatgat agcgcccgga agagagtcaa ttcagggtgg tgaatgtgaa accagtaacg
     2941 ttatacgatg tcgcagagta tgccggtgtc tcttatcaga ccgtttcccg cgtggtgaac
     3001 caggccagcc acgtttctgc gaaaacgcgg gaaaaagtgg aagcggcgat ggcggagctg
     3061 aattacattc ccaaccgcgt ggcacaacaa ctggcgggca aacagtcgtt gctgattggc
     3121 gttgccacct ccagtctggc cctgcacgcg ccgtcgcaaa ttgtcgcggc gattaaatct
     3181 cgcgccgatc aactgggtgc cagcgtggtg gtgtcgatgg tagaacgaag cggcgtcgaa
     3241 gcctgtaaag cggcggtgca caatcttctc gcgcaacgcg tcagtgggct gatcattaac
     3301 tatccgctgg atgaccagga tgccattgct gtggaagctg cctgcactaa tgttccggcg
     3361 ttatttcttg atgtctctga ccagacaccc atcaacagta ttattttctc ccatgaagac
     3421 ggtacgcgac tgggcgtgga gcatctggtc gcattgggtc accagcaaat cgcgctgtta
     3481 gcgggcccat taagttctgt ctcggcgcgt ctgcgtctgg ctggctggca taaatatctc
     3541 actcgcaatc aaattcagcc gatagcggaa cgggaaggcg actggagtgc catgtccggt
     3601 tttcaacaaa ccatgcaaat gctgaatgag ggcatcgttc ccactgcgat gctggttgcc
     3661 aacgatcaga tggcgctggg cgcaatgcgc gccattaccg agtccgggct gcgcgttggt
     3721 gcggatatct cggtagtggg atacgacgat accgaagaca gctcatgtta tatcccgccg
     3781 ttaaccacca tcaaacagga ttttcgcctg ctggggcaaa ccagcgtgga ccgcttgctg
     3841 caactctctc agggccaggc ggtgaagggc aatcagctgt tgcccgtctc actggtgaaa
     3901 agaaaaacca ccctggcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca
     3961 ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat
     4021 taatgtaagt tagctcactc attaggcacc gggatctcga ccgatgccct tgagagcctt
     4081 caacccagtc agctccttcc ggtgggcgcg gggcatgact atcgtcgccg cacttatgac
     4141 tgtcttcttt atcatgcaac tcgtaggaca ggtgccggca gcgctctggg tcattttcgg
     4201 cgaggaccgc tttcgctgga gcgcgacgat gatcggcctg tcgcttgcgg tattcggaat
     4261 cttgcacgcc ctcgctcaag ccttcgtcac tggtcccgcc accaaacgtt tcggcgagaa
     4321 gcaggccatt atcgccggca tggcggcccc acgggtgcgc atgatcgtgc tcctgtcgtt
     4381 gaggacccgg ctaggctggc ggggttgcct tactggttag cagaatgaat caccgatacg
     4441 cgagcgaacg tgaagcgact gctgctgcaa aacgtctgcg acctgagcaa caacatgaat
     4501 ggtcttcggt ttccgtgttt cgtaaagtct ggaaacgcgg aagtcagcgc cctgcaccat
     4561 tatgttccgg atctgcatcg caggatgctg ctggctaccc tgtggaacac ctacatctgt
     4621 attaacgaag cgctggcatt gaccctgagt gatttttctc tggtcccgcc gcatccatac
     4681 cgccagttgt ttaccctcac aacgttccag taaccgggca tgttcatcat cagtaacccg
     4741 tatcgtgagc atcctctctc gtttcatcgg tatcattacc cccatgaaca gaaatccccc
     4801 ttacacggag gcatcagtga ccaaacagga aaaaaccgcc cttaacatgg cccgctttat
     4861 cagaagccag acattaacgc ttctggagaa actcaacgag ctggacgcgg atgaacaggc
     4921 agacatctgt gaatcgcttc acgaccacgc tgatgagctt taccgcagct gcctcgcgcg
     4981 tttcggtgat gacggtgaaa acctctgaca catgcagctc ccggagacgg tcacagcttg
     5041 tctgtaagcg gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg
     5101 gtgtcggggc gcagccatga cccagtcacg tagcgatagc ggagtgtata ctggcttaac
     5161 tatgcggcat cagagcagat tgtactgaga gtgcaccata tatgcggtgt gaaataccgc
     5221 acagatgcgt aaggagaaaa taccgcatca ggcgctcttc cgcttcctcg ctcactgact
     5281 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
     5341 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     5401 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     5461 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     5521 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     5581 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     5641 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     5701 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     5761 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     5821 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagga
     5881 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     5941 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     6001 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     6061 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     6121 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     6181 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     6241 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     6301 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
     6361 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     6421 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     6481 ttaatagttt gcgcaacgtt gttgccattg ctgcaggcat cgtggtgtca cgctcgtcgt
     6541 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     6601 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     6661 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     6721 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     6781 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
     6841 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     6901 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     6961 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     7021 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caaattattg
     7081 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     7141 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctaaat tgtaagcgtt
     7201 aatgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc
     7261 ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg aacgtggcga
     7321 gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca
     7381 cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtcccatt
     7441 cgcca
//
