LOCUS       40924_30136        6362 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Bacterial vector encoding a C-terminal 10xHis-TVMV-MBP cassette, for
            high-throughput purification of recombinant proteins.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6362)
  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 6362)
  AUTHORS   Midwest Center for Structural Genomics
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 6362)
  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..6362
                     /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(213..1310)
                     /codon_start=1
                     /label=MBP
                     /note="maltose binding protein from E. coli"
                     /translation="KIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEE
                     KFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLI
                     AYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAA
                     DGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGET
                     AMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLE
                     NYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFW
                     YAVRTAVINAASGRQTVDEALKDAQT"
     CDS             complement(1311..1331)
                     /codon_start=1
                     /label=TVMV site
                     /note="tobacco vein mottling virus (TVMV) NIa protease 
                     recognition and cleavage site"
                     /translation="ETVRFQS"
     CDS             complement(1335..1361)
                     /codon_start=1
                     /label=9xHis
                     /note="9xHis affinity tag"
                     /translation="HHHHHHHHH"
     RBS             complement(1373..1393)
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    complement(1409..1433)
                     /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(1434..1452)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        1765..1842
                     /label=lacI promoter
     CDS             1843..2922
                     /codon_start=1
                     /label=lacI
                     /note="lac repressor"
                     /translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     protein_bind    2938..2959
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             3734..3922
                     /codon_start=1
                     /label=rop
                     /note="Rop protein, which maintains plasmids at low copy
                     number"
                     /translation="VTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     misc_feature    4027..4169
                     /label=bom
                     /note="basis of mobility region from pBR322"
     rep_origin      complement(4355..4943)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(5117..5974)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(5975..6078)
                     /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 tccttacgaa ttagtctgcg cgtctttcag ggcttcatcg
      241 acagtctgac gaccgctggc ggcgttgatc accgcagtac gcacggcata ccagaaagcg
      301 gacatctgcg ggatgttcgg catgatttca cctttctggg cgttttccat ggtggcggca
      361 atacgtggat ctttcgccaa ctcttcctcg taagacttca gcgctacggc acccagcggt
      421 ttgtctttat taaccgcttc cagaccttca tcagtcagca gatagttttc gaggaactct
      481 tttgccagct ctttgttcgg actggcggcg ttaatacctg cgctcagcac gccaacgaac
      541 ggtttggatg gttgaccctt gaaggtcggc agtaccgtta caccataatt cactttgctg
      601 gtgtcgatgt tggaccatgc ccacgggccg ttgatggtca tcgctgtttc gcctttatta
      661 aaggcagctt ctgcgatgga gtaatcggtg tctgcattca tgtgtttgtt tttaatcagg
      721 tcaaccagga aggtcagacc cgctttcgcg ccagcgttat ccacgcccac gtctttaatg
      781 tcgtacttgc cgttttcata cttgaacgca taacccccgt cagcagcaat cagcggccag
      841 gtgaagtacg gttcttgcag gttgaacatc agcgcgctct tacctttcgc tttcagttct
      901 ttatccagcg ccgggatctc ttcccaggtt tttggcgggt tcggcagcag atctttgtta
      961 taaatcagcg ataacgcttc aacagcgatc gggtaagcaa tcagcttgcc gttgtaacgt
     1021 acggcatccc aggtaaacgg atacagcttg tcctggaacg ctttgtccgg ggtgatttca
     1081 gccaacaggc cagattgagc gtagccacca aagcggtcgt gtgcccagaa gataatgtca
     1141 gggccatcgc cagttgccgc aacctgtggg aatttctctt ccagtttatc cggatgctca
     1201 acggtgactt taattccggt atctttctcg aatttcttac cgacttcagc gagaccgtta
     1261 tagcctttat cgccgttaat ccagattacc agtttacctt cttcgatttt agactggaaa
     1321 cgcacggttt cgtgatggtg gtgatgatga tgatggtggt gcccgggaga gactccttct
     1381 taaagttaaa caaaattatt tctagagggg aattgttatc cgctcacaat tcccctatag
     1441 tgagtcgtat taatttcgcg ggatcgagat cgatctcgat cctctacgcc ggacgcatcg
     1501 tggccggcat caccggcgcc acaggtgcgg ttgctggcgc ctatatcgcc gacatcaccg
     1561 atggggaaga tcgggctcgc cacttcgggc tcatgagcgc ttgtttcggc gtgggtatgg
     1621 tggcaggccc cgtggccggg ggactgttgg gcgccatctc cttgcatgca ccattccttg
     1681 cggcggcggt gctcaacggc ctcaacctac tactgggctg cttcctaatg caggagtcgc
     1741 ataagggaga gcgtcgagat cccggacacc atcgaatggc gcaaaacctt tcgcggtatg
     1801 gcatgatagc gcccggaaga gagtcaattc agggtggtga atgtgaaacc agtaacgtta
     1861 tacgatgtcg cagagtatgc cggtgtctct tatcagaccg tttcccgcgt ggtgaaccag
     1921 gccagccacg tttctgcgaa aacgcgggaa aaagtggaag cggcgatggc ggagctgaat
     1981 tacattccca accgcgtggc acaacaactg gcgggcaaac agtcgttgct gattggcgtt
     2041 gccacctcca gtctggccct gcacgcgccg tcgcaaattg tcgcggcgat taaatctcgc
     2101 gccgatcaac tgggtgccag cgtggtggtg tcgatggtag aacgaagcgg cgtcgaagcc
     2161 tgtaaagcgg cggtgcacaa tcttctcgcg caacgcgtca gtgggctgat cattaactat
     2221 ccgctggatg accaggatgc cattgctgtg gaagctgcct gcactaatgt tccggcgtta
     2281 tttcttgatg tctctgacca gacacccatc aacagtatta ttttctccca tgaagacggt
     2341 acgcgactgg gcgtggagca tctggtcgca ttgggtcacc agcaaatcgc gctgttagcg
     2401 ggcccattaa gttctgtctc ggcgcgtctg cgtctggctg gctggcataa atatctcact
     2461 cgcaatcaaa ttcagccgat agcggaacgg gaaggcgact ggagtgccat gtccggtttt
     2521 caacaaacca tgcaaatgct gaatgagggc atcgttccca ctgcgatgct ggttgccaac
     2581 gatcagatgg cgctgggcgc aatgcgcgcc attaccgagt ccgggctgcg cgttggtgcg
     2641 gatatctcgg tagtgggata cgacgatacc gaagacagct catgttatat cccgccgtta
     2701 accaccatca aacaggattt tcgcctgctg gggcaaacca gcgtggaccg cttgctgcaa
     2761 ctctctcagg gccaggcggt gaagggcaat cagctgttgc ccgtctcact ggtgaaaaga
     2821 aaaaccaccc tggcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta
     2881 atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa
     2941 tgtaagttag ctcactcatt aggcaccggg atctcgaccg atgcccttga gagccttcaa
     3001 cccagtcagc tccttccggt gggcgcgggg catgactatc gtcgccgcac ttatgactgt
     3061 cttctttatc atgcaactcg taggacaggt gccggcagcg ctctgggtca ttttcggcga
     3121 ggaccgcttt cgctggagcg cgacgatgat cggcctgtcg cttgcggtat tcggaatctt
     3181 gcacgccctc gctcaagcct tcgtcactgg tcccgccacc aaacgtttcg gcgagaagca
     3241 ggccattatc gccggcatgg cggccccacg ggtgcgcatg atcgtgctcc tgtcgttgag
     3301 gacccggcta ggctggcggg gttgccttac tggttagcag aatgaatcac cgatacgcga
     3361 gcgaacgtga agcgactgct gctgcaaaac gtctgcgacc tgagcaacaa catgaatggt
     3421 cttcggtttc cgtgtttcgt aaagtctgga aacgcggaag tcagcgccct gcaccattat
     3481 gttccggatc tgcatcgcag gatgctgctg gctaccctgt ggaacaccta catctgtatt
     3541 aacgaagcgc tggcattgac cctgagtgat ttttctctgg tcccgccgca tccataccgc
     3601 cagttgttta ccctcacaac gttccagtaa ccgggcatgt tcatcatcag taacccgtat
     3661 cgtgagcatc ctctctcgtt tcatcggtat cattaccccc atgaacagaa atccccctta
     3721 cacggaggca tcagtgacca aacaggaaaa aaccgccctt aacatggccc gctttatcag
     3781 aagccagaca ttaacgcttc tggagaaact caacgagctg gacgcggatg aacaggcaga
     3841 catctgtgaa tcgcttcacg accacgctga tgagctttac cgcagctgcc tcgcgcgttt
     3901 cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca cagcttgtct
     3961 gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg
     4021 tcggggcgca gccatgaccc agtcacgtag cgatagcgga gtgtatactg gcttaactat
     4081 gcggcatcag agcagattgt actgagagtg caccatatat gcggtgtgaa ataccgcaca
     4141 gatgcgtaag gagaaaatac cgcatcaggc gctcttccgc ttcctcgctc actgactcgc
     4201 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     4261 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     4321 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     4381 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     4441 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     4501 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     4561 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     4621 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     4681 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     4741 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag
     4801 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     4861 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     4921 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     4981 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     5041 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
     5101 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
     5161 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
     5221 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
     5281 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
     5341 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
     5401 atagtttgcg caacgttgtt gccattgctg caggcatcgt ggtgtcacgc tcgtcgtttg
     5461 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
     5521 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
     5581 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
     5641 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
     5701 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa
     5761 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
     5821 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
     5881 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
     5941 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa attattgaag
     6001 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     6061 acaaataggg gttccgcgca catttccccg aaaagtgcca cctaaattgt aagcgttaat
     6121 gtgaaccatc accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga
     6181 accctaaagg gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa
     6241 aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc
     6301 tgcgcgtaac caccacaccc gccgcgctta atgcgccgct acagggcgcg tcccattcgc
     6361 ca
//
