LOCUS       40924_30166        6457 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Bacterial vector encoding N-terminal 10xHis-MBP-TEV and C-terminal 
            TEV-6xHis, for high-throughput purification of recombinant proteins.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6457)
  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 6457)
  AUTHORS   Midwest Center for Structural Genomics
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 6457)
  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..6457
                     /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=TEV site
                     /note="tobacco etch virus (TEV) protease recognition and 
                     cleavage site"
                     /translation="ENLYFQS"
     CDS             complement(305..1402)
                     /codon_start=1
                     /label=MBP
                     /note="maltose binding protein from E. coli"
                     /translation="KIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEE
                     KFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLI
                     AYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAA
                     DGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGET
                     AMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLE
                     NYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFW
                     YAVRTAVINAASGRQTVDEALKDAQT"
     CDS             complement(1430..1456)
                     /codon_start=1
                     /label=9xHis
                     /note="9xHis affinity tag"
                     /translation="HHHHHHHHH"
     RBS             complement(1467..1489)
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    complement(1504..1528)
                     /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(1529..1547)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        1860..1937
                     /label=lacI promoter
     CDS             1938..3017
                     /codon_start=1
                     /label=lacI
                     /note="lac repressor"
                     /translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     protein_bind    3033..3054
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             3829..4017
                     /codon_start=1
                     /label=rop
                     /note="Rop protein, which maintains plasmids at low copy
                     number"
                     /translation="VTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     misc_feature    4122..4264
                     /label=bom
                     /note="basis of mobility region from pBR322"
     rep_origin      complement(4450..5038)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(5212..6069)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(6070..6173)
                     /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 agactttgga agtacaggtt ctcggtacct gggatatcgt
      301 aatcagtctg cgcgtctttc agggcttcat cgacagtctg acgaccgctg gcggcgttga
      361 tcaccgcagt acgcacggca taccagaaag cggacatctg cgggatgttc ggcatgattt
      421 cacctttctg ggcgttttcc atggtggcgg caatacgtgg atctttcgcc aactcttcct
      481 cgtaagactt cagcgctacg gcacccagcg gtttgtcttt attaaccgct tccagacctt
      541 catcagtcag cagatagttt tcgaggaact cttttgccag ctctttgttc ggactggcgg
      601 cgttaatacc tgcgctcagc acgccaacga acggtttgga tggttgaccc ttgaaggtcg
      661 gcagtaccgt tacaccataa ttcactttgc tggtgtcgat gttggaccat gcccacgggc
      721 cgttgatggt catcgctgtt tcgcctttat taaaggcagc ttctgcgatg gagtaatcgg
      781 tgtctgcatt catgtgtttg tttttaatca ggtcaaccag gaaggtcaga cccgctttcg
      841 cgccagcgtt atccacgccc acgtctttaa tgtcgtactt gccgttttca tacttgaacg
      901 cataaccccc gtcagcagca atcagcggcc aggtgaagta cggttcttgc aggttgaaca
      961 tcagcgcgct cttacctttc gctttcagtt ctttatccag cgccgggatc tcttcccagg
     1021 tttttggcgg gttcggcagc agatctttgt tataaatcag cgataacgct tcaacagcga
     1081 tcgggtaagc aatcagcttg ccgttgtaac gtacggcatc ccaggtaaac ggatacagct
     1141 tgtcctggaa cgctttgtcc ggggtgattt cagccaacag gccagattga gcgtagccac
     1201 caaagcggtc gtgtgcccag aagataatgt cagggccatc gccagttgcc gcaacctgtg
     1261 ggaatttctc ttccagttta tccggatgct caacggtgac tttaattccg gtatctttct
     1321 cgaatttctt accgacttca gcgagaccgt tatagccttt atcgccgtta atccagatta
     1381 ccagtttacc ttcttcgatt ttggtaccca gatctacacc agaagagtgg tggtggtgat
     1441 gatgatgatg atggtgcata tgtatatctc cttcttaaag ttaaacaaaa ttatttctag
     1501 aggggaattg ttatccgctc acaattcccc tatagtgagt cgtattaatt tcgcgggatc
     1561 gagatcgatc tcgatcctct acgccggacg catcgtggcc ggcatcaccg gcgccacagg
     1621 tgcggttgct ggcgcctata tcgccgacat caccgatggg gaagatcggg ctcgccactt
     1681 cgggctcatg agcgcttgtt tcggcgtggg tatggtggca ggccccgtgg ccgggggact
     1741 gttgggcgcc atctccttgc atgcaccatt ccttgcggcg gcggtgctca acggcctcaa
     1801 cctactactg ggctgcttcc taatgcagga gtcgcataag ggagagcgtc gagatcccgg
     1861 acaccatcga atggcgcaaa acctttcgcg gtatggcatg atagcgcccg gaagagagtc
     1921 aattcagggt ggtgaatgtg aaaccagtaa cgttatacga tgtcgcagag tatgccggtg
     1981 tctcttatca gaccgtttcc cgcgtggtga accaggccag ccacgtttct gcgaaaacgc
     2041 gggaaaaagt ggaagcggcg atggcggagc tgaattacat tcccaaccgc gtggcacaac
     2101 aactggcggg caaacagtcg ttgctgattg gcgttgccac ctccagtctg gccctgcacg
     2161 cgccgtcgca aattgtcgcg gcgattaaat ctcgcgccga tcaactgggt gccagcgtgg
     2221 tggtgtcgat ggtagaacga agcggcgtcg aagcctgtaa agcggcggtg cacaatcttc
     2281 tcgcgcaacg cgtcagtggg ctgatcatta actatccgct ggatgaccag gatgccattg
     2341 ctgtggaagc tgcctgcact aatgttccgg cgttatttct tgatgtctct gaccagacac
     2401 ccatcaacag tattattttc tcccatgaag acggtacgcg actgggcgtg gagcatctgg
     2461 tcgcattggg tcaccagcaa atcgcgctgt tagcgggccc attaagttct gtctcggcgc
     2521 gtctgcgtct ggctggctgg cataaatatc tcactcgcaa tcaaattcag ccgatagcgg
     2581 aacgggaagg cgactggagt gccatgtccg gttttcaaca aaccatgcaa atgctgaatg
     2641 agggcatcgt tcccactgcg atgctggttg ccaacgatca gatggcgctg ggcgcaatgc
     2701 gcgccattac cgagtccggg ctgcgcgttg gtgcggatat ctcggtagtg ggatacgacg
     2761 ataccgaaga cagctcatgt tatatcccgc cgttaaccac catcaaacag gattttcgcc
     2821 tgctggggca aaccagcgtg gaccgcttgc tgcaactctc tcagggccag gcggtgaagg
     2881 gcaatcagct gttgcccgtc tcactggtga aaagaaaaac caccctggcg cccaatacgc
     2941 aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc
     3001 gactggaaag cgggcagtga gcgcaacgca attaatgtaa gttagctcac tcattaggca
     3061 ccgggatctc gaccgatgcc cttgagagcc ttcaacccag tcagctcctt ccggtgggcg
     3121 cggggcatga ctatcgtcgc cgcacttatg actgtcttct ttatcatgca actcgtagga
     3181 caggtgccgg cagcgctctg ggtcattttc ggcgaggacc gctttcgctg gagcgcgacg
     3241 atgatcggcc tgtcgcttgc ggtattcgga atcttgcacg ccctcgctca agccttcgtc
     3301 actggtcccg ccaccaaacg tttcggcgag aagcaggcca ttatcgccgg catggcggcc
     3361 ccacgggtgc gcatgatcgt gctcctgtcg ttgaggaccc ggctaggctg gcggggttgc
     3421 cttactggtt agcagaatga atcaccgata cgcgagcgaa cgtgaagcga ctgctgctgc
     3481 aaaacgtctg cgacctgagc aacaacatga atggtcttcg gtttccgtgt ttcgtaaagt
     3541 ctggaaacgc ggaagtcagc gccctgcacc attatgttcc ggatctgcat cgcaggatgc
     3601 tgctggctac cctgtggaac acctacatct gtattaacga agcgctggca ttgaccctga
     3661 gtgatttttc tctggtcccg ccgcatccat accgccagtt gtttaccctc acaacgttcc
     3721 agtaaccggg catgttcatc atcagtaacc cgtatcgtga gcatcctctc tcgtttcatc
     3781 ggtatcatta cccccatgaa cagaaatccc ccttacacgg aggcatcagt gaccaaacag
     3841 gaaaaaaccg cccttaacat ggcccgcttt atcagaagcc agacattaac gcttctggag
     3901 aaactcaacg agctggacgc ggatgaacag gcagacatct gtgaatcgct tcacgaccac
     3961 gctgatgagc tttaccgcag ctgcctcgcg cgtttcggtg atgacggtga aaacctctga
     4021 cacatgcagc tcccggagac ggtcacagct tgtctgtaag cggatgccgg gagcagacaa
     4081 gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg gcgcagccat gacccagtca
     4141 cgtagcgata gcggagtgta tactggctta actatgcggc atcagagcag attgtactga
     4201 gagtgcacca tatatgcggt gtgaaatacc gcacagatgc gtaaggagaa aataccgcat
     4261 caggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg
     4321 agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc
     4381 aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
     4441 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
     4501 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
     4561 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
     4621 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
     4681 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
     4741 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
     4801 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
     4861 gtggtggcct aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa
     4921 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
     4981 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
     5041 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg
     5101 gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg
     5161 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt
     5221 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact
     5281 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat
     5341 gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg
     5401 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg
     5461 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat
     5521 tgctgcaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc
     5581 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt
     5641 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc
     5701 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga
     5761 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc
     5821 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa
     5881 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta
     5941 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg
     6001 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg
     6061 aatactcata ctcttccttt ttcaaattat tgaagcattt atcagggtta ttgtctcatg
     6121 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
     6181 ccccgaaaag tgccacctaa attgtaagcg ttaatgtgaa ccatcaccct aatcaagttt
     6241 tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag
     6301 agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc
     6361 gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc
     6421 gcttaatgcg ccgctacagg gcgcgtccca ttcgcca
//
