LOCUS       40924_30161        6418 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Bacterial vector encoding N-terminal MBP-TVMV 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 6418)
  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 6418)
  AUTHORS   Midwest Center for Structural Genomics
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 6418)
  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..6418
                     /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"
     promoter        1821..1898
                     /label=lacI promoter
     CDS             1899..2978
                     /codon_start=1
                     /label=lacI
                     /note="lac repressor"
                     /translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     protein_bind    2994..3015
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             3790..3978
                     /codon_start=1
                     /label=rop
                     /note="Rop protein, which maintains plasmids at low copy
                     number"
                     /translation="VTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     misc_feature    4083..4225
                     /label=bom
                     /note="basis of mobility region from pBR322"
     rep_origin      complement(4411..4999)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(5173..6030)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(6031..6134)
                     /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 catgcaccat tccttgcggc
     1741 ggcggtgctc aacggcctca acctactact gggctgcttc ctaatgcagg agtcgcataa
     1801 gggagagcgt cgagatcccg gacaccatcg aatggcgcaa aacctttcgc ggtatggcat
     1861 gatagcgccc ggaagagagt caattcaggg tggtgaatgt gaaaccagta acgttatacg
     1921 atgtcgcaga gtatgccggt gtctcttatc agaccgtttc ccgcgtggtg aaccaggcca
     1981 gccacgtttc tgcgaaaacg cgggaaaaag tggaagcggc gatggcggag ctgaattaca
     2041 ttcccaaccg cgtggcacaa caactggcgg gcaaacagtc gttgctgatt ggcgttgcca
     2101 cctccagtct ggccctgcac gcgccgtcgc aaattgtcgc ggcgattaaa tctcgcgccg
     2161 atcaactggg tgccagcgtg gtggtgtcga tggtagaacg aagcggcgtc gaagcctgta
     2221 aagcggcggt gcacaatctt ctcgcgcaac gcgtcagtgg gctgatcatt aactatccgc
     2281 tggatgacca ggatgccatt gctgtggaag ctgcctgcac taatgttccg gcgttatttc
     2341 ttgatgtctc tgaccagaca cccatcaaca gtattatttt ctcccatgaa gacggtacgc
     2401 gactgggcgt ggagcatctg gtcgcattgg gtcaccagca aatcgcgctg ttagcgggcc
     2461 cattaagttc tgtctcggcg cgtctgcgtc tggctggctg gcataaatat ctcactcgca
     2521 atcaaattca gccgatagcg gaacgggaag gcgactggag tgccatgtcc ggttttcaac
     2581 aaaccatgca aatgctgaat gagggcatcg ttcccactgc gatgctggtt gccaacgatc
     2641 agatggcgct gggcgcaatg cgcgccatta ccgagtccgg gctgcgcgtt ggtgcggata
     2701 tctcggtagt gggatacgac gataccgaag acagctcatg ttatatcccg ccgttaacca
     2761 ccatcaaaca ggattttcgc ctgctggggc aaaccagcgt ggaccgcttg ctgcaactct
     2821 ctcagggcca ggcggtgaag ggcaatcagc tgttgcccgt ctcactggtg aaaagaaaaa
     2881 ccaccctggc gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc
     2941 agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgta
     3001 agttagctca ctcattaggc accgggatct cgaccgatgc ccttgagagc cttcaaccca
     3061 gtcagctcct tccggtgggc gcggggcatg actatcgtcg ccgcacttat gactgtcttc
     3121 tttatcatgc aactcgtagg acaggtgccg gcagcgctct gggtcatttt cggcgaggac
     3181 cgctttcgct ggagcgcgac gatgatcggc ctgtcgcttg cggtattcgg aatcttgcac
     3241 gccctcgctc aagccttcgt cactggtccc gccaccaaac gtttcggcga gaagcaggcc
     3301 attatcgccg gcatggcggc cccacgggtg cgcatgatcg tgctcctgtc gttgaggacc
     3361 cggctaggct ggcggggttg ccttactggt tagcagaatg aatcaccgat acgcgagcga
     3421 acgtgaagcg actgctgctg caaaacgtct gcgacctgag caacaacatg aatggtcttc
     3481 ggtttccgtg tttcgtaaag tctggaaacg cggaagtcag cgccctgcac cattatgttc
     3541 cggatctgca tcgcaggatg ctgctggcta ccctgtggaa cacctacatc tgtattaacg
     3601 aagcgctggc attgaccctg agtgattttt ctctggtccc gccgcatcca taccgccagt
     3661 tgtttaccct cacaacgttc cagtaaccgg gcatgttcat catcagtaac ccgtatcgtg
     3721 agcatcctct ctcgtttcat cggtatcatt acccccatga acagaaatcc cccttacacg
     3781 gaggcatcag tgaccaaaca ggaaaaaacc gcccttaaca tggcccgctt tatcagaagc
     3841 cagacattaa cgcttctgga gaaactcaac gagctggacg cggatgaaca ggcagacatc
     3901 tgtgaatcgc ttcacgacca cgctgatgag ctttaccgca gctgcctcgc gcgtttcggt
     3961 gatgacggtg aaaacctctg acacatgcag ctcccggaga cggtcacagc ttgtctgtaa
     4021 gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg
     4081 ggcgcagcca tgacccagtc acgtagcgat agcggagtgt atactggctt aactatgcgg
     4141 catcagagca gattgtactg agagtgcacc atatatgcgg tgtgaaatac cgcacagatg
     4201 cgtaaggaga aaataccgca tcaggcgctc ttccgcttcc tcgctcactg actcgctgcg
     4261 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
     4321 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
     4381 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     4441 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     4501 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     4561 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     4621 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     4681 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     4741 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     4801 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt
     4861 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     4921 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     4981 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     5041 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     5101 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     5161 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     5221 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     5281 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
     5341 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     5401 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     5461 tttgcgcaac gttgttgcca ttgctgcagg catcgtggtg tcacgctcgt cgtttggtat
     5521 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     5581 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     5641 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     5701 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     5761 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     5821 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     5881 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     5941 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     6001 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaaatta ttgaagcatt
     6061 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     6121 ataggggttc cgcgcacatt tccccgaaaa gtgccaccta aattgtaagc gttaatgtga
     6181 accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc
     6241 taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga
     6301 agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg
     6361 cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtccc attcgcca
//
