LOCUS       40924_30146        6389 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Bacterial vector encoding a C-terminal TEV-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 6389)
  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 6389)
  AUTHORS   Midwest Center for Structural Genomics
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 6389)
  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..6389
                     /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"
     CDS             complement(1368..1388)
                     /codon_start=1
                     /label=TEV site
                     /note="tobacco etch virus (TEV) protease recognition and 
                     cleavage site"
                     /translation="ENLYFQS"
     RBS             complement(1400..1420)
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    complement(1436..1460)
                     /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(1461..1479)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        1792..1869
                     /label=lacI promoter
     CDS             1870..2949
                     /codon_start=1
                     /label=lacI
                     /note="lac repressor"
                     /translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     protein_bind    2965..2986
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             3761..3949
                     /codon_start=1
                     /label=rop
                     /note="Rop protein, which maintains plasmids at low copy
                     number"
                     /translation="VTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     misc_feature    4054..4196
                     /label=bom
                     /note="basis of mobility region from pBR322"
     rep_origin      complement(4382..4970)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(5144..6001)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(6002..6105)
                     /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 gcccggcgga ttggaagtac
     1381 aggttctccc cgggagagac tccttcttaa agttaaacaa aattatttct agaggggaat
     1441 tgttatccgc tcacaattcc cctatagtga gtcgtattaa tttcgcggga tcgagatcga
     1501 tctcgatcct ctacgccgga cgcatcgtgg ccggcatcac cggcgccaca ggtgcggttg
     1561 ctggcgccta tatcgccgac atcaccgatg gggaagatcg ggctcgccac ttcgggctca
     1621 tgagcgcttg tttcggcgtg ggtatggtgg caggccccgt ggccggggga ctgttgggcg
     1681 ccatctcctt gcatgcacca ttccttgcgg cggcggtgct caacggcctc aacctactac
     1741 tgggctgctt cctaatgcag gagtcgcata agggagagcg tcgagatccc ggacaccatc
     1801 gaatggcgca aaacctttcg cggtatggca tgatagcgcc cggaagagag tcaattcagg
     1861 gtggtgaatg tgaaaccagt aacgttatac gatgtcgcag agtatgccgg tgtctcttat
     1921 cagaccgttt cccgcgtggt gaaccaggcc agccacgttt ctgcgaaaac gcgggaaaaa
     1981 gtggaagcgg cgatggcgga gctgaattac attcccaacc gcgtggcaca acaactggcg
     2041 ggcaaacagt cgttgctgat tggcgttgcc acctccagtc tggccctgca cgcgccgtcg
     2101 caaattgtcg cggcgattaa atctcgcgcc gatcaactgg gtgccagcgt ggtggtgtcg
     2161 atggtagaac gaagcggcgt cgaagcctgt aaagcggcgg tgcacaatct tctcgcgcaa
     2221 cgcgtcagtg ggctgatcat taactatccg ctggatgacc aggatgccat tgctgtggaa
     2281 gctgcctgca ctaatgttcc ggcgttattt cttgatgtct ctgaccagac acccatcaac
     2341 agtattattt tctcccatga agacggtacg cgactgggcg tggagcatct ggtcgcattg
     2401 ggtcaccagc aaatcgcgct gttagcgggc ccattaagtt ctgtctcggc gcgtctgcgt
     2461 ctggctggct ggcataaata tctcactcgc aatcaaattc agccgatagc ggaacgggaa
     2521 ggcgactgga gtgccatgtc cggttttcaa caaaccatgc aaatgctgaa tgagggcatc
     2581 gttcccactg cgatgctggt tgccaacgat cagatggcgc tgggcgcaat gcgcgccatt
     2641 accgagtccg ggctgcgcgt tggtgcggat atctcggtag tgggatacga cgataccgaa
     2701 gacagctcat gttatatccc gccgttaacc accatcaaac aggattttcg cctgctgggg
     2761 caaaccagcg tggaccgctt gctgcaactc tctcagggcc aggcggtgaa gggcaatcag
     2821 ctgttgcccg tctcactggt gaaaagaaaa accaccctgg cgcccaatac gcaaaccgcc
     2881 tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa
     2941 agcgggcagt gagcgcaacg caattaatgt aagttagctc actcattagg caccgggatc
     3001 tcgaccgatg cccttgagag ccttcaaccc agtcagctcc ttccggtggg cgcggggcat
     3061 gactatcgtc gccgcactta tgactgtctt ctttatcatg caactcgtag gacaggtgcc
     3121 ggcagcgctc tgggtcattt tcggcgagga ccgctttcgc tggagcgcga cgatgatcgg
     3181 cctgtcgctt gcggtattcg gaatcttgca cgccctcgct caagccttcg tcactggtcc
     3241 cgccaccaaa cgtttcggcg agaagcaggc cattatcgcc ggcatggcgg ccccacgggt
     3301 gcgcatgatc gtgctcctgt cgttgaggac ccggctaggc tggcggggtt gccttactgg
     3361 ttagcagaat gaatcaccga tacgcgagcg aacgtgaagc gactgctgct gcaaaacgtc
     3421 tgcgacctga gcaacaacat gaatggtctt cggtttccgt gtttcgtaaa gtctggaaac
     3481 gcggaagtca gcgccctgca ccattatgtt ccggatctgc atcgcaggat gctgctggct
     3541 accctgtgga acacctacat ctgtattaac gaagcgctgg cattgaccct gagtgatttt
     3601 tctctggtcc cgccgcatcc ataccgccag ttgtttaccc tcacaacgtt ccagtaaccg
     3661 ggcatgttca tcatcagtaa cccgtatcgt gagcatcctc tctcgtttca tcggtatcat
     3721 tacccccatg aacagaaatc ccccttacac ggaggcatca gtgaccaaac aggaaaaaac
     3781 cgcccttaac atggcccgct ttatcagaag ccagacatta acgcttctgg agaaactcaa
     3841 cgagctggac gcggatgaac aggcagacat ctgtgaatcg cttcacgacc acgctgatga
     3901 gctttaccgc agctgcctcg cgcgtttcgg tgatgacggt gaaaacctct gacacatgca
     3961 gctcccggag acggtcacag cttgtctgta agcggatgcc gggagcagac aagcccgtca
     4021 gggcgcgtca gcgggtgttg gcgggtgtcg gggcgcagcc atgacccagt cacgtagcga
     4081 tagcggagtg tatactggct taactatgcg gcatcagagc agattgtact gagagtgcac
     4141 catatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgct
     4201 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     4261 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     4321 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     4381 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     4441 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     4501 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     4561 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     4621 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     4681 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     4741 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     4801 ctaactacgg ctacactaga aggacagtat ttggtatctg cgctctgctg aagccagtta
     4861 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     4921 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     4981 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     5041 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     5101 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     5161 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     5221 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     5281 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     5341 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     5401 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctgcag
     5461 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     5521 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     5581 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     5641 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     5701 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     5761 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     5821 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     5881 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     5941 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     6001 tactcttcct ttttcaaatt attgaagcat ttatcagggt tattgtctca tgagcggata
     6061 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
     6121 agtgccacct aaattgtaag cgttaatgtg aaccatcacc ctaatcaagt tttttggggt
     6181 cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac
     6241 ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta
     6301 gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg
     6361 cgccgctaca gggcgcgtcc cattcgcca
//
