LOCUS       40924_30216        6280 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Bacterial vector encoding a C-terminal TEV-6xHis cassette plus TVMV 
            protease, for high-throughput purification of recombinant proteins.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6280)
  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 6280)
  AUTHORS   Midwest Center for Structural Genomics
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 6280)
  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..6280
                     /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"
     RBS             complement(264..284)
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    complement(300..324)
                     /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(325..343)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        642..715
                     /label=PLtetO-1 promoter
                     /note="modified phage lambda PL promoter with tet operator
                     sites (Lutz and Bujard, 1997)"
     CDS             739..1446
                     /codon_start=1
                     /label=TVMV protease
                     /note="tobacco vein mottling virus NIa protease
                     (Nallamsetty et al., 2004)"
                     /translation="MSKALLKGVRDFNPISACVCLLENSSDGHSERLFGIGFGPYIIAN
                     QHLFRRNNGELTIKTMHGEFKVKNSTQLQMKPVEGRDIIVIKMAKDFPPFPQKLKFRQP
                     TIKDRVCMVSTNFQQKSVSSLVSESSHIVHKEDTSFWQHWITTKDGQCGSPLVSIIDGN
                     ILGIHSLTHTTNGSNYFVEFPEKFVATYLDAADGWCKNWKFNADKISWGSFTLVEDAPE
                     DDFMAKKTVAAIMD"
     terminator      1482..1568
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     promoter        1683..1760
                     /label=lacI promoter
     CDS             1761..2840
                     /codon_start=1
                     /label=lacI
                     /note="lac repressor"
                     /translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     protein_bind    2856..2877
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             3652..3840
                     /codon_start=1
                     /label=rop
                     /note="Rop protein, which maintains plasmids at low copy
                     number"
                     /translation="VTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     misc_feature    3945..4087
                     /label=bom
                     /note="basis of mobility region from pBR322"
     rep_origin      complement(4273..4861)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(5035..5892)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(5893..5996)
                     /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 agactccttc ttaaagttaa acaaaattat ttctagaggg
      301 gaattgttat ccgctcacaa ttcccctata gtgagtcgta ttaatttcgc gggatcgaga
      361 tcgatctcga tcctctacgc cggacgcatc gtggccggca tcaccggcgc cacaggtgcg
      421 gttgctggcg cctatatcgc cgacatcacc gatggggaag atcgggctcg ccacttcggg
      481 ctcatgagcg cttgtttcgg cgtgggtatg gtggcaggcc ccgtggccgg gggactgttg
      541 ggcgccatct ccttgcatgc gacgtctaag aaaccattat tatcatgaca ttaacctata
      601 aaaataggcg tatcacgagg ccctttcgtc ttcacctcga gtccctatca gtgatagaga
      661 ttgacatccc tatcagtgat agagatactg agcacatcag caggacgcac tgaccgaatt
      721 gatttaagga ggtaacatat gtctaaagct ttgctgaagg gcgtgcgcga ttttaatccg
      781 atctctgctt gcgtatgcct gctggaaaac tcctcggatg gtcatagtga acgtctgttt
      841 ggcattggtt ttggcccgta tatcattgcc aaccagcatc tgtttcgtcg taacaatggc
      901 gaactgacca tcaaaaccat gcatggtgaa ttcaaagtca aaaactctac ccagctgcag
      961 atgaaaccgg ttgaaggccg tgacattatc gttatcaaaa tggctaaaga cttcccgccg
     1021 ttcccgcaga aactgaaatt ccgtcagccg accatcaaag atcgtgtgtg catggtgtcc
     1081 accaactttc agcagaaaag cgtctcgagc ctggtgtctg aatcctctca cattgtgcat
     1141 aaagaagaca cttctttctg gcagcactgg atcaccacta aagatggcca gtgtggcagc
     1201 ccactagttt ccatcattga tggcaacatt ctgggcatcc acagcctgac tcataccacc
     1261 aacggtagca actacttcgt ggaatttccg gaaaaattcg tggcgactta tctagatgcc
     1321 gcggatggtt ggtgcaaaaa ctggaaattc aacgcggata aaatcagctg gggttccttt
     1381 accctggttg aagatgcgcc ggaagatgac ttcatggcca aaaaaactgt tgccgccatc
     1441 atggactaat aaagatccca tggtacgcgt gctagaggca tcaaataaaa cgaaaggctc
     1501 agtcgaaaga ctgggccttt cgttttatct gttgtttgtc ggtgaacgct ctcctgagta
     1561 ggacaaatcc gccgccctag agcatgcacc attccttgcg gcggcggtgc tcaacggcct
     1621 caacctacta ctgggctgct tcctaatgca ggagtcgcat aagggagagc gtcgagatcc
     1681 cggacaccat cgaatggcgc aaaacctttc gcggtatggc atgatagcgc ccggaagaga
     1741 gtcaattcag ggtggtgaat gtgaaaccag taacgttata cgatgtcgca gagtatgccg
     1801 gtgtctctta tcagaccgtt tcccgcgtgg tgaaccaggc cagccacgtt tctgcgaaaa
     1861 cgcgggaaaa agtggaagcg gcgatggcgg agctgaatta cattcccaac cgcgtggcac
     1921 aacaactggc gggcaaacag tcgttgctga ttggcgttgc cacctccagt ctggccctgc
     1981 acgcgccgtc gcaaattgtc gcggcgatta aatctcgcgc cgatcaactg ggtgccagcg
     2041 tggtggtgtc gatggtagaa cgaagcggcg tcgaagcctg taaagcggcg gtgcacaatc
     2101 ttctcgcgca acgcgtcagt gggctgatca ttaactatcc gctggatgac caggatgcca
     2161 ttgctgtgga agctgcctgc actaatgttc cggcgttatt tcttgatgtc tctgaccaga
     2221 cacccatcaa cagtattatt ttctcccatg aagacggtac gcgactgggc gtggagcatc
     2281 tggtcgcatt gggtcaccag caaatcgcgc tgttagcggg cccattaagt tctgtctcgg
     2341 cgcgtctgcg tctggctggc tggcataaat atctcactcg caatcaaatt cagccgatag
     2401 cggaacggga aggcgactgg agtgccatgt ccggttttca acaaaccatg caaatgctga
     2461 atgagggcat cgttcccact gcgatgctgg ttgccaacga tcagatggcg ctgggcgcaa
     2521 tgcgcgccat taccgagtcc gggctgcgcg ttggtgcgga tatctcggta gtgggatacg
     2581 acgataccga agacagctca tgttatatcc cgccgttaac caccatcaaa caggattttc
     2641 gcctgctggg gcaaaccagc gtggaccgct tgctgcaact ctctcagggc caggcggtga
     2701 agggcaatca gctgttgccc gtctcactgg tgaaaagaaa aaccaccctg gcgcccaata
     2761 cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt
     2821 cccgactgga aagcgggcag tgagcgcaac gcaattaatg taagttagct cactcattag
     2881 gcaccgggat ctcgaccgat gcccttgaga gccttcaacc cagtcagctc cttccggtgg
     2941 gcgcggggca tgactatcgt cgccgcactt atgactgtct tctttatcat gcaactcgta
     3001 ggacaggtgc cggcagcgct ctgggtcatt ttcggcgagg accgctttcg ctggagcgcg
     3061 acgatgatcg gcctgtcgct tgcggtattc ggaatcttgc acgccctcgc tcaagccttc
     3121 gtcactggtc ccgccaccaa acgtttcggc gagaagcagg ccattatcgc cggcatggcg
     3181 gccccacggg tgcgcatgat cgtgctcctg tcgttgagga cccggctagg ctggcggggt
     3241 tgccttactg gttagcagaa tgaatcaccg atacgcgagc gaacgtgaag cgactgctgc
     3301 tgcaaaacgt ctgcgacctg agcaacaaca tgaatggtct tcggtttccg tgtttcgtaa
     3361 agtctggaaa cgcggaagtc agcgccctgc accattatgt tccggatctg catcgcagga
     3421 tgctgctggc taccctgtgg aacacctaca tctgtattaa cgaagcgctg gcattgaccc
     3481 tgagtgattt ttctctggtc ccgccgcatc cataccgcca gttgtttacc ctcacaacgt
     3541 tccagtaacc gggcatgttc atcatcagta acccgtatcg tgagcatcct ctctcgtttc
     3601 atcggtatca ttacccccat gaacagaaat cccccttaca cggaggcatc agtgaccaaa
     3661 caggaaaaaa ccgcccttaa catggcccgc tttatcagaa gccagacatt aacgcttctg
     3721 gagaaactca acgagctgga cgcggatgaa caggcagaca tctgtgaatc gcttcacgac
     3781 cacgctgatg agctttaccg cagctgcctc gcgcgtttcg gtgatgacgg tgaaaacctc
     3841 tgacacatgc agctcccgga gacggtcaca gcttgtctgt aagcggatgc cgggagcaga
     3901 caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc ggggcgcagc catgacccag
     3961 tcacgtagcg atagcggagt gtatactggc ttaactatgc ggcatcagag cagattgtac
     4021 tgagagtgca ccatatatgc ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg
     4081 catcaggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg
     4141 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
     4201 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
     4261 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
     4321 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
     4381 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     4441 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
     4501 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     4561 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     4621 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     4681 gaagtggtgg cctaactacg gctacactag aaggacagta tttggtatct gcgctctgct
     4741 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     4801 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
     4861 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
     4921 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
     4981 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg
     5041 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
     5101 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc
     5161 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc
     5221 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
     5281 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
     5341 cattgctgca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
     5401 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
     5461 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
     5521 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
     5581 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
     5641 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
     5701 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
     5761 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
     5821 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
     5881 ttgaatactc atactcttcc tttttcaaat tattgaagca tttatcaggg ttattgtctc
     5941 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     6001 tttccccgaa aagtgccacc taaattgtaa gcgttaatgt gaaccatcac cctaatcaag
     6061 ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt
     6121 tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg
     6181 agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc
     6241 cgcgcttaat gcgccgctac agggcgcgtc ccattcgcca
//
