LOCUS       40924_30341        5963 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Compact bacterial vector encoding tRNA genes for rare Arg and Ile 
            codons, with an MBP-TVMV-6xHis-TEV leader for high-throughput 
            purification of recombinant proteins.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5963)
  AUTHORS   Eschenfeldt WH, Makowska-Grzyska M, Stols L, Donnelly MI, 
            Jedrzejczak R, Joachimiak A.
  TITLE     New LIC vectors for production of proteins from genes containing 
            rare codons.
  JOURNAL   J. Struct. Funct. Genomics 2013;14:135-44.
  PUBMED    24057978
REFERENCE   2  (bases 1 to 5963)
  AUTHORS   Midwest Center for Structural Genomics
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 5963)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "J. Struct. 
            Funct. Genomics"; date: "2013"; volume: "14"; pages: "135-44"
COMMENT     SGRef: number: 2; type: "Journal Article"
COMMENT     For ligation-independent cloning (LIC), linearize with SspI and 
            treat with T4 DNA polymerase plus dGTP.
FEATURES             Location/Qualifiers
     source          1..5963
                     /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(223..243)
                     /codon_start=1
                     /label=TEV site
                     /note="tobacco etch virus (TEV) protease recognition and 
                     cleavage site"
                     /translation="ENLYFQS"
     CDS             complement(268..285)
                     /codon_start=1
                     /label=6xHis
                     /note="6xHis affinity tag"
                     /translation="HHHHHH"
     CDS             complement(286..306)
                     /codon_start=1
                     /label=TVMV site
                     /note="tobacco vein mottling virus (TVMV) NIa protease 
                     recognition and cleavage site"
                     /translation="ETVRFQS"
     protein_bind    complement(309..333)
                     /label=attB1
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             complement(343..1443)
                     /codon_start=1
                     /label=MBP
                     /note="maltose binding protein from E. coli"
                     /translation="MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE
                     EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL
                     IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA
                     ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE
                     TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL
                     ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF
                     WYAVRTAVINAASGRQTVDEALKDAQT"
     RBS             complement(1451..1473)
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    complement(1488..1512)
                     /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(1513..1531)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     tRNA            complement(1806..1882)
                     /label=argU
     tRNA            complement(2152..2227)
                     /label=ileX
     promoter        2367..2444
                     /label=lacI promoter
     CDS             2445..3524
                     /codon_start=1
                     /label=lacI
                     /note="lac repressor"
                     /translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     protein_bind    3540..3561
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(3956..4544)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(4718..5575)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(5576..5679)
                     /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 tccgttatcc acttccaata ttggattgga agtacaggtt
      241 ctcggtaccc agatccacgc cagaagagtg atgatgatgg tggtgagact ggaaacgcac
      301 ggtttccgag cctgcttttt tgtacaaact tgtgatcgaa ttagtctgcg cgtctttcag
      361 ggcttcatcg acagtctgac gaccgctggc ggcgttgatc accgcagtac gcacggcata
      421 ccagaaagcg gacatctgcg ggatgttcgg catgatttca cctttctggg cgttttccat
      481 ggtggcggca atacgtggat ctttcgccaa ctcttcctcg taagacttca gcgctacggc
      541 acccagcggt ttgtctttat taaccgcttc cagaccttca tcagtcagca gatagttttc
      601 gaggaactct tttgccagct ctttgttcgg actggcggcg ttaatacctg cgctcagcac
      661 gccaacgaac ggtttggatg gttgaccctt gaaggtcggc agtaccgtta caccataatt
      721 cactttgctg gtgtcgatgt tggaccatgc ccacgggccg ttgatggtca tcgctgtttc
      781 gcctttatta aaggcagctt ctgcgatgga gtaatcggtg tctgcattca tgtgtttgtt
      841 tttaatcagg tcaaccagga aggtcagacc cgctttcgcg ccagcgttat ccacgcccac
      901 gtctttaatg tcgtacttgc cgttttcata cttgaacgca taacccccgt cagcagcaat
      961 cagcggccag gtgaagtacg gttcttgcag gttgaacatc agcgcgctct tacctttcgc
     1021 tttcagttct ttatccagcg ccgggatctc ttcccaggtt tttggcgggt tcggcagcag
     1081 atctttgtta taaatcagcg ataacgcttc aacagcgatc gggtaagcaa tcagcttgcc
     1141 gttgtaacgt acggcatccc aggtaaacgg atacagcttg tcctggaacg ctttgtccgg
     1201 ggtgatttca gccaacaggc cagattgagc gtagccacca aagcggtcgt gtgcccagaa
     1261 gataatgtca gggccatcgc cagttgccgc aacctgtggg aatttctctt ccagtttatc
     1321 cggatgctca acggtgactt taattccggt atctttctcg aatttcttac cgacttcagc
     1381 gagaccgtta tagcctttat cgccgttaat ccagattacc agtttacctt cttcgatttt
     1441 catatgtata tctccttctt aaagttaaac aaaattattt ctagagggga attgttatcc
     1501 gctcacaatt cccctatagt gagtcgtatt aatttcgcgg gatcgagatc gatctcgatc
     1561 ctctacgccg gacgcatcgt ggccggcatc accggtggat tcagtcaggc gtcccattat
     1621 cagtgcttca ggaaatgggc ggatgggagt ccatagaaat ggttcgtagg tatgctcacc
     1681 ttgcgcctaa tcatttgaca gagcatgcga ggaaaataga cgacattttt ggtgataatg
     1741 tcccaaatat gtcccactct ggaattatgg aggatataaa gaaggcgtaa ctgattgaat
     1801 tgtaatggcg cgccctgcag gattcgaacc tgcggcccac gacttagaag gtcgttgctc
     1861 tatccaactg agctaagggc gcgttgatac cgcaatgcgg tgtaatcgcg tgaattatac
     1921 ggtcaaccct tgctgagtca atggcttttg atctcgccgg cgccacaggt gcggttgctg
     1981 gcgcctatat cgccgacatc accgatgggg aagatcgggc tcgccacttc gggctcatga
     2041 gcgcttgttt cggcgtgggt atggtggcag gccccgtggc cgggggactg ttgggcgcca
     2101 tctccttgca tgcaaaaagg ctgacgattt ctcgtcagcc tttgctatat ctggtggccc
     2161 ctgctggact tgaaccagcg accaagcgat tatgagtcgc ctgctctaac cactgagcta
     2221 aggggccatg gtagcggatt ataaagtaac tccgtgtcgc aatccgcatg caccattcct
     2281 tgcggcggcg gtgctcaacg gcctcaacct actactgggc tgcttcctaa tgcaggagtc
     2341 gcataaggga gagcgtcgag atcccggaca ccatcgaatg gcgcaaaacc tttcgcggta
     2401 tggcatgata gcgcccggaa gagagtcaat tcagggtggt gaatgtgaaa ccagtaacgt
     2461 tatacgatgt cgcagagtat gccggtgtct cttatcagac cgtttcccgc gtggtgaacc
     2521 aggccagcca cgtttctgcg aaaacgcggg aaaaagtgga agcggcgatg gcggagctga
     2581 attacattcc caaccgcgtg gcacaacaac tggcgggcaa acagtcgttg ctgattggcg
     2641 ttgccacctc cagtctggcc ctgcacgcgc cgtcgcaaat tgtcgcggcg attaaatctc
     2701 gcgccgatca actgggtgcc agcgtggtgg tgtcgatggt agaacgaagc ggcgtcgaag
     2761 cctgtaaagc ggcggtgcac aatcttctcg cgcaacgcgt cagtgggctg atcattaact
     2821 atccgctgga tgaccaggat gccattgctg tggaagctgc ctgcactaat gttccggcgt
     2881 tatttcttga tgtctctgac cagacaccca tcaacagtat tattttctcc catgaagacg
     2941 gtacgcgact gggcgtggag catctggtcg cattgggtca ccagcaaatc gcgctgttag
     3001 cgggcccatt aagttctgtc tcggcgcgtc tgcgtctggc tggctggcat aaatatctca
     3061 ctcgcaatca aattcagccg atagcggaac gggaaggcga ctggagtgcc atgtccggtt
     3121 ttcaacaaac catgcaaatg ctgaatgagg gcatcgttcc cactgcgatg ctggttgcca
     3181 acgatcagat ggcgctgggc gcaatgcgcg ccattaccga gtccgggctg cgcgttggtg
     3241 cggatatctc ggtagtggga tacgacgata ccgaagacag ctcatgttat atcccgccgt
     3301 taaccaccat caaacaggat tttcgcctgc tggggcaaac cagcgtggac cgcttgctgc
     3361 aactctctca gggccaggcg gtgaagggca atcagctgtt gcccgtctca ctggtgaaaa
     3421 gaaaaaccac cctggcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat
     3481 taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt
     3541 aatgtaagtt agctcactca ttaggcaccg ggatctcgac cgatgccctt gagagccttc
     3601 aacccagtca gctccttccg gtgggcgcgg ggcatgacta cagtcacgta gcgatagcgg
     3661 agtgtatact ggcttaacta tgcggcatca gagcagattg tactgagagt gcaccatata
     3721 tgcggtgtga aataccgcac agatgcgtaa ggagaaaata ccgcatcagg cgctcttccg
     3781 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     3841 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     3901 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     3961 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     4021 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     4081 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     4141 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     4201 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     4261 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     4321 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     4381 acggctacac tagaaggaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     4441 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     4501 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     4561 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     4621 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     4681 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     4741 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     4801 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     4861 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     4921 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     4981 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct gcaggcatcg
     5041 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     5101 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     5161 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     5221 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     5281 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     5341 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     5401 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     5461 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     5521 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     5581 tcctttttca aattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
     5641 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc
     5701 acctaaattg taagcgttaa tgtgaaccat caccctaatc aagttttttg gggtcgaggt
     5761 gccgtaaagc actaaatcgg aaccctaaag ggagcccccg atttagagct tgacggggaa
     5821 agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc gctagggcgc
     5881 tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc
     5941 tacagggcgc gtcccattcg cca
//
