LOCUS       40924_30351        5911 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Compact bacterial vector encoding tRNA genes for rare Arg and Ile 
            codons, with a C-terminal TEV-10xHis-TVMV-MBP cassette.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5911)
  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 5911)
  AUTHORS   Midwest Center for Structural Genomics
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 5911)
  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 SmaI and 
            treat with T4 DNA polymerase plus dATP.
FEATURES             Location/Qualifiers
     source          1..5911
                     /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"
     tRNA            complement(1754..1830)
                     /label=argU
     tRNA            complement(2100..2175)
                     /label=ileX
     promoter        2315..2392
                     /label=lacI promoter
     CDS             2393..3472
                     /codon_start=1
                     /label=lacI
                     /note="lac repressor"
                     /translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     protein_bind    3488..3509
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(3904..4492)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(4666..5523)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(5524..5627)
                     /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 cggtggattc agtcaggcgt
     1561 cccattatca gtgcttcagg aaatgggcgg atgggagtcc atagaaatgg ttcgtaggta
     1621 tgctcacctt gcgcctaatc atttgacaga gcatgcgagg aaaatagacg acatttttgg
     1681 tgataatgtc ccaaatatgt cccactctgg aattatggag gatataaaga aggcgtaact
     1741 gattgaattg taatggcgcg ccctgcagga ttcgaacctg cggcccacga cttagaaggt
     1801 cgttgctcta tccaactgag ctaagggcgc gttgataccg caatgcggtg taatcgcgtg
     1861 aattatacgg tcaacccttg ctgagtcaat ggcttttgat ctcgccggcg ccacaggtgc
     1921 ggttgctggc gcctatatcg ccgacatcac cgatggggaa gatcgggctc gccacttcgg
     1981 gctcatgagc gcttgtttcg gcgtgggtat ggtggcaggc cccgtggccg ggggactgtt
     2041 gggcgccatc tccttgcatg caaaaaggct gacgatttct cgtcagcctt tgctatatct
     2101 ggtggcccct gctggacttg aaccagcgac caagcgatta tgagtcgcct gctctaacca
     2161 ctgagctaag gggccatggt agcggattat aaagtaactc cgtgtcgcaa tccgcatgca
     2221 ccattccttg cggcggcggt gctcaacggc ctcaacctac tactgggctg cttcctaatg
     2281 caggagtcgc ataagggaga gcgtcgagat cccggacacc atcgaatggc gcaaaacctt
     2341 tcgcggtatg gcatgatagc gcccggaaga gagtcaattc agggtggtga atgtgaaacc
     2401 agtaacgtta tacgatgtcg cagagtatgc cggtgtctct tatcagaccg tttcccgcgt
     2461 ggtgaaccag gccagccacg tttctgcgaa aacgcgggaa aaagtggaag cggcgatggc
     2521 ggagctgaat tacattccca accgcgtggc acaacaactg gcgggcaaac agtcgttgct
     2581 gattggcgtt gccacctcca gtctggccct gcacgcgccg tcgcaaattg tcgcggcgat
     2641 taaatctcgc gccgatcaac tgggtgccag cgtggtggtg tcgatggtag aacgaagcgg
     2701 cgtcgaagcc tgtaaagcgg cggtgcacaa tcttctcgcg caacgcgtca gtgggctgat
     2761 cattaactat ccgctggatg accaggatgc cattgctgtg gaagctgcct gcactaatgt
     2821 tccggcgtta tttcttgatg tctctgacca gacacccatc aacagtatta ttttctccca
     2881 tgaagacggt acgcgactgg gcgtggagca tctggtcgca ttgggtcacc agcaaatcgc
     2941 gctgttagcg ggcccattaa gttctgtctc ggcgcgtctg cgtctggctg gctggcataa
     3001 atatctcact cgcaatcaaa ttcagccgat agcggaacgg gaaggcgact ggagtgccat
     3061 gtccggtttt caacaaacca tgcaaatgct gaatgagggc atcgttccca ctgcgatgct
     3121 ggttgccaac gatcagatgg cgctgggcgc aatgcgcgcc attaccgagt ccgggctgcg
     3181 cgttggtgcg gatatctcgg tagtgggata cgacgatacc gaagacagct catgttatat
     3241 cccgccgtta accaccatca aacaggattt tcgcctgctg gggcaaacca gcgtggaccg
     3301 cttgctgcaa ctctctcagg gccaggcggt gaagggcaat cagctgttgc ccgtctcact
     3361 ggtgaaaaga aaaaccaccc tggcgcccaa tacgcaaacc gcctctcccc gcgcgttggc
     3421 cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca
     3481 acgcaattaa tgtaagttag ctcactcatt aggcaccggg atctcgaccg atgcccttga
     3541 gagccttcaa cccagtcagc tccttccggt gggcgcgggg catgactaca gtcacgtagc
     3601 gatagcggag tgtatactgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
     3661 accatatatg cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc gcatcaggcg
     3721 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
     3781 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     3841 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     3901 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     3961 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     4021 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     4081 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     4141 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     4201 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     4261 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     4321 gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc tgaagccagt
     4381 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     4441 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     4501 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
     4561 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
     4621 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
     4681 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
     4741 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
     4801 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
     4861 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     4921 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctgc
     4981 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
     5041 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     5101 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     5161 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
     5221 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
     5281 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     5341 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     5401 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     5461 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     5521 catactcttc ctttttcaaa ttattgaagc atttatcagg gttattgtct catgagcgga
     5581 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     5641 aaagtgccac ctaaattgta agcgttaatg tgaaccatca ccctaatcaa gttttttggg
     5701 gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg agcccccgat ttagagcttg
     5761 acggggaaag ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc
     5821 tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa
     5881 tgcgccgcta cagggcgcgt cccattcgcc a
//
