LOCUS       40924_30291        4775 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-6xHis cassette for high-throughput 
            purification of recombinant proteins.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4775)
  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 4775)
  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   3  (bases 1 to 4775)
  AUTHORS   Midwest Center for Structural Genomics
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 4775)
  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"; journalName: "J. Struct. 
            Funct. Genomics"; date: "2010"; volume: "11"; pages: "31-9"
COMMENT     SGRef: number: 3; 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..4775
                     /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"
     tRNA            complement(618..694)
                     /label=argU
     tRNA            complement(964..1039)
                     /label=ileX
     promoter        1179..1256
                     /label=lacI promoter
     CDS             1257..2336
                     /codon_start=1
                     /label=lacI
                     /note="lac repressor"
                     /translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     protein_bind    2352..2373
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(2768..3356)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(3530..4387)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(4388..4491)
                     /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 tcaccggtgg attcagtcag
      421 gcgtcccatt atcagtgctt caggaaatgg gcggatggga gtccatagaa atggttcgta
      481 ggtatgctca ccttgcgcct aatcatttga cagagcatgc gaggaaaata gacgacattt
      541 ttggtgataa tgtcccaaat atgtcccact ctggaattat ggaggatata aagaaggcgt
      601 aactgattga attgtaatgg cgcgccctgc aggattcgaa cctgcggccc acgacttaga
      661 aggtcgttgc tctatccaac tgagctaagg gcgcgttgat accgcaatgc ggtgtaatcg
      721 cgtgaattat acggtcaacc cttgctgagt caatggcttt tgatctcgcc ggcgccacag
      781 gtgcggttgc tggcgcctat atcgccgaca tcaccgatgg ggaagatcgg gctcgccact
      841 tcgggctcat gagcgcttgt ttcggcgtgg gtatggtggc aggccccgtg gccgggggac
      901 tgttgggcgc catctccttg catgcaaaaa ggctgacgat ttctcgtcag cctttgctat
      961 atctggtggc ccctgctgga cttgaaccag cgaccaagcg attatgagtc gcctgctcta
     1021 accactgagc taaggggcca tggtagcgga ttataaagta actccgtgtc gcaatccgca
     1081 tgcaccattc cttgcggcgg cggtgctcaa cggcctcaac ctactactgg gctgcttcct
     1141 aatgcaggag tcgcataagg gagagcgtcg agatcccgga caccatcgaa tggcgcaaaa
     1201 cctttcgcgg tatggcatga tagcgcccgg aagagagtca attcagggtg gtgaatgtga
     1261 aaccagtaac gttatacgat gtcgcagagt atgccggtgt ctcttatcag accgtttccc
     1321 gcgtggtgaa ccaggccagc cacgtttctg cgaaaacgcg ggaaaaagtg gaagcggcga
     1381 tggcggagct gaattacatt cccaaccgcg tggcacaaca actggcgggc aaacagtcgt
     1441 tgctgattgg cgttgccacc tccagtctgg ccctgcacgc gccgtcgcaa attgtcgcgg
     1501 cgattaaatc tcgcgccgat caactgggtg ccagcgtggt ggtgtcgatg gtagaacgaa
     1561 gcggcgtcga agcctgtaaa gcggcggtgc acaatcttct cgcgcaacgc gtcagtgggc
     1621 tgatcattaa ctatccgctg gatgaccagg atgccattgc tgtggaagct gcctgcacta
     1681 atgttccggc gttatttctt gatgtctctg accagacacc catcaacagt attattttct
     1741 cccatgaaga cggtacgcga ctgggcgtgg agcatctggt cgcattgggt caccagcaaa
     1801 tcgcgctgtt agcgggccca ttaagttctg tctcggcgcg tctgcgtctg gctggctggc
     1861 ataaatatct cactcgcaat caaattcagc cgatagcgga acgggaaggc gactggagtg
     1921 ccatgtccgg ttttcaacaa accatgcaaa tgctgaatga gggcatcgtt cccactgcga
     1981 tgctggttgc caacgatcag atggcgctgg gcgcaatgcg cgccattacc gagtccgggc
     2041 tgcgcgttgg tgcggatatc tcggtagtgg gatacgacga taccgaagac agctcatgtt
     2101 atatcccgcc gttaaccacc atcaaacagg attttcgcct gctggggcaa accagcgtgg
     2161 accgcttgct gcaactctct cagggccagg cggtgaaggg caatcagctg ttgcccgtct
     2221 cactggtgaa aagaaaaacc accctggcgc ccaatacgca aaccgcctct ccccgcgcgt
     2281 tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag
     2341 cgcaacgcaa ttaatgtaag ttagctcact cattaggcac cgggatctcg accgatgccc
     2401 ttgagagcct tcaacccagt cagctccttc cggtgggcgc ggggcatgac tacagtcacg
     2461 tagcgatagc ggagtgtata ctggcttaac tatgcggcat cagagcagat tgtactgaga
     2521 gtgcaccata tatgcggtgt gaaataccgc acagatgcgt aaggagaaaa taccgcatca
     2581 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
     2641 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     2701 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     2761 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     2821 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     2881 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     2941 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     3001 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     3061 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     3121 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     3181 ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc
     3241 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     3301 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     3361 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     3421 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     3481 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     3541 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     3601 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     3661 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     3721 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     3781 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     3841 ctgcaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     3901 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     3961 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     4021 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     4081 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     4141 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     4201 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     4261 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     4321 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     4381 tactcatact cttccttttt caaattattg aagcatttat cagggttatt gtctcatgag
     4441 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc
     4501 ccgaaaagtg ccacctaaat tgtaagcgtt aatgtgaacc atcaccctaa tcaagttttt
     4561 tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag
     4621 cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg
     4681 gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc
     4741 ttaatgcgcc gctacagggc gcgtcccatt cgcca
//
