LOCUS       40924_30286        4748 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Compact bacterial vector encoding tRNA genes for rare Arg and Ile 
            codons, with a C-terminal 6xHis tag for high-throughput purification
            of recombinant proteins.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4748)
  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 4748)
  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 4748)
  AUTHORS   Midwest Center for Structural Genomics
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 4748)
  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..4748
                     /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"
     RBS             complement(237..257)
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    complement(273..297)
                     /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(298..316)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     tRNA            complement(591..667)
                     /label=argU
     tRNA            complement(937..1012)
                     /label=ileX
     promoter        1152..1229
                     /label=lacI promoter
     CDS             1230..2309
                     /codon_start=1
                     /label=lacI
                     /note="lac repressor"
                     /translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     protein_bind    2325..2346
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(2741..3329)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(3503..4360)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(4361..4464)
                     /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 gagagactcc
      241 ttcttaaagt taaacaaaat tatttctaga ggggaattgt tatccgctca caattcccct
      301 atagtgagtc gtattaattt cgcgggatcg agatcgatct cgatcctcta cgccggacgc
      361 atcgtggccg gcatcaccgg tggattcagt caggcgtccc attatcagtg cttcaggaaa
      421 tgggcggatg ggagtccata gaaatggttc gtaggtatgc tcaccttgcg cctaatcatt
      481 tgacagagca tgcgaggaaa atagacgaca tttttggtga taatgtccca aatatgtccc
      541 actctggaat tatggaggat ataaagaagg cgtaactgat tgaattgtaa tggcgcgccc
      601 tgcaggattc gaacctgcgg cccacgactt agaaggtcgt tgctctatcc aactgagcta
      661 agggcgcgtt gataccgcaa tgcggtgtaa tcgcgtgaat tatacggtca acccttgctg
      721 agtcaatggc ttttgatctc gccggcgcca caggtgcggt tgctggcgcc tatatcgccg
      781 acatcaccga tggggaagat cgggctcgcc acttcgggct catgagcgct tgtttcggcg
      841 tgggtatggt ggcaggcccc gtggccgggg gactgttggg cgccatctcc ttgcatgcaa
      901 aaaggctgac gatttctcgt cagcctttgc tatatctggt ggcccctgct ggacttgaac
      961 cagcgaccaa gcgattatga gtcgcctgct ctaaccactg agctaagggg ccatggtagc
     1021 ggattataaa gtaactccgt gtcgcaatcc gcatgcacca ttccttgcgg cggcggtgct
     1081 caacggcctc aacctactac tgggctgctt cctaatgcag gagtcgcata agggagagcg
     1141 tcgagatccc ggacaccatc gaatggcgca aaacctttcg cggtatggca tgatagcgcc
     1201 cggaagagag tcaattcagg gtggtgaatg tgaaaccagt aacgttatac gatgtcgcag
     1261 agtatgccgg tgtctcttat cagaccgttt cccgcgtggt gaaccaggcc agccacgttt
     1321 ctgcgaaaac gcgggaaaaa gtggaagcgg cgatggcgga gctgaattac attcccaacc
     1381 gcgtggcaca acaactggcg ggcaaacagt cgttgctgat tggcgttgcc acctccagtc
     1441 tggccctgca cgcgccgtcg caaattgtcg cggcgattaa atctcgcgcc gatcaactgg
     1501 gtgccagcgt ggtggtgtcg atggtagaac gaagcggcgt cgaagcctgt aaagcggcgg
     1561 tgcacaatct tctcgcgcaa cgcgtcagtg ggctgatcat taactatccg ctggatgacc
     1621 aggatgccat tgctgtggaa gctgcctgca ctaatgttcc ggcgttattt cttgatgtct
     1681 ctgaccagac acccatcaac agtattattt tctcccatga agacggtacg cgactgggcg
     1741 tggagcatct ggtcgcattg ggtcaccagc aaatcgcgct gttagcgggc ccattaagtt
     1801 ctgtctcggc gcgtctgcgt ctggctggct ggcataaata tctcactcgc aatcaaattc
     1861 agccgatagc ggaacgggaa ggcgactgga gtgccatgtc cggttttcaa caaaccatgc
     1921 aaatgctgaa tgagggcatc gttcccactg cgatgctggt tgccaacgat cagatggcgc
     1981 tgggcgcaat gcgcgccatt accgagtccg ggctgcgcgt tggtgcggat atctcggtag
     2041 tgggatacga cgataccgaa gacagctcat gttatatccc gccgttaacc accatcaaac
     2101 aggattttcg cctgctgggg caaaccagcg tggaccgctt gctgcaactc tctcagggcc
     2161 aggcggtgaa gggcaatcag ctgttgcccg tctcactggt gaaaagaaaa accaccctgg
     2221 cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac
     2281 gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt aagttagctc
     2341 actcattagg caccgggatc tcgaccgatg cccttgagag ccttcaaccc agtcagctcc
     2401 ttccggtggg cgcggggcat gactacagtc acgtagcgat agcggagtgt atactggctt
     2461 aactatgcgg catcagagca gattgtactg agagtgcacc atatatgcgg tgtgaaatac
     2521 cgcacagatg cgtaaggaga aaataccgca tcaggcgctc ttccgcttcc tcgctcactg
     2581 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     2641 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     2701 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     2761 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     2821 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     2881 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     2941 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     3001 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     3061 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     3121 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     3181 ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     3241 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     3301 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     3361 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     3421 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     3481 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     3541 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     3601 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     3661 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     3721 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     3781 cagttaatag tttgcgcaac gttgttgcca ttgctgcagg catcgtggtg tcacgctcgt
     3841 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     3901 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     3961 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     4021 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     4081 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
     4141 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     4201 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     4261 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     4321 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaaatta
     4381 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa
     4441 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccaccta aattgtaagc
     4501 gttaatgtga accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa
     4561 atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg
     4621 cgagaaagga agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg
     4681 tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtccc
     4741 attcgcca
//
