LOCUS       40924_30361        6227 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Compact bacterial vector encoding tRNA genes for rare Arg and Ile 
            codons, with a NusA-TVMV-6xHis-Strep-Tag(R) II-TEV leader.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6227)
  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 6227)
  AUTHORS   Midwest Center for Structural Genomics
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 6227)
  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..6227
                     /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(155..175)
                     /codon_start=1
                     /label=TEV site
                     /note="tobacco etch virus (TEV) protease recognition and 
                     cleavage site"
                     /translation="ENLYFQS"
     CDS             complement(176..199)
                     /codon_start=1
                     /label=Strep-Tag II
                     /note="peptide that binds Strep-Tactin(R), an engineered 
                     form
                     of streptavidin"
                     /translation="WSHPQFEK"
     CDS             complement(200..217)
                     /codon_start=1
                     /label=6xHis
                     /note="6xHis affinity tag"
                     /translation="HHHHHH"
     CDS             complement(218..238)
                     /codon_start=1
                     /label=TVMV site
                     /note="tobacco vein mottling virus (TVMV) NIa protease 
                     recognition and cleavage site"
                     /translation="ETVRFQS"
     CDS             complement(248..1708)
                     /codon_start=1
                     /label=NusA
                     /note="transcription elongation protein (N utilization
                     substance protein A)"
                     /translation="MNKEILAVVEAVSNEKALPREKIFEALESALATATKKKYEQEIDV
                     RVQIDRKSGDFDTFRRWLVVDEVTQPTKEITLEAARYEDESLNLGDYVEDQIESVTFDR
                     ITTQTAKQVIVQKVREAERAMVVDQFREHEGEIITGVVKKVNRDNISLDLGNNAEAVIL
                     REDMLPRENFRPGDRVRGVLYSVRPEARGAQLFVTRSKPEMLIELFRIEVPEIGEEVIE
                     IKAAARDPGSRAKIAVKTNDKRIDPVGACVGMRGARVQAVSTELGGERIDIVLWDDNPA
                     QFVINAMAPADVASIVVDEDKHTMDIAVEAGNLAQAIGRNGQNVRLASQLSGWELNVMT
                     VDDLQAKHQAEAHAAIDTFTKYLDIDEDFATVLVEEGFSTLEELAYVPMKELLEIEGLD
                     EPTVEALRERAKNALATIAQAQEESLGDNKPADDLLNLEGVDRDLAFKLAARGVCTLED
                     LAEQGIDDLADIEGLTDEKAGALIMAARN"
     RBS             complement(1716..1738)
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    complement(1752..1776)
                     /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(1777..1795)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     tRNA            complement(2070..2146)
                     /label=argU
     tRNA            complement(2416..2491)
                     /label=ileX
     promoter        2631..2708
                     /label=lacI promoter
     CDS             2709..3788
                     /codon_start=1
                     /label=lacI
                     /note="lac repressor"
                     /translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     protein_bind    3804..3825
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(4220..4808)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(4982..5839)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(5840..5943)
                     /label=AmpR promoter
ORIGIN
        1 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa
       61 ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt
      121 tgttagcagc cggatgttat ccacttccaa tattggattg gaagtacagg ttctccttct
      181 cgaactgtgg atgagaccag tgatgatgat ggtggtgaga ctggaaacgc acggtttcgt
      241 tgttgttatt acgggcagcc ataatcagtg ctccggcttt ttcgtcggtc aacccttcga
      301 tatcagccag atcatcaatg ccctgttcgg cgagatcttc cagcgtacaa acgccacggg
      361 cggccagttt gaatgccaaa tcacgatcta ccccttcaag gttcagcaga tcgtcagccg
      421 gtttgttatc accgaggctt tcttcctggg cctgtgcaat ggtggccagt gcatttttag
      481 cacgctcgcg cagtgcttca acggtcggct catcaaggcc ttcgatttcc aacagctctt
      541 tcatcggcac ataggccaat tcttccagcg tcgagaagcc ttcttctacc agaacagtcg
      601 cgaagtcttc gtcgatgtcg agatatttgg tgaaggtgtc gatcgctgcg tgcgcttccg
      661 cctgatgctt agcttgcagg tcgtcaacgg tcatcacgtt gagttcccaa ccgctcagtt
      721 gcgaagccag acgcacgttc tgaccgttac ggccaatcgc ctgcgccaga ttaccggctt
      781 caacggcgat gtccatggtg tgtttatctt catccaccac gatagaagca acgtctgccg
      841 gtgccattgc gttaatcacg aactgcgccg ggttatcatc ccacaggacg atatcgatac
      901 gctcgccacc cagttcagta gacaccgcct gaacacgcgc gccacgcata cctacgcaag
      961 cacctaccgg atcgatacgt ttatcgttgg ttttcaccgc gattttcgca cgagaacccg
     1021 gatcgcgagc cgctgcttta atttcaatca cttcttcgcc gatttctggc acttcaatac
     1081 ggaacagttc gatcagcatt tccggcttgg aacgagtgac gaacagttgc gcgccacgcg
     1141 cttccgggcg aacggaatag agcacgccac gaacgcggtc gccagggcgg aagttttcac
     1201 gcggcagcat atcttcgcgc aggatcacgg cttcagcgtt gttgcccaga tccagagaga
     1261 tgttgtcgcg gtttactttt ttcaccacgc cggtgatgat ttcaccttcg tgttcacgga
     1321 actgatcaac caccatcgca cgttcggctt cacgcacttt ctgcacgata acctgttttg
     1381 ccgtctgggt agtgatacgg tcaaaggtaa cagactcaat ctgatcttca acgtaatcgc
     1441 ccaggttcag gctttcatct tcataacgtg cggcttcaag ggtgatttcc ttggtcggct
     1501 gggtgacttc atcaacaact aaccagcgac ggaaagtgtc aaaatcaccg cttttgcgat
     1561 cgatctgtac gcggacgtcg atctcttgtt catatttttt ctttgttgct gtcgccagcg
     1621 cgctttccaa tgcttcgaaa atcttctcgc gaggtagcgc cttttcattg gatacggctt
     1681 caactacagc caaaatttct ttgttcatat gtatatctcc ttcttaaagt taaacaaaat
     1741 tattctagag gggaattgtt atccgctcac aattccccta tagtgagtcg tattaatttc
     1801 gcgggatcga gatcgatctc gatcctctac gccggacgca tcgtggccgg catcaccggt
     1861 ggattcagtc aggcgtccca ttatcagtgc ttcaggaaat gggcggatgg gagtccatag
     1921 aaatggttcg taggtatgct caccttgcgc ctaatcattt gacagagcat gcgaggaaaa
     1981 tagacgacat ttttggtgat aatgtcccaa atatgtccca ctctggaatt atggaggata
     2041 taaagaaggc gtaactgatt gaattgtaat ggcgcgccct gcaggattcg aacctgcggc
     2101 ccacgactta gaaggtcgtt gctctatcca actgagctaa gggcgcgttg ataccgcaat
     2161 gcggtgtaat cgcgtgaatt atacggtcaa cccttgctga gtcaatggct tttgatctcg
     2221 ccggcgccac aggtgcggtt gctggcgcct atatcgccga catcaccgat ggggaagatc
     2281 gggctcgcca cttcgggctc atgagcgctt gtttcggcgt gggtatggtg gcaggccccg
     2341 tggccggggg actgttgggc gccatctcct tgcatgcaaa aaggctgacg atttctcgtc
     2401 agcctttgct atatctggtg gcccctgctg gacttgaacc agcgaccaag cgattatgag
     2461 tcgcctgctc taaccactga gctaaggggc catggtagcg gattataaag taactccgtg
     2521 tcgcaatccg catgcaccat tccttgcggc ggcggtgctc aacggcctca acctactact
     2581 gggctgcttc ctaatgcagg agtcgcataa gggagagcgt cgagatcccg gacaccatcg
     2641 aatggcgcaa aacctttcgc ggtatggcat gatagcgccc ggaagagagt caattcaggg
     2701 tggtgaatgt gaaaccagta acgttatacg atgtcgcaga gtatgccggt gtctcttatc
     2761 agaccgtttc ccgcgtggtg aaccaggcca gccacgtttc tgcgaaaacg cgggaaaaag
     2821 tggaagcggc gatggcggag ctgaattaca ttcccaaccg cgtggcacaa caactggcgg
     2881 gcaaacagtc gttgctgatt ggcgttgcca cctccagtct ggccctgcac gcgccgtcgc
     2941 aaattgtcgc ggcgattaaa tctcgcgccg atcaactggg tgccagcgtg gtggtgtcga
     3001 tggtagaacg aagcggcgtc gaagcctgta aagcggcggt gcacaatctt ctcgcgcaac
     3061 gcgtcagtgg gctgatcatt aactatccgc tggatgacca ggatgccatt gctgtggaag
     3121 ctgcctgcac taatgttccg gcgttatttc ttgatgtctc tgaccagaca cccatcaaca
     3181 gtattatttt ctcccatgaa gacggtacgc gactgggcgt ggagcatctg gtcgcattgg
     3241 gtcaccagca aatcgcgctg ttagcgggcc cattaagttc tgtctcggcg cgtctgcgtc
     3301 tggctggctg gcataaatat ctcactcgca atcaaattca gccgatagcg gaacgggaag
     3361 gcgactggag tgccatgtcc ggttttcaac aaaccatgca aatgctgaat gagggcatcg
     3421 ttcccactgc gatgctggtt gccaacgatc agatggcgct gggcgcaatg cgcgccatta
     3481 ccgagtccgg gctgcgcgtt ggtgcggata tctcggtagt gggatacgac gataccgaag
     3541 acagctcatg ttatatcccg ccgttaacca ccatcaaaca ggattttcgc ctgctggggc
     3601 aaaccagcgt ggaccgcttg ctgcaactct ctcagggcca ggcggtgaag ggcaatcagc
     3661 tgttgcccgt ctcactggtg aaaagaaaaa ccaccctggc gcccaatacg caaaccgcct
     3721 ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa
     3781 gcgggcagtg agcgcaacgc aattaatgta agttagctca ctcattaggc accgggatct
     3841 cgaccgatgc ccttgagagc cttcaaccca gtcagctcct tccggtgggc gcggggcatg
     3901 actacagtca cgtagcgata gcggagtgta tactggctta actatgcggc atcagagcag
     3961 attgtactga gagtgcacca tatatgcggt gtgaaatacc gcacagatgc gtaaggagaa
     4021 aataccgcat caggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     4081 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     4141 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     4201 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     4261 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     4321 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     4381 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     4441 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     4501 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     4561 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     4621 agttcttgaa gtggtggcct aactacggct acactagaag gacagtattt ggtatctgcg
     4681 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     4741 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     4801 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     4861 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     4921 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     4981 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     5041 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     5101 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     5161 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     5221 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     5281 ttgttgccat tgctgcaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     5341 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     5401 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     5461 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     5521 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     5581 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     5641 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     5701 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     5761 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     5821 ggaaatgttg aatactcata ctcttccttt ttcaaattat tgaagcattt atcagggtta
     5881 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
     5941 gcgcacattt ccccgaaaag tgccacctaa attgtaagcg ttaatgtgaa ccatcaccct
     6001 aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc
     6061 cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag
     6121 cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca
     6181 cacccgccgc gcttaatgcg ccgctacagg gcgcgtccca ttcgcca
//
