LOCUS       40924_30356        5940 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Compact bacterial vector encoding N-terminal MBP-TVMV and C-terminal
            TEV-6xHis, plus tRNA genes for rare Arg and Ile codons.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5940)
  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 5940)
  AUTHORS   Midwest Center for Structural Genomics
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 5940)
  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..5940
                     /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"
     CDS             complement(263..283)
                     /codon_start=1
                     /label=TVMV site
                     /note="tobacco vein mottling virus (TVMV) NIa protease 
                     recognition and cleavage site"
                     /translation="ETVRFQS"
     protein_bind    complement(286..310)
                     /label=attB1
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             complement(320..1420)
                     /codon_start=1
                     /label=MBP
                     /note="maltose binding protein from E. coli"
                     /translation="MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE
                     EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL
                     IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA
                     ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE
                     TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL
                     ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF
                     WYAVRTAVINAASGRQTVDEALKDAQT"
     RBS             complement(1428..1450)
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    complement(1465..1489)
                     /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(1490..1508)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     tRNA            complement(1783..1859)
                     /label=argU
     tRNA            complement(2129..2204)
                     /label=ileX
     promoter        2344..2421
                     /label=lacI promoter
     CDS             2422..3501
                     /codon_start=1
                     /label=lacI
                     /note="lac repressor"
                     /translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     protein_bind    3517..3538
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(3933..4521)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(4695..5552)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(5553..5656)
                     /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 agactctgga aacgcacggt ttccgagcct gcttttttgt
      301 acaaacttgt gatcgaatta gtctgcgcgt ctttcagggc ttcatcgaca gtctgacgac
      361 cgctggcggc gttgatcacc gcagtacgca cggcatacca gaaagcggac atctgcggga
      421 tgttcggcat gatttcacct ttctgggcgt tttccatggt ggcggcaata cgtggatctt
      481 tcgccaactc ttcctcgtaa gacttcagcg ctacggcacc cagcggtttg tctttattaa
      541 ccgcttccag accttcatca gtcagcagat agttttcgag gaactctttt gccagctctt
      601 tgttcggact ggcggcgtta atacctgcgc tcagcacgcc aacgaacggt ttggatggtt
      661 gacccttgaa ggtcggcagt accgttacac cataattcac tttgctggtg tcgatgttgg
      721 accatgccca cgggccgttg atggtcatcg ctgtttcgcc tttattaaag gcagcttctg
      781 cgatggagta atcggtgtct gcattcatgt gtttgttttt aatcaggtca accaggaagg
      841 tcagacccgc tttcgcgcca gcgttatcca cgcccacgtc tttaatgtcg tacttgccgt
      901 tttcatactt gaacgcataa cccccgtcag cagcaatcag cggccaggtg aagtacggtt
      961 cttgcaggtt gaacatcagc gcgctcttac ctttcgcttt cagttcttta tccagcgccg
     1021 ggatctcttc ccaggttttt ggcgggttcg gcagcagatc tttgttataa atcagcgata
     1081 acgcttcaac agcgatcggg taagcaatca gcttgccgtt gtaacgtacg gcatcccagg
     1141 taaacggata cagcttgtcc tggaacgctt tgtccggggt gatttcagcc aacaggccag
     1201 attgagcgta gccaccaaag cggtcgtgtg cccagaagat aatgtcaggg ccatcgccag
     1261 ttgccgcaac ctgtgggaat ttctcttcca gtttatccgg atgctcaacg gtgactttaa
     1321 ttccggtatc tttctcgaat ttcttaccga cttcagcgag accgttatag cctttatcgc
     1381 cgttaatcca gattaccagt ttaccttctt cgattttcat atgtatatct ccttcttaaa
     1441 gttaaacaaa attatttcta gaggggaatt gttatccgct cacaattccc ctatagtgag
     1501 tcgtattaat ttcgcgggat cgagatcgat ctcgatcctc tacgccggac gcatcgtggc
     1561 cggcatcacc ggtggattca gtcaggcgtc ccattatcag tgcttcagga aatgggcgga
     1621 tgggagtcca tagaaatggt tcgtaggtat gctcaccttg cgcctaatca tttgacagag
     1681 catgcgagga aaatagacga catttttggt gataatgtcc caaatatgtc ccactctgga
     1741 attatggagg atataaagaa ggcgtaactg attgaattgt aatggcgcgc cctgcaggat
     1801 tcgaacctgc ggcccacgac ttagaaggtc gttgctctat ccaactgagc taagggcgcg
     1861 ttgataccgc aatgcggtgt aatcgcgtga attatacggt caacccttgc tgagtcaatg
     1921 gcttttgatc tcgccggcgc cacaggtgcg gttgctggcg cctatatcgc cgacatcacc
     1981 gatggggaag atcgggctcg ccacttcggg ctcatgagcg cttgtttcgg cgtgggtatg
     2041 gtggcaggcc ccgtggccgg gggactgttg ggcgccatct ccttgcatgc aaaaaggctg
     2101 acgatttctc gtcagccttt gctatatctg gtggcccctg ctggacttga accagcgacc
     2161 aagcgattat gagtcgcctg ctctaaccac tgagctaagg ggccatggta gcggattata
     2221 aagtaactcc gtgtcgcaat ccgcatgcac cattccttgc ggcggcggtg ctcaacggcc
     2281 tcaacctact actgggctgc ttcctaatgc aggagtcgca taagggagag cgtcgagatc
     2341 ccggacacca tcgaatggcg caaaaccttt cgcggtatgg catgatagcg cccggaagag
     2401 agtcaattca gggtggtgaa tgtgaaacca gtaacgttat acgatgtcgc agagtatgcc
     2461 ggtgtctctt atcagaccgt ttcccgcgtg gtgaaccagg ccagccacgt ttctgcgaaa
     2521 acgcgggaaa aagtggaagc ggcgatggcg gagctgaatt acattcccaa ccgcgtggca
     2581 caacaactgg cgggcaaaca gtcgttgctg attggcgttg ccacctccag tctggccctg
     2641 cacgcgccgt cgcaaattgt cgcggcgatt aaatctcgcg ccgatcaact gggtgccagc
     2701 gtggtggtgt cgatggtaga acgaagcggc gtcgaagcct gtaaagcggc ggtgcacaat
     2761 cttctcgcgc aacgcgtcag tgggctgatc attaactatc cgctggatga ccaggatgcc
     2821 attgctgtgg aagctgcctg cactaatgtt ccggcgttat ttcttgatgt ctctgaccag
     2881 acacccatca acagtattat tttctcccat gaagacggta cgcgactggg cgtggagcat
     2941 ctggtcgcat tgggtcacca gcaaatcgcg ctgttagcgg gcccattaag ttctgtctcg
     3001 gcgcgtctgc gtctggctgg ctggcataaa tatctcactc gcaatcaaat tcagccgata
     3061 gcggaacggg aaggcgactg gagtgccatg tccggttttc aacaaaccat gcaaatgctg
     3121 aatgagggca tcgttcccac tgcgatgctg gttgccaacg atcagatggc gctgggcgca
     3181 atgcgcgcca ttaccgagtc cgggctgcgc gttggtgcgg atatctcggt agtgggatac
     3241 gacgataccg aagacagctc atgttatatc ccgccgttaa ccaccatcaa acaggatttt
     3301 cgcctgctgg ggcaaaccag cgtggaccgc ttgctgcaac tctctcaggg ccaggcggtg
     3361 aagggcaatc agctgttgcc cgtctcactg gtgaaaagaa aaaccaccct ggcgcccaat
     3421 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt
     3481 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtaagttagc tcactcatta
     3541 ggcaccggga tctcgaccga tgcccttgag agccttcaac ccagtcagct ccttccggtg
     3601 ggcgcggggc atgactacag tcacgtagcg atagcggagt gtatactggc ttaactatgc
     3661 ggcatcagag cagattgtac tgagagtgca ccatatatgc ggtgtgaaat accgcacaga
     3721 tgcgtaagga gaaaataccg catcaggcgc tcttccgctt cctcgctcac tgactcgctg
     3781 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta
     3841 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc
     3901 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
     3961 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
     4021 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
     4081 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt
     4141 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
     4201 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
     4261 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
     4321 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta
     4381 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
     4441 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
     4501 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag
     4561 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc
     4621 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact
     4681 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt
     4741 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta
     4801 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta
     4861 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc
     4921 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat
     4981 agtttgcgca acgttgttgc cattgctgca ggcatcgtgg tgtcacgctc gtcgtttggt
     5041 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg
     5101 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca
     5161 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta
     5221 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg
     5281 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact
     5341 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg
     5401 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt
     5461 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga
     5521 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaaat tattgaagca
     5581 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     5641 aaataggggt tccgcgcaca tttccccgaa aagtgccacc taaattgtaa gcgttaatgt
     5701 gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac
     5761 cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag
     5821 gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg
     5881 cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc ccattcgcca
//
