LOCUS       40924_6197        7515 bp DNA     circular SYN 17-DEC-2018
DEFINITION  Bacterial expression vector pBEN1-SGC, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7515)
  AUTHORS   Savitsky P, Bray J, Cooper CDO., Marsden BD, Mahajan P, 
            Burgess-Brown NA, Gileadi O.
  TITLE     High-throughput production of human proteins for crystallization: 
            the SGC experience
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7515)
  AUTHORS   Gileadi O, Savitsky P, Burgess-Brown NA, Oppermann U, Ng S.
  TITLE     Direct Submission
  JOURNAL   Submitted (08-FEB-2010) SGC, University of Oxford, ORCRB, Roosevelt 
            Drive, Oxford OX3 7DQ, UK
REFERENCE   3  (bases 1 to 7515)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 7515)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (08-FEB-2010) SGC, University of Oxford, ORCRB, Roosevelt Drive, 
            Oxford OX3 7DQ, UK"
COMMENT     SGRef: number: 3; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..7515
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             11..136
                     /codon_start=1
                     /label=SET1
                     /note="solubility-enhancement tag derived from T7 phage
                     gene 10B"
                     /translation="DPEEASVTSTEETLTPAQEAARTRAANKARKEAELAAATAEQ"
     CDS             143..253
                     /codon_start=1
                     /label=SBP
                     /note="streptavidin-binding peptide"
                     /translation="DEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP"
     CDS             284..304
                     /codon_start=1
                     /label=TEV site
                     /note="tobacco etch virus (TEV) protease recognition and 
                     cleavage site"
                     /translation="ENLYFQS"
     promoter        320..765
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             766..2184
                     /codon_start=1
                     /label=SacB
                     /note="secreted levansucrase that renders bacterial growth 
                     sensitive to sucrose"
                     /translation="MNIKKFAKQATVLTFTTALLAGGATQAFAKETNQKPYKETYGISH
                     ITRHDMLQIPEQQKNEKYKVPEFDSSTIKNISSAKGLDVWDSWPLQNTDGTVANYHGYH
                     IVFALAGDPKNADDTSIYMFYQKVGETSIDSWKNAGRVFKDSDKFDANDSILKDQTQEW
                     SGSATFTSDGKIRLFYTDFSGKHYGKQTLTTAQVNVSASDSSLNINGVEDYKSIFDGDG
                     KTYQNVQQFIDEGNYSSGDNHTLRDPHYVEDKGHKYLVFEANTGTEDGYQGEESLFNKA
                     YYGKSTSFFRQESQKLLQSDKKRTAELANGALGMIELNDDYTLKKVMKPLIASNTVTDE
                     IERANVFKMNGKWYLFTDSRGSKMTIDGITSNDIYMLGYVSNSLTGPYKPLNKTGLVLK
                     MDLDPNDVTFTYSHFAVPQAKGNNVVITSYMTNRGFYADKQSTFAPSFLLNIKGKKTSV
                     VKDSILEQGQLTVNK"
     CDS             2295..2312
                     /codon_start=1
                     /label=6xHis
                     /note="6xHis affinity tag"
                     /translation="HHHHHH"
     terminator      2379..2426
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2463..2918
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     CDS             complement(3014..3826)
                     /codon_start=1
                     /label=KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     rep_origin      3948..4536
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     misc_feature    complement(4722..4864)
                     /label=bom
                     /note="basis of mobility region from pBR322"
     CDS             complement(4969..5157)
                     /codon_start=1
                     /label=rop
                     /note="Rop protein, which maintains plasmids at low copy
                     number"
                     /translation="VTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     protein_bind    complement(5932..5953)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             complement(5969..7048)
                     /codon_start=1
                     /label=lacI
                     /note="lac repressor"
                     /translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     promoter        complement(7049..7126)
                     /label=lacI promoter
     promoter        7435..7453
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    7454..7478
                     /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)."
     RBS             7493..7515
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 tatacatatg gaccccgaag aggcgagtgt tacttcaaca gaagaaacct taacgccagc
       61 acaggaggcc gcacgcaccc gcgctgctaa caaagcccga aaggaagctg agttggctgc
      121 tgccaccgct gaacaaacta gtgacgagaa gaccaccggc tggcggggcg gccacgtggt
      181 ggagggcctg gccggcgagc tggagcagct gcgggccagg ctggagcacc accctcaggg
      241 ccagcgggag ccctccggcg gctgcaagct gggcctgggt accgagaacc tgtacttcca
      301 atccatggag accgacgtcc acatatacct gccgttcact attatttagt gaaatgagat
      361 attatgatat tttctgaatt gtgattaaaa aggcaacttt atgcccatgc aacagaaact
      421 ataaaaaata cagagaatga aaagaaacag atagattttt tagttcttta ggcccgtagt
      481 ctgcaaatcc ttttatgatt ttctatcaaa caaaagagga aaatagacca gttgcaatcc
      541 aaacgagagt ctaatagaat gaggtcgaaa agtaaatcgc gcgggtttgt tactgataaa
      601 gcaggcaaga cctaaaatgt gtaaagggca aagtgtatac tttggcgtca ccccttacat
      661 attttaggtc tttttttatt gtgcgtaact aacttgccat cttcaaacag gagggctgga
      721 agaagcagac cgctaacaca gtacataaaa aaggagacat gaacgatgaa catcaaaaag
      781 tttgcaaaac aagcaacagt attaaccttt actaccgcac tgctggcagg aggcgcaact
      841 caagcgtttg cgaaagaaac gaaccaaaag ccatataagg aaacatacgg catttcccat
      901 attacacgcc atgatatgct gcaaatccct gaacagcaaa aaaatgaaaa atataaagtt
      961 cctgagttcg attcgtccac aattaaaaat atctcttctg caaaaggcct ggacgtttgg
     1021 gacagctggc cattacaaaa cactgacggc actgtcgcaa actatcacgg ctaccacatc
     1081 gtctttgcat tagccggaga tcctaaaaat gcggatgaca catcgattta catgttctat
     1141 caaaaagtcg gcgaaacttc tattgacagc tggaaaaacg ctggccgcgt ctttaaagac
     1201 agcgacaaat tcgatgcaaa tgattctatc ctaaaagacc aaacacaaga atggtcaggt
     1261 tcagccacat ttacatctga cggaaaaatc cgtttattct acactgattt ctccggtaaa
     1321 cattacggca aacaaacact gacaactgca caagttaacg tatcagcatc agacagctct
     1381 ttgaacatca acggtgtaga ggattataaa tcaatctttg acggtgacgg aaaaacgtat
     1441 caaaatgtac agcagttcat cgatgaaggc aactacagct caggcgacaa ccatacgctg
     1501 agagatcctc actacgtaga agataaaggc cacaaatact tagtatttga agcaaacact
     1561 ggaactgaag atggctacca aggcgaagaa tctttattta acaaagcata ctatggcaaa
     1621 agcacatcat tcttccgtca agaaagtcaa aaacttctgc aaagcgataa aaaacgcacg
     1681 gctgagttag caaacggcgc tctcggtatg attgagctaa acgatgatta cacactgaaa
     1741 aaagtgatga aaccgctgat tgcatctaac acagtaacag atgaaattga acgcgcgaac
     1801 gtctttaaaa tgaacggcaa atggtacctg ttcactgact cccgcggatc aaaaatgacg
     1861 attgacggca ttacgtctaa cgatatttac atgcttggtt atgtttctaa ttctttaact
     1921 ggcccataca agccgctgaa caaaactggc cttgtgttaa aaatggatct tgatcctaac
     1981 gatgtaacct ttacttactc acacttcgct gtacctcaag cgaaaggaaa caatgtcgtg
     2041 attacaagct atatgacaaa cagaggattc tacgcagaca aacaatcaac gtttgcgcct
     2101 agcttcctgc tgaacatcaa aggcaagaaa acatctgttg tcaaagacag catccttgaa
     2161 caaggacaat taacagttaa caaataaaaa cgcaaaagaa aatgccgata tcctattggc
     2221 attgacggtc tccagtaaag gtggatacgg atccgaattc gagctccgtc gacaagcttg
     2281 cggccgcact cgagcaccac caccaccacc actgagatcc ggctgctaac aaagcccgaa
     2341 aggaagctga gttggctgct gccaccgctg agcaataact agcataaccc cttggggcct
     2401 ctaaacgggt cttgaggggt tttttgctga aaggaggaac tatatccgga ttggcgaatg
     2461 ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
     2521 cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
     2581 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
     2641 tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg
     2701 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag
     2761 tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt
     2821 ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt
     2881 taacgcgaat tttaacaaaa tattaacgtt tacaatttca ggtggcactt ttcggggaaa
     2941 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     3001 gaattaattc ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta ttcatatcag
     3061 gattatcaat accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga
     3121 ggcagttcca taggatggca agatcctggt atcggtctgc gattccgact cgtccaacat
     3181 caatacaacc tattaatttc ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat
     3241 gagtgacgac tgaatccggt gagaatggca aaagtttatg catttctttc cagacttgtt
     3301 caacaggcca gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa ccgttattca
     3361 ttcgtgattg cgcctgagcg agacgaaata cgcgatcgct gttaaaagga caattacaaa
     3421 caggaatcga atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata ttttcacctg
     3481 aatcaggata ttcttctaat acctggaatg ctgttttccc ggggatcgca gtggtgagta
     3541 accatgcatc atcaggagta cggataaaat gcttgatggt cggaagaggc ataaattccg
     3601 tcagccagtt tagtctgacc atctcatctg taacatcatt ggcaacgcta cctttgccat
     3661 gtttcagaaa caactctggc gcatcgggct tcccatacaa tcgatagatt gtcgcacctg
     3721 attgcccgac attatcgcga gcccatttat acccatataa atcagcatcc atgttggaat
     3781 ttaatcgcgg cctagagcaa gacgtttccc gttgaatatg gctcataaca ccccttgtat
     3841 tactgtttat gtaagcagac agttttattg ttcatgacca aaatccctta acgtgagttt
     3901 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     3961 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     4021 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     4081 ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     4141 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     4201 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     4261 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     4321 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     4381 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     4441 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     4501 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     4561 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
     4621 tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg
     4681 accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc
     4741 cttacgcatc tgtgcggtat ttcacaccgc atatatggtg cactctcagt acaatctgct
     4801 ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact gggtcatggc
     4861 tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc
     4921 atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc
     4981 gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga
     5041 ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca gaagcgttaa
     5101 tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt tggtcactga
     5161 tgcctccgtg taagggggat ttctgttcat gggggtaatg ataccgatga aacgagagag
     5221 gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac gttgtgaggg
     5281 taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg gtcaatgcca
     5341 gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc ctgcgatgca
     5401 gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt acgaaacacg
     5461 gaaaccgaag accattcatg ttgttgctca ggtcgcagac gttttgcagc agcagtcgct
     5521 tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac cccgccagcc
     5581 tagccgggtc ctcaacgaca ggagcacgat catgcgcacc cgtggggccg ccatgccggc
     5641 gataatggcc tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga aggcttgagc
     5701 gagggcgtgc aagattccga ataccgcaag cgacaggccg atcatcgtcg cgctccagcg
     5761 aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc acctgtccta cgagttgcat
     5821 gataaagaag acagtcataa gtgcggcgac gatagtcatg ccccgcgccc accggaagga
     5881 gctgactggg ttgaaggctc tcaagggcat cggtcgagat cccggtgcct aatgagtgag
     5941 ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     6001 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca
     6061 gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc accgcctggc
     6121 cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga aaatcctgtt
     6181 tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg tatcccacta
     6241 ccgagatatc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg
     6301 ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc agcatttgca
     6361 tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct atcggctgaa
     6421 tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc gagacagaac
     6481 ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc
     6541 ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc tggtcagaga
     6601 catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg gcatcctggt
     6661 catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg
     6721 ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg ctggcaccca
     6781 gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc agggccagac
     6841 tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt gccacgcggt
     6901 tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa
     6961 cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg gcatactctg
     7021 cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc tcttccgggc
     7081 gctatcatgc cataccgcga aaggttttgc gccattcgat ggtgtccggg atctcgacgc
     7141 tctcccttat gcgactcctg cattaggaag cagcccagta gtaggttgag gccgttgagc
     7201 accgccgccg caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc cccggccacg
     7261 gggcctgcca ccatacccac gccgaaacaa gcgctcatga gcccgaagtg gcgagcccga
     7321 tcttccccat cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt ggcgccggtg
     7381 atgccggcca cgatgcgtcc ggcgtagagg atcgagatct cgatcccgcg aaattaatac
     7441 gactcactat aggggaattg tgagcggata acaattcccc tctagaaata attttgttta
     7501 actttaagaa ggaga
//
