LOCUS       40924_33252        7284 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Expression vector pNIC28-Bsa4, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7284)
  AUTHORS   Stols L, Gu M, Dieckman L, Raffen R, Collart FR, Donnelly MI.
  TITLE     A new vector for high-throughput, ligation-independent cloning 
            encoding a tobacco etch virus protease cleavage site
  JOURNAL   Protein Expr. Purif. 25 (1), 8-15 (2002)
  PUBMED    12071693
REFERENCE   2  (bases 1 to 7284)
  AUTHORS   Keates T, Cooper CD, Savitsky P, Allerston CK, Phillips C, 
            Hammarstrom M, Daga N, Berridge G, Mahajan P, Burgess-Brown NA, 
            Muller S, Graslund S, Gileadi O.
  TITLE     Expressing the human proteome for affinity proteomics: optimising 
            expression of soluble protein domains and in vivo biotinylation
  JOURNAL   N Biotechnol (2011) In press
  PUBMED    22027370
REFERENCE   3  (bases 1 to 7284)
  AUTHORS   Gileadi O, Burgess-Brown N, Loppnau P.
  TITLE     Direct Submission
  JOURNAL   Submitted (25-DEC-2006) Structural Genomics Consortium, University 
            of Oxford, Botnar Research Centre, Oxford OX3 7LD, UK
REFERENCE   4  (bases 1 to 7284)
  TITLE     Direct Submission
REFERENCE   5  (bases 1 to 7284)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Protein 
            Expr. Purif."; date: "2002"; volume: "25"; issue: "1"; pages: "8-15"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "N 
            Biotechnol (2011) In press"
COMMENT     SGRef: number: 3; type: "Journal Article"; journalName: "Submitted 
            (25-DEC-2006) Structural Genomics Consortium, University of Oxford, 
            Botnar Research Centre, Oxford OX3 7LD, UK"
COMMENT     SGRef: number: 4; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..7284
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /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             59..81
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             92..109
                     /label=6xHis
                     /note="6xHis affinity tag"
     CDS             134..154
                     /label=TEV site
                     /note="tobacco etch virus (TEV) protease recognition and 
                     cleavage site"
     promoter        170..615
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             616..2034
                     /label=SacB
                     /note="secreted levansucrase that renders bacterial growth 
                     sensitive to sucrose"
     misc_feature    complement(2083..2097)
                     /note="3'-LIC sequence; add complement to downstream PCR
                     primer"
     CDS             2145..2162
                     /label=6xHis
                     /note="6xHis affinity tag"
     terminator      2229..2276
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2313..2768
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     CDS             complement(2864..3676)
                     /label=KanR
                     /note="aminoglycoside phosphotransferase"
     rep_origin      3798..4386
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     misc_feature    complement(4572..4714)
                     /label=bom
                     /note="basis of mobility region from pBR322"
     CDS             complement(4819..5007)
                     /label=rop
                     /note="Rop protein, which maintains plasmids at low copy
                     number"
     protein_bind    complement(5782..5803)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             complement(5819..6898)
                     /label=lacI
                     /note="lac repressor"
     promoter        complement(6899..6976)
                     /label=lacI promoter
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atatacatat gcaccatcat catcatcatt cttctggtgt
      121 agatctgggt accgagaacc tgtacttcca atccatggag accgacgtcc acatatacct
      181 gccgttcact attatttagt gaaatgagat attatgatat tttctgaatt gtgattaaaa
      241 aggcaacttt atgcccatgc aacagaaact ataaaaaata cagagaatga aaagaaacag
      301 atagattttt tagttcttta ggcccgtagt ctgcaaatcc ttttatgatt ttctatcaaa
      361 caaaagagga aaatagacca gttgcaatcc aaacgagagt ctaatagaat gaggtcgaaa
      421 agtaaatcgc gcgggtttgt tactgataaa gcaggcaaga cctaaaatgt gtaaagggca
      481 aagtgtatac tttggcgtca ccccttacat attttaggtc tttttttatt gtgcgtaact
      541 aacttgccat cttcaaacag gagggctgga agaagcagac cgctaacaca gtacataaaa
      601 aaggagacat gaacgatgaa catcaaaaag tttgcaaaac aagcaacagt attaaccttt
      661 actaccgcac tgctggcagg aggcgcaact caagcgtttg cgaaagaaac gaaccaaaag
      721 ccatataagg aaacatacgg catttcccat attacacgcc atgatatgct gcaaatccct
      781 gaacagcaaa aaaatgaaaa atataaagtt cctgagttcg attcgtccac aattaaaaat
      841 atctcttctg caaaaggcct ggacgtttgg gacagctggc cattacaaaa cactgacggc
      901 actgtcgcaa actatcacgg ctaccacatc gtctttgcat tagccggaga tcctaaaaat
      961 gcggatgaca catcgattta catgttctat caaaaagtcg gcgaaacttc tattgacagc
     1021 tggaaaaacg ctggccgcgt ctttaaagac agcgacaaat tcgatgcaaa tgattctatc
     1081 ctaaaagacc aaacacaaga atggtcaggt tcagccacat ttacatctga cggaaaaatc
     1141 cgtttattct acactgattt ctccggtaaa cattacggca aacaaacact gacaactgca
     1201 caagttaacg tatcagcatc agacagctct ttgaacatca acggtgtaga ggattataaa
     1261 tcaatctttg acggtgacgg aaaaacgtat caaaatgtac agcagttcat cgatgaaggc
     1321 aactacagct caggcgacaa ccatacgctg agagatcctc actacgtaga agataaaggc
     1381 cacaaatact tagtatttga agcaaacact ggaactgaag atggctacca aggcgaagaa
     1441 tctttattta acaaagcata ctatggcaaa agcacatcat tcttccgtca agaaagtcaa
     1501 aaacttctgc aaagcgataa aaaacgcacg gctgagttag caaacggcgc tctcggtatg
     1561 attgagctaa acgatgatta cacactgaaa aaagtgatga aaccgctgat tgcatctaac
     1621 acagtaacag atgaaattga acgcgcgaac gtctttaaaa tgaacggcaa atggtacctg
     1681 ttcactgact cccgcggatc aaaaatgacg attgacggca ttacgtctaa cgatatttac
     1741 atgcttggtt atgtttctaa ttctttaact ggcccataca agccgctgaa caaaactggc
     1801 cttgtgttaa aaatggatct tgatcctaac gatgtaacct ttacttactc acacttcgct
     1861 gtacctcaag cgaaaggaaa caatgtcgtg attacaagct atatgacaaa cagaggattc
     1921 tacgcagaca aacaatcaac gtttgcgcct agcttcctgc tgaacatcaa aggcaagaaa
     1981 acatctgttg tcaaagacag catccttgaa caaggacaat taacagttaa caaataaaaa
     2041 cgcaaaagaa aatgccgata tcctattggc attgacggtc tccagtaaag gtggatacgg
     2101 atccgaattc gagctccgtc gacaagcttg cggccgcact cgagcaccac caccaccacc
     2161 actgagatcc ggctgctaac aaagcccgaa aggaagctga gttggctgct gccaccgctg
     2221 agcaataact agcataaccc cttggggcct ctaaacgggt cttgaggggt tttttgctga
     2281 aaggaggaac tatatccgga ttggcgaatg ggacgcgccc tgtagcggcg cattaagcgc
     2341 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc
     2401 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
     2461 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
     2521 acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
     2581 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
     2641 caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg
     2701 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt
     2761 tacaatttca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt
     2821 ctaaatacat tcaaatatgt atccgctcat gaattaattc ttagaaaaac tcatcgagca
     2881 tcaaatgaaa ctgcaattta ttcatatcag gattatcaat accatatttt tgaaaaagcc
     2941 gtttctgtaa tgaaggagaa aactcaccga ggcagttcca taggatggca agatcctggt
     3001 atcggtctgc gattccgact cgtccaacat caatacaacc tattaatttc ccctcgtcaa
     3061 aaataaggtt atcaagtgag aaatcaccat gagtgacgac tgaatccggt gagaatggca
     3121 aaagtttatg catttctttc cagacttgtt caacaggcca gccattacgc tcgtcatcaa
     3181 aatcactcgc atcaaccaaa ccgttattca ttcgtgattg cgcctgagcg agacgaaata
     3241 cgcgatcgct gttaaaagga caattacaaa caggaatcga atgcaaccgg cgcaggaaca
     3301 ctgccagcgc atcaacaata ttttcacctg aatcaggata ttcttctaat acctggaatg
     3361 ctgttttccc ggggatcgca gtggtgagta accatgcatc atcaggagta cggataaaat
     3421 gcttgatggt cggaagaggc ataaattccg tcagccagtt tagtctgacc atctcatctg
     3481 taacatcatt ggcaacgcta cctttgccat gtttcagaaa caactctggc gcatcgggct
     3541 tcccatacaa tcgatagatt gtcgcacctg attgcccgac attatcgcga gcccatttat
     3601 acccatataa atcagcatcc atgttggaat ttaatcgcgg cctagagcaa gacgtttccc
     3661 gttgaatatg gctcataaca ccccttgtat tactgtttat gtaagcagac agttttattg
     3721 ttcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     3781 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     3841 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     3901 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg
     3961 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     4021 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     4081 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     4141 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     4201 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     4261 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     4321 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     4381 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     4441 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag
     4501 tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa
     4561 gcggaagagc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc
     4621 atatatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatacac
     4681 tccgctatcg ctacgtgact gggtcatggc tgcgccccga cacccgccaa cacccgctga
     4741 cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc
     4801 cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg
     4861 gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc
     4921 cagctcgttg agtttctcca gaagcgttaa tgtctggctt ctgataaagc gggccatgtt
     4981 aagggcggtt ttttcctgtt tggtcactga tgcctccgtg taagggggat ttctgttcat
     5041 gggggtaatg ataccgatga aacgagagag gatgctcacg atacgggtta ctgatgatga
     5101 acatgcccgg ttactggaac gttgtgaggg taaacaactg gcggtatgga tgcggcggga
     5161 ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc
     5221 acagggtagc cagcagcatc ctgcgatgca gatccggaac ataatggtgc agggcgctga
     5281 cttccgcgtt tccagacttt acgaaacacg gaaaccgaag accattcatg ttgttgctca
     5341 ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt
     5401 ctgctaacca gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca ggagcacgat
     5461 catgcgcacc cgtggggccg ccatgccggc gataatggcc tgcttctcgc cgaaacgttt
     5521 ggtggcggga ccagtgacga aggcttgagc gagggcgtgc aagattccga ataccgcaag
     5581 cgacaggccg atcatcgtcg cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag
     5641 cgctgccggc acctgtccta cgagttgcat gataaagaag acagtcataa gtgcggcgac
     5701 gatagtcatg ccccgcgccc accggaagga gctgactggg ttgaaggctc tcaagggcat
     5761 cggtcgagat cccggtgcct aatgagtgag ctaacttaca ttaattgcgt tgcgctcact
     5821 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc
     5881 ggggagaggc ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc agtgagacgg
     5941 gcaacagctg attgcccttc accgcctggc cctgagagag ttgcagcaag cggtccacgc
     6001 tggtttgccc cagcaggcga aaatcctgtt tgatggtggt taacggcggg atataacatg
     6061 agctgtcttc ggtatcgtcg tatcccacta ccgagatatc cgcaccaacg cgcagcccgg
     6121 actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc agcatcgcag
     6181 tgggaacgat gccctcattc agcatttgca tggtttgttg aaaaccggac atggcactcc
     6241 agtcgccttc ccgttccgct atcggctgaa tttgattgcg agtgagatat ttatgccagc
     6301 cagccagacg cagacgcgcc gagacagaac ttaatgggcc cgctaacagc gcgatttgct
     6361 ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa
     6421 taatactgtt gatgggtgtc tggtcagaga catcaagaaa taacgccgga acattagtgc
     6481 aggcagcttc cacagcaatg gcatcctggt catccagcgg atagttaatg atcagcccac
     6541 tgacgcgttg cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt
     6601 ctaccatcga caccaccacg ctggcaccca gttgatcggc gcgagattta atcgccgcga
     6661 caatttgcga cggcgcgtgc agggccagac tggaggtggc aacgccaatc agcaacgact
     6721 gtttgcccgc cagttgttgt gccacgcggt tgggaatgta attcagctcc gccatcgccg
     6781 cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa
     6841 cggtctgata agagacaccg gcatactctg cgacatcgta taacgttact ggtttcacat
     6901 tcaccaccct gaattgactc tcttccgggc gctatcatgc cataccgcga aaggttttgc
     6961 gccattcgat ggtgtccggg atctcgacgc tctcccttat gcgactcctg cattaggaag
     7021 cagcccagta gtaggttgag gccgttgagc accgccgccg caaggaatgg tgcatgcaag
     7081 gagatggcgc ccaacagtcc cccggccacg gggcctgcca ccatacccac gccgaaacaa
     7141 gcgctcatga gcccgaagtg gcgagcccga tcttccccat cggtgatgtc ggcgatatag
     7201 gcgccagcaa ccgcacctgt ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg
     7261 atcgagatct cgatcccgcg aaat
//
