LOCUS       40924_33207        7335 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Expression vector pNIC-Bio2, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7335)
  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 29 (5), 515-525 (2012)
  PUBMED    22027370
REFERENCE   2  (bases 1 to 7335)
  AUTHORS   Graslund S, Hammarstrom M, Gileadi O.
  TITLE     Direct Submission
  JOURNAL   Submitted (03-MAY-2011) Structural Genomics Consortium, SGC, 
            University of Oxford, ORCRB, Roosevelt Drive, Oxford OX3 7DQ, UK
REFERENCE   3  (bases 1 to 7335)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 7335)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "N 
            Biotechnol"; date: "2012"; volume: "29"; issue: "5"; pages: 
            "515-525"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (03-MAY-2011) Structural Genomics Consortium, SGC, University of 
            Oxford, ORCRB, Roosevelt Drive, Oxford OX3 7DQ, UK"
COMMENT     SGRef: number: 3; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..7335
                     /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..118
                     /label=9xHis
                     /note="9xHis 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(2084..2097)
                     /note="3'-LIC sequence; add complement to downstream PCR
                     primer"
     CDS             2099..2143
                     /label=AviTag(TM)
                     /note="peptide tag that allows for enzymatic biotinylation"
     CDS             2196..2213
                     /label=6xHis
                     /note="6xHis affinity tag"
     terminator      2280..2327
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2364..2819
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     CDS             complement(2915..3727)
                     /label=KanR
                     /note="aminoglycoside phosphotransferase"
     rep_origin      3849..4437
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     misc_feature    complement(4623..4765)
                     /label=bom
                     /note="basis of mobility region from pBR322"
     CDS             complement(4870..5058)
                     /label=rop
                     /note="Rop protein, which maintains plasmids at low copy
                     number"
     protein_bind    complement(5833..5854)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             complement(5870..6949)
                     /label=lacI
                     /note="lac repressor"
     promoter        complement(6950..7027)
                     /label=lacI promoter
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atatacatat gcaccatcat catcatcatc accatcatca
      121 cgatctgggt 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 atatcaagtt cctgaattcg attcgtccac aattaaaaat
      841 atctcttctg caaaaggcct ggacgtttgg gacagctggc cattacaaaa cgctgacggc
      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 gtttgcgcca agcttcctgc tgaacatcaa aggcaagaaa
     1981 acatctgttg tcaaagacag catccttgaa caaggacaat taacagttaa caaataaaaa
     2041 cgcaaaagaa aatgccgata tcctattggc attgacggtc tccagtaaag gtggatacgg
     2101 cctgaatgat atctttgaag cgcagaagat tgaatggcat gaatgataag gatccgaatt
     2161 cgagctccgt cgacaagctt gcggccgcac tcgagcacca ccaccaccac cactgagatc
     2221 cggctgctaa caaagcccga aaggaagctg agttggctgc tgccaccgct gagcaataac
     2281 tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg aaaggaggaa
     2341 ctatatccgg attggcgaat gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     2401 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
     2461 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     2521 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta
     2581 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     2641 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat
     2701 ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa
     2761 tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt ttacaatttc
     2821 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     2881 ttcaaatatg tatccgctca tgaattaatt cttagaaaaa ctcatcgagc atcaaatgaa
     2941 actgcaattt attcatatca ggattatcaa taccatattt ttgaaaaagc cgtttctgta
     3001 atgaaggaga aaactcaccg aggcagttcc ataggatggc aagatcctgg tatcggtctg
     3061 cgattccgac tcgtccaaca tcaatacaac ctattaattt cccctcgtca aaaataaggt
     3121 tatcaagtga gaaatcacca tgagtgacga ctgaatccgg tgagaatggc aaaagtttat
     3181 gcatttcttt ccagacttgt tcaacaggcc agccattacg ctcgtcatca aaatcactcg
     3241 catcaaccaa accgttattc attcgtgatt gcgcctgagc gagacgaaat acgcgatcgc
     3301 tgttaaaagg acaattacaa acaggaatcg aatgcaaccg gcgcaggaac actgccagcg
     3361 catcaacaat attttcacct gaatcaggat attcttctaa tacctggaat gctgttttcc
     3421 cggggatcgc agtggtgagt aaccatgcat catcaggagt acggataaaa tgcttgatgg
     3481 tcggaagagg cataaattcc gtcagccagt ttagtctgac catctcatct gtaacatcat
     3541 tggcaacgct acctttgcca tgtttcagaa acaactctgg cgcatcgggc ttcccataca
     3601 atcgatagat tgtcgcacct gattgcccga cattatcgcg agcccattta tacccatata
     3661 aatcagcatc catgttggaa tttaatcgcg gcctagagca agacgtttcc cgttgaatat
     3721 ggctcataac accccttgta ttactgttta tgtaagcaga cagttttatt gttcatgacc
     3781 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     3841 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     3901 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     3961 actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc
     4021 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     4081 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     4141 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     4201 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     4261 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     4321 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     4381 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     4441 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     4501 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat
     4561 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag
     4621 cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg catatatggt
     4681 gcactctcag tacaatctgc tctgatgccg catagttaag ccagtataca ctccgctatc
     4741 gctacgtgac tgggtcatgg ctgcgccccg acacccgcca acacccgctg acgcgccctg
     4801 acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg
     4861 catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc
     4921 atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt tcatccgcgt ccagctcgtt
     4981 gagtttctcc agaagcgtta atgtctggct tctgataaag cgggccatgt taagggcggt
     5041 tttttcctgt ttggtcactg atgcctccgt gtaaggggga tttctgttca tgggggtaat
     5101 gataccgatg aaacgagaga ggatgctcac gatacgggtt actgatgatg aacatgcccg
     5161 gttactggaa cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg accagagaaa
     5221 aatcactcag ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc cacagggtag
     5281 ccagcagcat cctgcgatgc agatccggaa cataatggtg cagggcgctg acttccgcgt
     5341 ttccagactt tacgaaacac ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga
     5401 cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat tctgctaacc
     5461 agtaaggcaa ccccgccagc ctagccgggt cctcaacgac aggagcacga tcatgcgcac
     5521 ccgtggggcc gccatgccgg cgataatggc ctgcttctcg ccgaaacgtt tggtggcggg
     5581 accagtgacg aaggcttgag cgagggcgtg caagattccg aataccgcaa gcgacaggcc
     5641 gatcatcgtc gcgctccagc gaaagcggtc ctcgccgaaa atgacccaga gcgctgccgg
     5701 cacctgtcct acgagttgca tgataaagaa gacagtcata agtgcggcga cgatagtcat
     5761 gccccgcgcc caccggaagg agctgactgg gttgaaggct ctcaagggca tcggtcgaga
     5821 tcccggtgcc taatgagtga gctaacttac attaattgcg ttgcgctcac tgcccgcttt
     5881 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg
     5941 cggtttgcgt attgggcgcc agggtggttt ttcttttcac cagtgagacg ggcaacagct
     6001 gattgccctt caccgcctgg ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc
     6061 ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg gatataacat gagctgtctt
     6121 cggtatcgtc gtatcccact accgagatat ccgcaccaac gcgcagcccg gactcggtaa
     6181 tggcgcgcat tgcgcccagc gccatctgat cgttggcaac cagcatcgca gtgggaacga
     6241 tgccctcatt cagcatttgc atggtttgtt gaaaaccgga catggcactc cagtcgcctt
     6301 cccgttccgc tatcggctga atttgattgc gagtgagata tttatgccag ccagccagac
     6361 gcagacgcgc cgagacagaa cttaatgggc ccgctaacag cgcgatttgc tggtgaccca
     6421 atgcgaccag atgctccacg cccagtcgcg taccgtcttc atgggagaaa ataatactgt
     6481 tgatgggtgt ctggtcagag acatcaagaa ataacgccgg aacattagtg caggcagctt
     6541 ccacagcaat ggcatcctgg tcatccagcg gatagttaat gatcagccca ctgacgcgtt
     6601 gcgcgagaag attgtgcacc gccgctttac aggcttcgac gccgcttcgt tctaccatcg
     6661 acaccaccac gctggcaccc agttgatcgg cgcgagattt aatcgccgcg acaatttgcg
     6721 acggcgcgtg cagggccaga ctggaggtgg caacgccaat cagcaacgac tgtttgcccg
     6781 ccagttgttg tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc gcttccactt
     6841 tttcccgcgt tttcgcagaa acgtggctgg cctggttcac cacgcgggaa acggtctgat
     6901 aagagacacc ggcatactct gcgacatcgt ataacgttac tggtttcaca ttcaccaccc
     6961 tgaattgact ctcttccggg cgctatcatg ccataccgcg aaaggttttg cgccattcga
     7021 tggtgtccgg gatctcgacg ctctccctta tgcgactcct gcattaggaa gcagcccagt
     7081 agtaggttga ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa ggagatggcg
     7141 cccaacagtc ccccggccac ggggcctgcc accataccca cgccgaaaca agcgctcatg
     7201 agcccgaagt ggcgagcccg atcttcccca tcggtgatgt cggcgatata ggcgccagca
     7261 accgcacctg tggcgccggt gatgccggcc acgatgcgtc cggcgtagag gatcgagatc
     7321 tcgatcccgc gaaat
//
