LOCUS       40924_33212        7335 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Expression vector pNIC-Bio3 plasmid pNIC-Bio3, 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   Burgess-Brown NA, Gileadi O.
  TITLE     Direct Submission
  JOURNAL   Submitted (27-SEP-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 
            (27-SEP-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"
     RBS             12..34
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             45..62
                     /codon_start=1
                     /label=6xHis
                     /note="6xHis affinity tag"
                     /translation="HHHHHH"
     CDS             87..107
                     /codon_start=1
                     /label=TEV site
                     /note="tobacco etch virus (TEV) protease recognition and 
                     cleavage site"
                     /translation="ENLYFQS"
     promoter        123..568
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             569..1987
                     /codon_start=1
                     /label=SacB
                     /note="secreted levansucrase that renders bacterial growth 
                     sensitive to sucrose"
                     /translation="MNIKKFAKQATVLTFTTALLAGGATQAFAKETNQKPYKETYGISH
                     ITRHDMLQIPEQQKNEKYQVPEFDSSTIKNISSAKGLDVWDSWPLQNADGTVANYHGYH
                     IVFALAGDPKNADDTSIYMFYQKVGETSIDSWKNAGRVFKDSDKFDANDSILKDQTQEW
                     SGSATFTSDGKIRLFYTDFSGKHYGKQTLTTAQVNVSASDSSLNINGVEDYKSIFDGDG
                     KTYQNVQQFIDEGNYSSGDNHTLRDPHYVEDKGHKYLVFEANTGTEDGYQGEESLFNKA
                     YYGKSTSFFRQESQKLLQSDKKRTAELANGALGMIELNDDYTLKKVMKPLIASNTVTDE
                     IERANVFKMNGKWYLFTDSRGSKMTIDGITSNDIYMLGYVSNSLTGPYKPLNKTGLVLK
                     MDLDPNDVTFTYSHFAVPQAKGNNVVITSYMTNRGFYADKQSTFAPSFLLNIKGKKTSV
                     VKDSILEQGQLTVNK"
     CDS             2052..2096
                     /codon_start=1
                     /label=AviTag(TM)
                     /note="peptide tag that allows for enzymatic biotinylation"
                     /translation="GLNDIFEAQKIEWHE"
     CDS             2149..2166
                     /codon_start=1
                     /label=6xHis
                     /note="6xHis affinity tag"
                     /translation="HHHHHH"
     terminator      2233..2280
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2317..2772
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     CDS             complement(2868..3680)
                     /codon_start=1
                     /label=KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     rep_origin      3802..4390
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     misc_feature    complement(4576..4718)
                     /label=bom
                     /note="basis of mobility region from pBR322"
     CDS             complement(4823..5011)
                     /codon_start=1
                     /label=rop
                     /note="Rop protein, which maintains plasmids at low copy
                     number"
                     /translation="VTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     protein_bind    complement(5786..5807)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             complement(5823..6902)
                     /codon_start=1
                     /label=lacI
                     /note="lac repressor"
                     /translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     promoter        complement(6903..6980)
                     /label=lacI promoter
     promoter        7289..7307
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    7308..7332
                     /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)."
ORIGIN
        1 ctagaaataa ttttgtttaa ctttaagaag gagatataca tatgcaccat catcatcatc
       61 attcttctgg tgtagatctg ggtaccgaga acctgtactt ccaatccatg gagaccgacg
      121 tccacatata cctgccgttc actattattt agtgaaatga gatattatga tattttctga
      181 attgtgatta aaaaggcaac tttatgccca tgcaacagaa actataaaaa atacagagaa
      241 tgaaaagaaa cagatagatt ttttagttct ttaggcccgt agtctgcaaa tccttttatg
      301 attttctatc aaacaaaaga ggaaaataga ccagttgcaa tccaaacgag agtctaatag
      361 aatgaggtcg aaaagtaaat cgcgcgggtt tgttactgat aaagcaggca agacctaaaa
      421 tgtgtaaagg gcaaagtgta tactttggcg tcacccctta catattttag gtcttttttt
      481 attgtgcgta actaacttgc catcttcaaa caggagggct ggaagaagca gaccgctaac
      541 acagtacata aaaaaggaga catgaacgat gaacatcaaa aagtttgcaa aacaagcaac
      601 agtattaacc tttactaccg cactgctggc aggaggcgca actcaagcgt ttgcgaaaga
      661 aacgaaccaa aagccatata aggaaacata cggcatttcc catattacac gccatgatat
      721 gctgcaaatc cctgaacagc aaaaaaatga aaaatatcaa gttcctgaat tcgattcgtc
      781 cacaattaaa aatatctctt ctgcaaaagg cctggacgtt tgggacagct ggccattaca
      841 aaacgctgac ggcactgtcg caaactatca cggctaccac atcgtctttg cattagccgg
      901 agatcctaaa aatgcggatg acacatcgat ttacatgttc tatcaaaaag tcggcgaaac
      961 ttctattgac agctggaaaa acgctggccg cgtctttaaa gacagcgaca aattcgatgc
     1021 aaatgattct atcctaaaag accaaacaca agaatggtca ggttcagcca catttacatc
     1081 tgacggaaaa atccgtttat tctacactga tttctccggt aaacattacg gcaaacaaac
     1141 actgacaact gcacaagtta acgtatcagc atcagacagc tctttgaaca tcaacggtgt
     1201 agaggattat aaatcaatct ttgacggtga cggaaaaacg tatcaaaatg tacagcagtt
     1261 catcgatgaa ggcaactaca gctcaggcga caaccatacg ctgagagatc ctcactacgt
     1321 agaagataaa ggccacaaat acttagtatt tgaagcaaac actggaactg aagatggcta
     1381 ccaaggcgaa gaatctttat ttaacaaagc atactatggc aaaagcacat cattcttccg
     1441 tcaagaaagt caaaaacttc tgcaaagcga taaaaaacgc acggctgagt tagcaaacgg
     1501 cgctctcggt atgattgagc taaacgatga ttacacactg aaaaaagtga tgaaaccgct
     1561 gattgcatct aacacagtaa cagatgaaat tgaacgcgcg aacgtcttta aaatgaacgg
     1621 caaatggtac ctgttcactg actcccgcgg atcaaaaatg acgattgacg gcattacgtc
     1681 taacgatatt tacatgcttg gttatgtttc taattcttta actggcccat acaagccgct
     1741 gaacaaaact ggccttgtgt taaaaatgga tcttgatcct aacgatgtaa cctttactta
     1801 ctcacacttc gctgtacctc aagcgaaagg aaacaatgtc gtgattacaa gctatatgac
     1861 aaacagagga ttctacgcag acaaacaatc aacgtttgcg ccaagcttcc tgctgaacat
     1921 caaaggcaag aaaacatctg ttgtcaaaga cagcatcctt gaacaaggac aattaacagt
     1981 taacaaataa aaacgcaaaa gaaaatgccg atatcctatt ggcattgacg gtctccagta
     2041 aaggtggata cggcctgaat gatatctttg aagcgcagaa gattgaatgg catgaatgat
     2101 aaggatccga attcgagctc cgtcgacaag cttgcggccg cactcgagca ccaccaccac
     2161 caccactgag atccggctgc taacaaagcc cgaaaggaag ctgagttggc tgctgccacc
     2221 gctgagcaat aactagcata accccttggg gcctctaaac gggtcttgag gggttttttg
     2281 ctgaaaggag gaactatatc cggattggcg aatgggacgc gccctgtagc ggcgcattaa
     2341 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc
     2401 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
     2461 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
     2521 aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc
     2581 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa
     2641 cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct
     2701 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa
     2761 cgtttacaat ttcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
     2821 ttttctaaat acattcaaat atgtatccgc tcatgaatta attcttagaa aaactcatcg
     2881 agcatcaaat gaaactgcaa tttattcata tcaggattat caataccata tttttgaaaa
     2941 agccgtttct gtaatgaagg agaaaactca ccgaggcagt tccataggat ggcaagatcc
     3001 tggtatcggt ctgcgattcc gactcgtcca acatcaatac aacctattaa tttcccctcg
     3061 tcaaaaataa ggttatcaag tgagaaatca ccatgagtga cgactgaatc cggtgagaat
     3121 ggcaaaagtt tatgcatttc tttccagact tgttcaacag gccagccatt acgctcgtca
     3181 tcaaaatcac tcgcatcaac caaaccgtta ttcattcgtg attgcgcctg agcgagacga
     3241 aatacgcgat cgctgttaaa aggacaatta caaacaggaa tcgaatgcaa ccggcgcagg
     3301 aacactgcca gcgcatcaac aatattttca cctgaatcag gatattcttc taatacctgg
     3361 aatgctgttt tcccggggat cgcagtggtg agtaaccatg catcatcagg agtacggata
     3421 aaatgcttga tggtcggaag aggcataaat tccgtcagcc agtttagtct gaccatctca
     3481 tctgtaacat cattggcaac gctacctttg ccatgtttca gaaacaactc tggcgcatcg
     3541 ggcttcccat acaatcgata gattgtcgca cctgattgcc cgacattatc gcgagcccat
     3601 ttatacccat ataaatcagc atccatgttg gaatttaatc gcggcctaga gcaagacgtt
     3661 tcccgttgaa tatggctcat aacacccctt gtattactgt ttatgtaagc agacagtttt
     3721 attgttcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     3781 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     3841 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     3901 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta
     3961 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     4021 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     4081 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     4141 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     4201 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     4261 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     4321 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     4381 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     4441 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt
     4501 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga
     4561 ggaagcggaa gagcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca
     4621 ccgcatatat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat
     4681 acactccgct atcgctacgt gactgggtca tggctgcgcc ccgacacccg ccaacacccg
     4741 ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg
     4801 tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgaggcagc
     4861 tgcggtaaag ctcatcagcg tggtcgtgaa gcgattcaca gatgtctgcc tgttcatccg
     4921 cgtccagctc gttgagtttc tccagaagcg ttaatgtctg gcttctgata aagcgggcca
     4981 tgttaagggc ggttttttcc tgtttggtca ctgatgcctc cgtgtaaggg ggatttctgt
     5041 tcatgggggt aatgataccg atgaaacgag agaggatgct cacgatacgg gttactgatg
     5101 atgaacatgc ccggttactg gaacgttgtg agggtaaaca actggcggta tggatgcggc
     5161 gggaccagag aaaaatcact cagggtcaat gccagcgctt cgttaataca gatgtaggtg
     5221 ttccacaggg tagccagcag catcctgcga tgcagatccg gaacataatg gtgcagggcg
     5281 ctgacttccg cgtttccaga ctttacgaaa cacggaaacc gaagaccatt catgttgttg
     5341 ctcaggtcgc agacgttttg cagcagcagt cgcttcacgt tcgctcgcgt atcggtgatt
     5401 cattctgcta accagtaagg caaccccgcc agcctagccg ggtcctcaac gacaggagca
     5461 cgatcatgcg cacccgtggg gccgccatgc cggcgataat ggcctgcttc tcgccgaaac
     5521 gtttggtggc gggaccagtg acgaaggctt gagcgagggc gtgcaagatt ccgaataccg
     5581 caagcgacag gccgatcatc gtcgcgctcc agcgaaagcg gtcctcgccg aaaatgaccc
     5641 agagcgctgc cggcacctgt cctacgagtt gcatgataaa gaagacagtc ataagtgcgg
     5701 cgacgatagt catgccccgc gcccaccgga aggagctgac tgggttgaag gctctcaagg
     5761 gcatcggtcg agatcccggt gcctaatgag tgagctaact tacattaatt gcgttgcgct
     5821 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
     5881 gcgcggggag aggcggtttg cgtattgggc gccagggtgg tttttctttt caccagtgag
     5941 acgggcaaca gctgattgcc cttcaccgcc tggccctgag agagttgcag caagcggtcc
     6001 acgctggttt gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa
     6061 catgagctgt cttcggtatc gtcgtatccc actaccgaga tatccgcacc aacgcgcagc
     6121 ccggactcgg taatggcgcg cattgcgccc agcgccatct gatcgttggc aaccagcatc
     6181 gcagtgggaa cgatgccctc attcagcatt tgcatggttt gttgaaaacc ggacatggca
     6241 ctccagtcgc cttcccgttc cgctatcggc tgaatttgat tgcgagtgag atatttatgc
     6301 cagccagcca gacgcagacg cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt
     6361 tgctggtgac ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag
     6421 aaaataatac tgttgatggg tgtctggtca gagacatcaa gaaataacgc cggaacatta
     6481 gtgcaggcag cttccacagc aatggcatcc tggtcatcca gcggatagtt aatgatcagc
     6541 ccactgacgc gttgcgcgag aagattgtgc accgccgctt tacaggcttc gacgccgctt
     6601 cgttctacca tcgacaccac cacgctggca cccagttgat cggcgcgaga tttaatcgcc
     6661 gcgacaattt gcgacggcgc gtgcagggcc agactggagg tggcaacgcc aatcagcaac
     6721 gactgtttgc ccgccagttg ttgtgccacg cggttgggaa tgtaattcag ctccgccatc
     6781 gccgcttcca ctttttcccg cgttttcgca gaaacgtggc tggcctggtt caccacgcgg
     6841 gaaacggtct gataagagac accggcatac tctgcgacat cgtataacgt tactggtttc
     6901 acattcacca ccctgaattg actctcttcc gggcgctatc atgccatacc gcgaaaggtt
     6961 ttgcgccatt cgatggtgtc cgggatctcg acgctctccc ttatgcgact cctgcattag
     7021 gaagcagccc agtagtaggt tgaggccgtt gagcaccgcc gccgcaagga atggtgcatg
     7081 caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac ccacgccgaa
     7141 acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat
     7201 ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta
     7261 gaggatcgag atctcgatcc cgcgaaatta atacgactca ctatagggga attgtgagcg
     7321 gataacaatt cccct
//
