LOCUS       40924_33242        7393 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Expression vector pNIC-NHT-CTB, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7393)
  AUTHORS   Savitsky PA, Gileadi O.
  TITLE     Direct Submission
  JOURNAL   Submitted (13-DEC-2011) Structural Genomics Consortium, University 
            of Oxford, Old Road Campus Research Building, Roosevelt Drive, 
            Oxford, Oxfordshire OX3 7DQ, UK
REFERENCE   2  (bases 1 to 7393)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 7393)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Submitted 
            (13-DEC-2011) Structural Genomics Consortium, University of Oxford, 
            Old Road Campus Research Building, Roosevelt Drive, Oxford, 
            Oxfordshire OX3 7DQ, UK"
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..7393
                     /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
                     /codon_start=1
                     /label=6xHis
                     /note="6xHis affinity tag"
                     /translation="HHHHHH"
     CDS             134..154
                     /codon_start=1
                     /label=TEV site
                     /note="tobacco etch virus (TEV) protease recognition and 
                     cleavage site"
                     /translation="ENLYFQS"
     promoter        170..615
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             616..2034
                     /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             2084..2104
                     /codon_start=1
                     /label=TEV site
                     /note="tobacco etch virus (TEV) protease recognition and 
                     cleavage site"
                     /translation="ENLYFQS"
     CDS             2111..2155
                     /codon_start=1
                     /label=AviTag(TM)
                     /note="peptide tag that allows for enzymatic biotinylation"
                     /translation="GLNDIFEAQKIEWHE"
     CDS             2181..2204
                     /codon_start=1
                     /label=FLAG
                     /note="FLAG(R) epitope tag, followed by an enterokinase
                     cleavage site"
                     /translation="DYKDDDDK"
     CDS             2254..2271
                     /codon_start=1
                     /label=6xHis
                     /note="6xHis affinity tag"
                     /translation="HHHHHH"
     terminator      2338..2385
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2422..2877
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     CDS             complement(2973..3785)
                     /codon_start=1
                     /label=KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     rep_origin      3907..4495
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     misc_feature    complement(4681..4823)
                     /label=bom
                     /note="basis of mobility region from pBR322"
     CDS             complement(4928..5116)
                     /codon_start=1
                     /label=rop
                     /note="Rop protein, which maintains plasmids at low copy
                     number"
                     /translation="VTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     protein_bind    complement(5891..5912)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             complement(5928..7007)
                     /codon_start=1
                     /label=lacI
                     /note="lac repressor"
                     /translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     promoter        complement(7008..7085)
                     /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 tccgagaatt tgtatttcca
     2101 atccggtggc ggtctgaacg acatcttcga ggctcagaaa atcgaatggc acgaatgaca
     2161 gtaaaggtgg atacggatct gattacaagg atgacgacga taagtgaaga tccgaattcg
     2221 agctccgtcg acaagcttgc ggccgcactc gagcaccacc accaccacca ctgagatccg
     2281 gctgctaaca aagcccgaaa ggaagctgag ttggctgctg ccaccgctga gcaataacta
     2341 gcataacccc ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa aggaggaact
     2401 atatccggat tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
     2461 tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt
     2521 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
     2581 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg
     2641 gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg
     2701 agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct
     2761 cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg
     2821 agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttcag
     2881 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
     2941 caaatatgta tccgctcatg aattaattct tagaaaaact catcgagcat caaatgaaac
     3001 tgcaatttat tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat
     3061 gaaggagaaa actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg
     3121 attccgactc gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta
     3181 tcaagtgaga aatcaccatg agtgacgact gaatccggtg agaatggcaa aagtttatgc
     3241 atttctttcc agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca
     3301 tcaaccaaac cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg
     3361 ttaaaaggac aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca
     3421 tcaacaatat tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg
     3481 gggatcgcag tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc
     3541 ggaagaggca taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg
     3601 gcaacgctac ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat
     3661 cgatagattg tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa
     3721 tcagcatcca tgttggaatt taatcgcggc ctagagcaag acgtttcccg ttgaatatgg
     3781 ctcataacac cccttgtatt actgtttatg taagcagaca gttttattgt tcatgaccaa
     3841 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     3901 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     3961 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     4021 tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca
     4081 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     4141 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     4201 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     4261 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     4321 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     4381 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     4441 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     4501 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt
     4561 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac
     4621 cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg
     4681 cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca tatatggtgc
     4741 actctcagta caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc
     4801 tacgtgactg ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac
     4861 gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca
     4921 tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat
     4981 cagcgtggtc gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga
     5041 gtttctccag aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt
     5101 tttcctgttt ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga
     5161 taccgatgaa acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt
     5221 tactggaacg ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa
     5281 tcactcaggg tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc
     5341 agcagcatcc tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt
     5401 ccagacttta cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg
     5461 ttttgcagca gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag
     5521 taaggcaacc ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc
     5581 gtggggccgc catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac
     5641 cagtgacgaa ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga
     5701 tcatcgtcgc gctccagcga aagcggtcct cgccgaaaat gacccagagc gctgccggca
     5761 cctgtcctac gagttgcatg ataaagaaga cagtcataag tgcggcgacg atagtcatgc
     5821 cccgcgccca ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc
     5881 ccggtgccta atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc
     5941 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     6001 gtttgcgtat tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga
     6061 ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc
     6121 agcaggcgaa aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg
     6181 gtatcgtcgt atcccactac cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg
     6241 gcgcgcattg cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg
     6301 ccctcattca gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc
     6361 cgttccgcta tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc
     6421 agacgcgccg agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat
     6481 gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg
     6541 atgggtgtct ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc
     6601 acagcaatgg catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc
     6661 gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac
     6721 accaccacgc tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac
     6781 ggcgcgtgca gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc
     6841 agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt
     6901 tcccgcgttt tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa
     6961 gagacaccgg catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg
     7021 aattgactct cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg
     7081 gtgtccggga tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag
     7141 taggttgagg ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc
     7201 caacagtccc ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag
     7261 cccgaagtgg cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac
     7321 cgcacctgtg gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatctc
     7381 gatcccgcga aat
//
