LOCUS       40924_33227        7260 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Expression vector pNIC-CTHF, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7260)
  AUTHORS   Gileadi O, Burgess-Brown N, Loppnau P.
  TITLE     Vectors for Ligation-independent Cloning (LIC)
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7260)
  AUTHORS   Gileadi O, Burgess-Brown N.
  TITLE     Direct Submission
  JOURNAL   Submitted (27-DEC-2006) Structural Genomics Consortium, University 
            of Oxford, Botnar Research Centre, Oxford OX3 7LD, UK
REFERENCE   3  (bases 1 to 7260)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 7260)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (27-DEC-2006) Structural Genomics Consortium, University of Oxford, 
            Botnar Research Centre, Oxford OX3 7LD, UK"
COMMENT     SGRef: number: 3; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..7260
                     /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)"
     promoter        96..528
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             529..1947
                     /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             2009..2029
                     /codon_start=1
                     /label=TEV site
                     /note="tobacco etch virus (TEV) protease recognition and 
                     cleavage site"
                     /translation="ENLYFQS"
     CDS             2030..2047
                     /codon_start=1
                     /label=6xHis
                     /note="6xHis affinity tag"
                     /translation="HHHHHH"
     CDS             2048..2071
                     /codon_start=1
                     /label=FLAG
                     /note="FLAG(R) epitope tag, followed by an enterokinase
                     cleavage site"
                     /translation="DYKDDDDK"
     CDS             2121..2138
                     /codon_start=1
                     /label=6xHis
                     /note="6xHis affinity tag"
                     /translation="HHHHHH"
     terminator      2205..2252
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2289..2744
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     CDS             complement(2840..3652)
                     /codon_start=1
                     /label=KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     rep_origin      3774..4362
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     misc_feature    complement(4548..4690)
                     /label=bom
                     /note="basis of mobility region from pBR322"
     CDS             complement(4795..4983)
                     /codon_start=1
                     /label=rop
                     /note="Rop protein, which maintains plasmids at low copy
                     number"
                     /translation="VTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     protein_bind    complement(5758..5779)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             complement(5795..6874)
                     /codon_start=1
                     /label=lacI
                     /note="lac repressor"
                     /translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     promoter        complement(6875..6952)
                     /label=lacI promoter
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaacct taagaaggag atatactatg caggtcgttc actattattt agtgaaatga
      121 gatattatga tattttctga attgtgatta aaaaggcaac tttatgccca tgcaacagaa
      181 actataaaaa atacagagaa tgaaaagaaa cagatagatt ttttagttct ttaggcccgt
      241 agtctgcaaa tccttttatg attttctatc aaacaaaaga ggaaaataga ccagttgcaa
      301 tccaaacgag agtctaatag aatgaggtcg aaaagtaaat cgcgcgggtt tgttactgat
      361 aaagcaggca agacctaaaa tgtgtaaagg gcaaagtgta tactttggcg tcacccctta
      421 catattttag gtcttttttt attgtgcgta actaacttgc catcttcaaa caggagggct
      481 ggaagaagca gaccgctaac acagtacata aaaaaggaga catgaacgat gaacatcaaa
      541 aagtttgcaa aacaagcaac agtattaacc tttactaccg cactgctggc aggaggcgca
      601 actcaagcgt ttgcgaaaga aacgaaccaa aagccatata aggaaacata cggcatttcc
      661 catattacac gccatgatat gctgcaaatc cctgaacagc aaaaaaatga aaaatataaa
      721 gttcctgagt tcgattcgtc cacaattaaa aatatctctt ctgcaaaagg cctggacgtt
      781 tgggacagct ggccattaca aaacactgac ggcactgtcg caaactatca cggctaccac
      841 atcgtctttg cattagccgg agatcctaaa aatgcggatg acacatcgat ttacatgttc
      901 tatcaaaaag tcggcgaaac ttctattgac agctggaaaa acgctggccg cgtctttaaa
      961 gacagcgaca aattcgatgc aaatgattct atcctaaaag accaaacaca agaatggtca
     1021 ggttcagcca catttacatc tgacggaaaa atccgtttat tctacactga tttctccggt
     1081 aaacattacg gcaaacaaac actgacaact gcacaagtta acgtatcagc atcagacagc
     1141 tctttgaaca tcaacggtgt agaggattat aaatcaatct ttgacggtga cggaaaaacg
     1201 tatcaaaatg tacagcagtt catcgatgaa ggcaactaca gctcaggcga caaccatacg
     1261 ctgagagatc ctcactacgt agaagataaa ggccacaaat acttagtatt tgaagcaaac
     1321 actggaactg aagatggcta ccaaggcgaa gaatctttat ttaacaaagc atactatggc
     1381 aaaagcacat cattcttccg tcaagaaagt caaaaacttc tgcaaagcga taaaaaacgc
     1441 acggctgagt tagcaaacgg cgctctcggt atgattgagc taaacgatga ttacacactg
     1501 aaaaaagtga tgaaaccgct gattgcatct aacacagtaa cagatgaaat tgaacgcgcg
     1561 aacgtcttta aaatgaacgg caaatggtac ctgttcactg actcccgcgg atcaaaaatg
     1621 acgattgacg gcattacgtc taacgatatt tacatgcttg gttatgtttc taattcttta
     1681 actggcccat acaagccgct gaacaaaact ggccttgtgt taaaaatgga tcttgatcct
     1741 aacgatgtaa cctttactta ctcacacttc gctgtacctc aagcgaaagg aaacaatgtc
     1801 gtgattacaa gctatatgac aaacagagga ttctacgcag acaaacaatc aacgtttgcg
     1861 cctagcttcc tgctgaacat caaaggcaag aaaacatctg ttgtcaaaga cagcatcctt
     1921 gaacaaggac aattaacagt taacaaataa aaacgcaaaa gaaaatgccg atatcctatt
     1981 ggcattgacg tcaggtggca cacctgcaga gaacctctac ttccaatcgc accatcatca
     2041 ccaccatgat tacaaggatg acgacgataa gtgaggatcc gaattcgagc tccgtcgaca
     2101 agcttgcggc cgcactcgag caccaccacc accaccactg agatccggct gctaacaaag
     2161 cccgaaagga agctgagttg gctgctgcca ccgctgagca ataactagca taaccccttg
     2221 gggcctctaa acgggtcttg aggggttttt tgctgaaagg aggaactata tccggattgg
     2281 cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
     2341 cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt
     2401 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt
     2461 ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg
     2521 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt
     2581 taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt
     2641 tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca
     2701 aaaatttaac gcgaatttta acaaaatatt aacgtttaca atttcaggtg gcacttttcg
     2761 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     2821 gctcatgaat taattcttag aaaaactcat cgagcatcaa atgaaactgc aatttattca
     2881 tatcaggatt atcaatacca tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact
     2941 caccgaggca gttccatagg atggcaagat cctggtatcg gtctgcgatt ccgactcgtc
     3001 caacatcaat acaacctatt aatttcccct cgtcaaaaat aaggttatca agtgagaaat
     3061 caccatgagt gacgactgaa tccggtgaga atggcaaaag tttatgcatt tctttccaga
     3121 cttgttcaac aggccagcca ttacgctcgt catcaaaatc actcgcatca accaaaccgt
     3181 tattcattcg tgattgcgcc tgagcgagac gaaatacgcg atcgctgtta aaaggacaat
     3241 tacaaacagg aatcgaatgc aaccggcgca ggaacactgc cagcgcatca acaatatttt
     3301 cacctgaatc aggatattct tctaatacct ggaatgctgt tttcccgggg atcgcagtgg
     3361 tgagtaacca tgcatcatca ggagtacgga taaaatgctt gatggtcgga agaggcataa
     3421 attccgtcag ccagtttagt ctgaccatct catctgtaac atcattggca acgctacctt
     3481 tgccatgttt cagaaacaac tctggcgcat cgggcttccc atacaatcga tagattgtcg
     3541 cacctgattg cccgacatta tcgcgagccc atttataccc atataaatca gcatccatgt
     3601 tggaatttaa tcgcggccta gagcaagacg tttcccgttg aatatggctc ataacacccc
     3661 ttgtattact gtttatgtaa gcagacagtt ttattgttca tgaccaaaat cccttaacgt
     3721 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     3781 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     3841 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     3901 gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac
     3961 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     4021 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     4081 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     4141 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     4201 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     4261 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     4321 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     4381 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
     4441 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc
     4501 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat
     4561 tttctcctta cgcatctgtg cggtatttca caccgcatat atggtgcact ctcagtacaa
     4621 tctgctctga tgccgcatag ttaagccagt atacactccg ctatcgctac gtgactgggt
     4681 catggctgcg ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct
     4741 cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt
     4801 ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa agctcatcag cgtggtcgtg
     4861 aagcgattca cagatgtctg cctgttcatc cgcgtccagc tcgttgagtt tctccagaag
     4921 cgttaatgtc tggcttctga taaagcgggc catgttaagg gcggtttttt cctgtttggt
     4981 cactgatgcc tccgtgtaag ggggatttct gttcatgggg gtaatgatac cgatgaaacg
     5041 agagaggatg ctcacgatac gggttactga tgatgaacat gcccggttac tggaacgttg
     5101 tgagggtaaa caactggcgg tatggatgcg gcgggaccag agaaaaatca ctcagggtca
     5161 atgccagcgc ttcgttaata cagatgtagg tgttccacag ggtagccagc agcatcctgc
     5221 gatgcagatc cggaacataa tggtgcaggg cgctgacttc cgcgtttcca gactttacga
     5281 aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc gcagacgttt tgcagcagca
     5341 gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc taaccagtaa ggcaaccccg
     5401 ccagcctagc cgggtcctca acgacaggag cacgatcatg cgcacccgtg gggccgccat
     5461 gccggcgata atggcctgct tctcgccgaa acgtttggtg gcgggaccag tgacgaaggc
     5521 ttgagcgagg gcgtgcaaga ttccgaatac cgcaagcgac aggccgatca tcgtcgcgct
     5581 ccagcgaaag cggtcctcgc cgaaaatgac ccagagcgct gccggcacct gtcctacgag
     5641 ttgcatgata aagaagacag tcataagtgc ggcgacgata gtcatgcccc gcgcccaccg
     5701 gaaggagctg actgggttga aggctctcaa gggcatcggt cgagatcccg gtgcctaatg
     5761 agtgagctaa cttacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
     5821 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
     5881 gcgccagggt ggtttttctt ttcaccagtg agacgggcaa cagctgattg cccttcaccg
     5941 cctggccctg agagagttgc agcaagcggt ccacgctggt ttgccccagc aggcgaaaat
     6001 cctgtttgat ggtggttaac ggcgggatat aacatgagct gtcttcggta tcgtcgtatc
     6061 ccactaccga gatatccgca ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc
     6121 ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc tcattcagca
     6181 tttgcatggt ttgttgaaaa ccggacatgg cactccagtc gccttcccgt tccgctatcg
     6241 gctgaatttg attgcgagtg agatatttat gccagccagc cagacgcaga cgcgccgaga
     6301 cagaacttaa tgggcccgct aacagcgcga tttgctggtg acccaatgcg accagatgct
     6361 ccacgcccag tcgcgtaccg tcttcatggg agaaaataat actgttgatg ggtgtctggt
     6421 cagagacatc aagaaataac gccggaacat tagtgcaggc agcttccaca gcaatggcat
     6481 cctggtcatc cagcggatag ttaatgatca gcccactgac gcgttgcgcg agaagattgt
     6541 gcaccgccgc tttacaggct tcgacgccgc ttcgttctac catcgacacc accacgctgg
     6601 cacccagttg atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg
     6661 ccagactgga ggtggcaacg ccaatcagca acgactgttt gcccgccagt tgttgtgcca
     6721 cgcggttggg aatgtaattc agctccgcca tcgccgcttc cactttttcc cgcgttttcg
     6781 cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt ctgataagag acaccggcat
     6841 actctgcgac atcgtataac gttactggtt tcacattcac caccctgaat tgactctctt
     6901 ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca ttcgatggtg tccgggatct
     6961 cgacgctctc ccttatgcga ctcctgcatt aggaagcagc ccagtagtag gttgaggccg
     7021 ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga tggcgcccaa cagtcccccg
     7081 gccacggggc ctgccaccat acccacgccg aaacaagcgc tcatgagccc gaagtggcga
     7141 gcccgatctt ccccatcggt gatgtcggcg atataggcgc cagcaaccgc acctgtggcg
     7201 ccggtgatgc cggccacgat gcgtccggcg tagaggatcg agatctcgat cccgcgaaat
//
