LOCUS       40924_33217        7218 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Expression vector pNIC-CH, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7218)
  AUTHORS   Gileadi O, Burgess-Brown N, Loppnau P.
  TITLE     Vectors for Ligation-independent Cloning (LIC)
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7218)
  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 7218)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 7218)
  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            "Unpublished"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..7218
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        253..271
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    272..296
                     /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             311..333
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     promoter        348..780
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             781..2199
                     /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             2264..2281
                     /codon_start=1
                     /label=6xHis
                     /note="6xHis affinity tag"
                     /translation="HHHHHH"
     CDS             2331..2348
                     /codon_start=1
                     /label=6xHis
                     /note="6xHis affinity tag"
                     /translation="HHHHHH"
     terminator      2415..2462
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2499..2954
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     CDS             complement(3050..3862)
                     /codon_start=1
                     /label=KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     rep_origin      3984..4572
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     misc_feature    complement(4758..4900)
                     /label=bom
                     /note="basis of mobility region from pBR322"
     CDS             complement(5005..5193)
                     /codon_start=1
                     /label=rop
                     /note="Rop protein, which maintains plasmids at low copy
                     number"
                     /translation="VTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     protein_bind    complement(5968..5989)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             complement(6005..7084)
                     /codon_start=1
                     /label=lacI
                     /note="lac repressor"
                     /translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     promoter        complement(7085..7162)
                     /label=lacI promoter
ORIGIN
        1 aggttgaggc cgttgagcac cgccgccgca aggaatggtg catgcaagga gatggcgccc
       61 aacagtcccc cggccacggg gcctgccacc atacccacgc cgaaacaagc gctcatgagc
      121 ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg cgatataggc gccagcaacc
      181 gcacctgtgg cgccggtgat gccggccacg atgcgtccgg cgtagaggat cgagatctcg
      241 atcccgcgaa attaatacga ctcactatag gggaattgtg agcggataac aattcccctc
      301 tagaaataat tttgtttaac cttaagaagg agatatacta tgcaggtcgt tcactattat
      361 ttagtgaaat gagatattat gatattttct gaattgtgat taaaaaggca actttatgcc
      421 catgcaacag aaactataaa aaatacagag aatgaaaaga aacagataga ttttttagtt
      481 ctttaggccc gtagtctgca aatcctttta tgattttcta tcaaacaaaa gaggaaaata
      541 gaccagttgc aatccaaacg agagtctaat agaatgaggt cgaaaagtaa atcgcgcggg
      601 tttgttactg ataaagcagg caagacctaa aatgtgtaaa gggcaaagtg tatactttgg
      661 cgtcacccct tacatatttt aggtcttttt ttattgtgcg taactaactt gccatcttca
      721 aacaggaggg ctggaagaag cagaccgcta acacagtaca taaaaaagga gacatgaacg
      781 atgaacatca aaaagtttgc aaaacaagca acagtattaa cctttactac cgcactgctg
      841 gcaggaggcg caactcaagc gtttgcgaaa gaaacgaacc aaaagccata taaggaaaca
      901 tacggcattt cccatattac acgccatgat atgctgcaaa tccctgaaca gcaaaaaaat
      961 gaaaaatata aagttcctga gttcgattcg tccacaatta aaaatatctc ttctgcaaaa
     1021 ggcctggacg tttgggacag ctggccatta caaaacactg acggcactgt cgcaaactat
     1081 cacggctacc acatcgtctt tgcattagcc ggagatccta aaaatgcgga tgacacatcg
     1141 atttacatgt tctatcaaaa agtcggcgaa acttctattg acagctggaa aaacgctggc
     1201 cgcgtcttta aagacagcga caaattcgat gcaaatgatt ctatcctaaa agaccaaaca
     1261 caagaatggt caggttcagc cacatttaca tctgacggaa aaatccgttt attctacact
     1321 gatttctccg gtaaacatta cggcaaacaa acactgacaa ctgcacaagt taacgtatca
     1381 gcatcagaca gctctttgaa catcaacggt gtagaggatt ataaatcaat ctttgacggt
     1441 gacggaaaaa cgtatcaaaa tgtacagcag ttcatcgatg aaggcaacta cagctcaggc
     1501 gacaaccata cgctgagaga tcctcactac gtagaagata aaggccacaa atacttagta
     1561 tttgaagcaa acactggaac tgaagatggc taccaaggcg aagaatcttt atttaacaaa
     1621 gcatactatg gcaaaagcac atcattcttc cgtcaagaaa gtcaaaaact tctgcaaagc
     1681 gataaaaaac gcacggctga gttagcaaac ggcgctctcg gtatgattga gctaaacgat
     1741 gattacacac tgaaaaaagt gatgaaaccg ctgattgcat ctaacacagt aacagatgaa
     1801 attgaacgcg cgaacgtctt taaaatgaac ggcaaatggt acctgttcac tgactcccgc
     1861 ggatcaaaaa tgacgattga cggcattacg tctaacgata tttacatgct tggttatgtt
     1921 tctaattctt taactggccc atacaagccg ctgaacaaaa ctggccttgt gttaaaaatg
     1981 gatcttgatc ctaacgatgt aacctttact tactcacact tcgctgtacc tcaagcgaaa
     2041 ggaaacaatg tcgtgattac aagctatatg acaaacagag gattctacgc agacaaacaa
     2101 tcaacgtttg cgcctagctt cctgctgaac atcaaaggca agaaaacatc tgttgtcaaa
     2161 gacagcatcc ttgaacaagg acaattaaca gttaacaaat aaaaacgcaa aagaaaatgc
     2221 cgatatccta ttggcattga cgtcaggtgg cacacctgca gcgcaccatc atcaccacca
     2281 ttgaggatcc gaattcgagc tccgtcgaca agcttgcggc cgcactcgag caccaccacc
     2341 accaccactg agatccggct gctaacaaag cccgaaagga agctgagttg gctgctgcca
     2401 ccgctgagca ataactagca taaccccttg gggcctctaa acgggtcttg aggggttttt
     2461 tgctgaaagg aggaactata tccggattgg cgaatgggac gcgccctgta gcggcgcatt
     2521 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
     2581 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     2641 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     2701 caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
     2761 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
     2821 aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc
     2881 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt
     2941 aacgtttaca atttcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt
     3001 atttttctaa atacattcaa atatgtatcc gctcatgaat taattcttag aaaaactcat
     3061 cgagcatcaa atgaaactgc aatttattca tatcaggatt atcaatacca tatttttgaa
     3121 aaagccgttt ctgtaatgaa ggagaaaact caccgaggca gttccatagg atggcaagat
     3181 cctggtatcg gtctgcgatt ccgactcgtc caacatcaat acaacctatt aatttcccct
     3241 cgtcaaaaat aaggttatca agtgagaaat caccatgagt gacgactgaa tccggtgaga
     3301 atggcaaaag tttatgcatt tctttccaga cttgttcaac aggccagcca ttacgctcgt
     3361 catcaaaatc actcgcatca accaaaccgt tattcattcg tgattgcgcc tgagcgagac
     3421 gaaatacgcg atcgctgtta aaaggacaat tacaaacagg aatcgaatgc aaccggcgca
     3481 ggaacactgc cagcgcatca acaatatttt cacctgaatc aggatattct tctaatacct
     3541 ggaatgctgt tttcccgggg atcgcagtgg tgagtaacca tgcatcatca ggagtacgga
     3601 taaaatgctt gatggtcgga agaggcataa attccgtcag ccagtttagt ctgaccatct
     3661 catctgtaac atcattggca acgctacctt tgccatgttt cagaaacaac tctggcgcat
     3721 cgggcttccc atacaatcga tagattgtcg cacctgattg cccgacatta tcgcgagccc
     3781 atttataccc atataaatca gcatccatgt tggaatttaa tcgcggccta gagcaagacg
     3841 tttcccgttg aatatggctc ataacacccc ttgtattact gtttatgtaa gcagacagtt
     3901 ttattgttca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     3961 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     4021 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     4081 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg
     4141 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     4201 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     4261 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     4321 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     4381 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     4441 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     4501 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     4561 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     4621 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc
     4681 tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc
     4741 gaggaagcgg aagagcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca
     4801 caccgcatat atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt
     4861 atacactccg ctatcgctac gtgactgggt catggctgcg ccccgacacc cgccaacacc
     4921 cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac
     4981 cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca
     5041 gctgcggtaa agctcatcag cgtggtcgtg aagcgattca cagatgtctg cctgttcatc
     5101 cgcgtccagc tcgttgagtt tctccagaag cgttaatgtc tggcttctga taaagcgggc
     5161 catgttaagg gcggtttttt cctgtttggt cactgatgcc tccgtgtaag ggggatttct
     5221 gttcatgggg gtaatgatac cgatgaaacg agagaggatg ctcacgatac gggttactga
     5281 tgatgaacat gcccggttac tggaacgttg tgagggtaaa caactggcgg tatggatgcg
     5341 gcgggaccag agaaaaatca ctcagggtca atgccagcgc ttcgttaata cagatgtagg
     5401 tgttccacag ggtagccagc agcatcctgc gatgcagatc cggaacataa tggtgcaggg
     5461 cgctgacttc cgcgtttcca gactttacga aacacggaaa ccgaagacca ttcatgttgt
     5521 tgctcaggtc gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga
     5581 ttcattctgc taaccagtaa ggcaaccccg ccagcctagc cgggtcctca acgacaggag
     5641 cacgatcatg cgcacccgtg gggccgccat gccggcgata atggcctgct tctcgccgaa
     5701 acgtttggtg gcgggaccag tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac
     5761 cgcaagcgac aggccgatca tcgtcgcgct ccagcgaaag cggtcctcgc cgaaaatgac
     5821 ccagagcgct gccggcacct gtcctacgag ttgcatgata aagaagacag tcataagtgc
     5881 ggcgacgata gtcatgcccc gcgcccaccg gaaggagctg actgggttga aggctctcaa
     5941 gggcatcggt cgagatcccg gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg
     6001 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
     6061 acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg
     6121 agacgggcaa cagctgattg cccttcaccg cctggccctg agagagttgc agcaagcggt
     6181 ccacgctggt ttgccccagc aggcgaaaat cctgtttgat ggtggttaac ggcgggatat
     6241 aacatgagct gtcttcggta tcgtcgtatc ccactaccga gatatccgca ccaacgcgca
     6301 gcccggactc ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca
     6361 tcgcagtggg aacgatgccc tcattcagca tttgcatggt ttgttgaaaa ccggacatgg
     6421 cactccagtc gccttcccgt tccgctatcg gctgaatttg attgcgagtg agatatttat
     6481 gccagccagc cagacgcaga cgcgccgaga cagaacttaa tgggcccgct aacagcgcga
     6541 tttgctggtg acccaatgcg accagatgct ccacgcccag tcgcgtaccg tcttcatggg
     6601 agaaaataat actgttgatg ggtgtctggt cagagacatc aagaaataac gccggaacat
     6661 tagtgcaggc agcttccaca gcaatggcat cctggtcatc cagcggatag ttaatgatca
     6721 gcccactgac gcgttgcgcg agaagattgt gcaccgccgc tttacaggct tcgacgccgc
     6781 ttcgttctac catcgacacc accacgctgg cacccagttg atcggcgcga gatttaatcg
     6841 ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca
     6901 acgactgttt gcccgccagt tgttgtgcca cgcggttggg aatgtaattc agctccgcca
     6961 tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc
     7021 gggaaacggt ctgataagag acaccggcat actctgcgac atcgtataac gttactggtt
     7081 tcacattcac caccctgaat tgactctctt ccgggcgcta tcatgccata ccgcgaaagg
     7141 ttttgcgcca ttcgatggtg tccgggatct cgacgctctc ccttatgcga ctcctgcatt
     7201 aggaagcagc ccagtagt
//
