LOCUS       40924_33222        7321 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Expression vector pNIC-CTB10H, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7321)
  AUTHORS   Savitsky P.
  TITLE     Direct Submission
  JOURNAL   Submitted (25-APR-2016) Structural Genomics Consortium, University 
            of Oxford, Old Road Campus Research Building, Roosevelt Drive, 
            Oxford, Oxfordshire OX3 7DQ, UK
REFERENCE   2  (bases 1 to 7321)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 7321)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Submitted 
            (25-APR-2016) 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..7321
                     /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)"
     protein_bind    complement(113..146)
                     /label=loxP
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        161..606
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             607..2025
                     /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             2098..2118
                     /codon_start=1
                     /label=TEV site
                     /note="tobacco etch virus (TEV) protease recognition and 
                     cleavage site"
                     /translation="ENLYFQS"
     CDS             2125..2169
                     /codon_start=1
                     /label=AviTag(TM)
                     /note="peptide tag that allows for enzymatic biotinylation"
                     /translation="GLNDIFEAQKIEWHE"
     CDS             2170..2196
                     /codon_start=1
                     /label=9xHis
                     /note="9xHis affinity tag"
                     /translation="HHHHHHHHH"
     terminator      2266..2313
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2350..2805
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     CDS             complement(2901..3713)
                     /codon_start=1
                     /label=KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     rep_origin      3835..4423
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     misc_feature    complement(4609..4751)
                     /label=bom
                     /note="basis of mobility region from pBR322"
     CDS             complement(4856..5044)
                     /codon_start=1
                     /label=rop
                     /note="Rop protein, which maintains plasmids at low copy
                     number"
                     /translation="VTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     protein_bind    complement(5819..5840)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             complement(5856..6935)
                     /codon_start=1
                     /label=lacI
                     /note="lac repressor"
                     /translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     promoter        complement(6936..7013)
                     /label=lacI promoter
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaacct taagaaggag atatactgct agcttctcct cctgaaagat ccataacttc
      121 gtatagcata cattatacga agttatgcgg ccgcgacgtc cacatatacc tgccgttcac
      181 tattatttag tgaaatgaga tattatgata ttttctgaat tgtgattaaa aaggcaactt
      241 tatgcccatg caacagaaac tataaaaaat acagagaatg aaaagaaaca gatagatttt
      301 ttagttcttt aggcccgtag tctgcaaatc cttttatgat tttctatcaa acaaaagagg
      361 aaaatagacc agttgcaatc caaacgagag tctaatagaa tgaggtcgaa aagtaaatcg
      421 cgcgggtttg ttactgataa agcaggcaag acctaaaatg tgtaaagggc aaagtgtata
      481 ctttggcgtc accccttaca tattttaggt ctttttttat tgtgcgtaac taacttgcca
      541 tcttcaaaca ggagggctgg aagaagcaga ccgctaacac agtacataaa aaaggagaca
      601 tgaacgatga acatcaaaaa gtttgcaaaa caagcaacag tattaacctt tactaccgca
      661 ctgctggcag gaggcgcaac tcaagcgttt gcgaaagaaa cgaaccaaaa gccatataag
      721 gaaacatacg gcatttccca tattacacgc catgatatgc tgcaaatccc tgaacagcaa
      781 aaaaatgaaa aatatcaagt tcctgaattc gattcgtcca caattaaaaa tatctcttct
      841 gcaaaaggcc tggacgtttg ggacagctgg ccattacaaa acgctgacgg cactgtcgca
      901 aactatcacg gctaccacat cgtctttgca ttagccggag atcctaaaaa tgcggatgac
      961 acatcgattt acatgttcta tcaaaaagtc ggcgaaactt ctattgacag ctggaaaaac
     1021 gctggccgcg tctttaaaga cagcgacaaa ttcgatgcaa atgattctat cctaaaagac
     1081 caaacacaag aatggtcagg ttcagccaca tttacatctg acggaaaaat ccgtttattc
     1141 tacactgatt tctccggtaa acattacggc aaacaaacac tgacaactgc acaagttaac
     1201 gtatcagcat cagacagctc tttgaacatc aacggtgtag aggattataa atcaatcttt
     1261 gacggtgacg gaaaaacgta tcaaaatgta cagcagttca tcgatgaagg caactacagc
     1321 tcaggcgaca accatacgct gagagatcct cactacgtag aagataaagg ccacaaatac
     1381 ttagtatttg aagcaaacac tggaactgaa gatggctacc aaggcgaaga atctttattt
     1441 aacaaagcat actatggcaa aagcacatca ttcttccgtc aagaaagtca aaaacttctg
     1501 caaagcgata aaaaacgcac ggctgagtta gcaaacggcg ctctcggtat gattgagcta
     1561 aacgatgatt acacactgaa aaaagtgatg aaaccgctga ttgcatctaa cacagtaaca
     1621 gatgaaattg aacgcgcgaa cgtctttaaa atgaacggca aatggtacct gttcactgac
     1681 tcccgcggat caaaaatgac gattgacggc attacgtcta acgatattta catgcttggt
     1741 tatgtttcta attctttaac tggcccatac aagccgctga acaaaactgg ccttgtgtta
     1801 aaaatggatc ttgatcctaa cgatgtaacc tttacttact cacacttcgc tgtacctcaa
     1861 gcgaaaggaa acaatgtcgt gattacaagc tatatgacaa acagaggatt ctacgcagac
     1921 aaacaatcaa cgtttgcgcc aagcttcctg ctgaacatca aaggcaagaa aacatctgtt
     1981 gtcaaagaca gcatccttga acaaggacaa ttaacagtta acaaataaaa acgcaaaaga
     2041 aaatgccgat atcctattgg cattgacgtc aggtggcact tttcgaggag tttactagag
     2101 aacctctact tccaatcggg tggcggtctg aacgacatct tcgaggctca gaaaatcgaa
     2161 tggcacgaac atcatcatca tcaccaccac caccaccact gagatccggc tgctaacaaa
     2221 gcccgaaagg aagctgagtt ggctgctgcc accgctgagc aataactagc ataacccctt
     2281 ggggcctcta aacgggtctt gaggggtttt ttgctgaaag gaggaactat atccggattg
     2341 gcgaatggga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca
     2401 gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct
     2461 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt
     2521 tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac
     2581 gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct
     2641 ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt
     2701 ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac
     2761 aaaaatttaa cgcgaatttt aacaaaatat taacgtttac aatttcaggt ggcacttttc
     2821 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc
     2881 cgctcatgaa ttaattctta gaaaaactca tcgagcatca aatgaaactg caatttattc
     2941 atatcaggat tatcaatacc atatttttga aaaagccgtt tctgtaatga aggagaaaac
     3001 tcaccgaggc agttccatag gatggcaaga tcctggtatc ggtctgcgat tccgactcgt
     3061 ccaacatcaa tacaacctat taatttcccc tcgtcaaaaa taaggttatc aagtgagaaa
     3121 tcaccatgag tgacgactga atccggtgag aatggcaaaa gtttatgcat ttctttccag
     3181 acttgttcaa caggccagcc attacgctcg tcatcaaaat cactcgcatc aaccaaaccg
     3241 ttattcattc gtgattgcgc ctgagcgaga cgaaatacgc gatcgctgtt aaaaggacaa
     3301 ttacaaacag gaatcgaatg caaccggcgc aggaacactg ccagcgcatc aacaatattt
     3361 tcacctgaat caggatattc ttctaatacc tggaatgctg ttttcccggg gatcgcagtg
     3421 gtgagtaacc atgcatcatc aggagtacgg ataaaatgct tgatggtcgg aagaggcata
     3481 aattccgtca gccagtttag tctgaccatc tcatctgtaa catcattggc aacgctacct
     3541 ttgccatgtt tcagaaacaa ctctggcgca tcgggcttcc catacaatcg atagattgtc
     3601 gcacctgatt gcccgacatt atcgcgagcc catttatacc catataaatc agcatccatg
     3661 ttggaattta atcgcggcct agagcaagac gtttcccgtt gaatatggct cataacaccc
     3721 cttgtattac tgtttatgta agcagacagt tttattgttc atgaccaaaa tcccttaacg
     3781 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     3841 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     3901 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     3961 agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa
     4021 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     4081 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     4141 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     4201 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     4261 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     4321 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     4381 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     4441 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc
     4501 ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag
     4561 ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta
     4621 ttttctcctt acgcatctgt gcggtatttc acaccgcata tatggtgcac tctcagtaca
     4681 atctgctctg atgccgcata gttaagccag tatacactcc gctatcgcta cgtgactggg
     4741 tcatggctgc gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc
     4801 tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt
     4861 tttcaccgtc atcaccgaaa cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt
     4921 gaagcgattc acagatgtct gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa
     4981 gcgttaatgt ctggcttctg ataaagcggg ccatgttaag ggcggttttt tcctgtttgg
     5041 tcactgatgc ctccgtgtaa gggggatttc tgttcatggg ggtaatgata ccgatgaaac
     5101 gagagaggat gctcacgata cgggttactg atgatgaaca tgcccggtta ctggaacgtt
     5161 gtgagggtaa acaactggcg gtatggatgc ggcgggacca gagaaaaatc actcagggtc
     5221 aatgccagcg cttcgttaat acagatgtag gtgttccaca gggtagccag cagcatcctg
     5281 cgatgcagat ccggaacata atggtgcagg gcgctgactt ccgcgtttcc agactttacg
     5341 aaacacggaa accgaagacc attcatgttg ttgctcaggt cgcagacgtt ttgcagcagc
     5401 agtcgcttca cgttcgctcg cgtatcggtg attcattctg ctaaccagta aggcaacccc
     5461 gccagcctag ccgggtcctc aacgacagga gcacgatcat gcgcacccgt ggggccgcca
     5521 tgccggcgat aatggcctgc ttctcgccga aacgtttggt ggcgggacca gtgacgaagg
     5581 cttgagcgag ggcgtgcaag attccgaata ccgcaagcga caggccgatc atcgtcgcgc
     5641 tccagcgaaa gcggtcctcg ccgaaaatga cccagagcgc tgccggcacc tgtcctacga
     5701 gttgcatgat aaagaagaca gtcataagtg cggcgacgat agtcatgccc cgcgcccacc
     5761 ggaaggagct gactgggttg aaggctctca agggcatcgg tcgagatccc ggtgcctaat
     5821 gagtgagcta acttacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     5881 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     5941 ggcgccaggg tggtttttct tttcaccagt gagacgggca acagctgatt gcccttcacc
     6001 gcctggccct gagagagttg cagcaagcgg tccacgctgg tttgccccag caggcgaaaa
     6061 tcctgtttga tggtggttaa cggcgggata taacatgagc tgtcttcggt atcgtcgtat
     6121 cccactaccg agatatccgc accaacgcgc agcccggact cggtaatggc gcgcattgcg
     6181 cccagcgcca tctgatcgtt ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc
     6241 atttgcatgg tttgttgaaa accggacatg gcactccagt cgccttcccg ttccgctatc
     6301 ggctgaattt gattgcgagt gagatattta tgccagccag ccagacgcag acgcgccgag
     6361 acagaactta atgggcccgc taacagcgcg atttgctggt gacccaatgc gaccagatgc
     6421 tccacgccca gtcgcgtacc gtcttcatgg gagaaaataa tactgttgat gggtgtctgg
     6481 tcagagacat caagaaataa cgccggaaca ttagtgcagg cagcttccac agcaatggca
     6541 tcctggtcat ccagcggata gttaatgatc agcccactga cgcgttgcgc gagaagattg
     6601 tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta ccatcgacac caccacgctg
     6661 gcacccagtt gatcggcgcg agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg
     6721 gccagactgg aggtggcaac gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc
     6781 acgcggttgg gaatgtaatt cagctccgcc atcgccgctt ccactttttc ccgcgttttc
     6841 gcagaaacgt ggctggcctg gttcaccacg cgggaaacgg tctgataaga gacaccggca
     6901 tactctgcga catcgtataa cgttactggt ttcacattca ccaccctgaa ttgactctct
     6961 tccgggcgct atcatgccat accgcgaaag gttttgcgcc attcgatggt gtccgggatc
     7021 tcgacgctct cccttatgcg actcctgcat taggaagcag cccagtagta ggttgaggcc
     7081 gttgagcacc gccgccgcaa ggaatggtgc atgcaaggag atggcgccca acagtccccc
     7141 ggccacgggg cctgccacca tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg
     7201 agcccgatct tccccatcgg tgatgtcggc gatataggcg ccagcaaccg cacctgtggc
     7261 gccggtgatg ccggccacga tgcgtccggc gtagaggatc gagatctcga tcccgcgaaa
     7321 t
//
