LOCUS       62056_12320        8011 bp DNA     circular SYN 03-APR-2019
DEFINITION  Expression vector pHGT-Bio, complete sequence.
ACCESSION   MH763823
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8011)
  AUTHORS   Gileadi O.
  TITLE     Direct Submission
  JOURNAL   Submitted (17-AUG-2018) Structural Genomics Consortium, SGC, 
            University of Oxford, ORCRB, Roosevelt Drive, Oxford OX3 7DQ, UK
REFERENCE   2  (bases 1 to 8011)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Submitted 
            (17-AUG-2018) Structural Genomics Consortium, SGC, University of 
            Oxford, ORCRB, Roosevelt Drive, Oxford OX3 7DQ, UK"
COMMENT     ##Assembly-Data-START##
            Sequencing Technology :: Sanger dideoxy sequencing 
            ##Assembly-Data-END##
FEATURES             Location/Qualifiers
     source          1..8011
                     /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             116..769
                     /codon_start=1
                     /label=GST
                     /note="glutathione S-transferase from Schistosoma
                     japonicum"
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGADHPPK"
     CDS             797..817
                     /codon_start=1
                     /label=TEV site
                     /note="tobacco etch virus (TEV) protease recognition and 
                     cleavage site"
                     /translation="ENLYFQS"
     promoter        833..1278
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             1279..2697
                     /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             2762..2806
                     /codon_start=1
                     /label=AviTag(TM)
                     /note="peptide tag that allows for enzymatic biotinylation"
                     /translation="GLNDIFEAQKIEWHE"
     CDS             2872..2889
                     /codon_start=1
                     /label=6xHis
                     /note="6xHis affinity tag"
                     /translation="HHHHHH"
     terminator      2956..3003
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3040..3495
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     CDS             complement(3591..4403)
                     /codon_start=1
                     /label=KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     rep_origin      4525..5113
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     misc_feature    complement(5299..5441)
                     /label=bom
                     /note="basis of mobility region from pBR322"
     CDS             complement(5546..5734)
                     /codon_start=1
                     /label=rop
                     /note="Rop protein, which maintains plasmids at low copy
                     number"
                     /translation="VTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     protein_bind    complement(6509..6530)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             complement(6546..7625)
                     /codon_start=1
                     /label=lacI
                     /note="lac repressor"
                     /translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     promoter        complement(7626..7703)
                     /label=lacI promoter
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atatacatat gcaccatcat catcatcatt cttctatgtc
      121 ccctatacta ggttattgga aaattaaggg ccttgtgcaa cccactcgac ttcttttgga
      181 atatcttgaa gaaaaatatg aagagcattt gtatgagcgc gatgaaggtg ataaatggcg
      241 aaacaaaaag tttgaattgg gtttggagtt tcccaatctt ccttattata ttgatggtga
      301 tgttaaatta acacagtcta tggccatcat acgttatata gctgacaagc acaacatgtt
      361 gggtggttgt ccaaaagagc gtgcagagat ttcaatgctt gaaggagcgg ttttggatat
      421 tagatacggt gtttcgagaa ttgcatatag taaagacttt gaaactctca aagttgattt
      481 tcttagcaag ctacctgaaa tgctgaaaat gttcgaagat cgtttatgtc ataaaacata
      541 tttaaatggt gatcatgtaa cccatcctga cttcatgttg tatgacgctc ttgatgttgt
      601 tttatacatg gacccaatgt gcctggatgc gttcccaaaa ttagtttgtt ttaaaaaacg
      661 tattgaagct atcccacaaa ttgataagta cttgaaatcc agcaagtata tagcatggcc
      721 tttgcagggc tggcaagcca cgtttggtgg tgccgaccat cctccaaaat cgagctcagg
      781 tgtagatctg ggtaccgaga acctgtactt ccaatccatg gagaccgacg tccacatata
      841 cctgccgttc actattattt agtgaaatga gatattatga tattttctga attgtgatta
      901 aaaaggcaac tttatgccca tgcaacagaa actataaaaa atacagagaa tgaaaagaaa
      961 cagatagatt ttttagttct ttaggcccgt agtctgcaaa tccttttatg attttctatc
     1021 aaacaaaaga ggaaaataga ccagttgcaa tccaaacgag agtctaatag aatgaggtcg
     1081 aaaagtaaat cgcgcgggtt tgttactgat aaagcaggca agacctaaaa tgtgtaaagg
     1141 gcaaagtgta tactttggcg tcacccctta catattttag gtcttttttt attgtgcgta
     1201 actaacttgc catcttcaaa caggagggct ggaataagca gaccgctaac acagtacata
     1261 aaaaaggaga catgaacgat gaacatcaaa aagtttgcaa aacaagcaac agtattaacc
     1321 tttactaccg cactgctggc aggaggcgca actcaagcgt ttgcgaaaga aacgaaccaa
     1381 aagccatata aggaaacata cggcatttcc catattacac gccatgatat gctgcaaatc
     1441 cctgaacagc aaaaaaatga aaaatataaa gttcctgagt tcgattcgtc cacaattaaa
     1501 aatatctctt ctgcaaaagg cctggacgtt tgggacagct ggccattaca aaacactgac
     1561 ggcactgtcg caaactatca cggctaccac atcgtctttg cattagccgg agatcctaaa
     1621 aatgcggatg acacatcgat ttacatgttc tatcaaaaag tcggcgaaac ttctattgac
     1681 agctggaaaa acgctggccg cgtctttaaa gacagcgaca aattcgatgc aaatgattct
     1741 atcctaaaag accaaacaca agaatggtca ggttcagcca catttacatc tgacggaaaa
     1801 atccgtttat tctacactga tttctccggt aaacattacg gcaaacaaac actgacaact
     1861 gcacaagtta acgtatcagc atcagacagc tctttgaaca tcaacggtgt agaggattat
     1921 aaatcaatct ttgacggtga cggaaaaacg tatcaaaatg tacagcagtt catcgatgaa
     1981 ggcaactaca gctcaggcga caaccatacg ctgagagatc ctcactacgt agaagataaa
     2041 ggccacaaat acttagtatt tgaagcaaac actggaactg aagatggcta ccaaggcgaa
     2101 gaatctttat ttaacaaagc atactatggc aaaagcacat cattcttccg tcaagaaagt
     2161 caaaaacttc tgcaaagcga taaaaaacgc acggctgagt tagcaaacgg cgctctcggt
     2221 atgattgagc taaacgatga ttacacactg aaaaaagtga tgaaaccgct gattgcatct
     2281 aacacagtaa cagatgaaat tgaacgcgcg aacgtcttta aaatgaacgg caaatggtac
     2341 ctgttcactg actcccgcgg atcaaaaatg acgattgacg gcattacgtc taacgatatt
     2401 tacatgcttg gttatgtttc taattcttta actggcccat acaagccgct gaacaaaact
     2461 ggccttgtgt taaaaatgga tcttgatcct aacgatgtaa cctttactta ctcacacttc
     2521 gctgtacctc aagcgaaagg aaacaatgtc gtgattacaa gctatatgac aaacagagga
     2581 ttctacgcag acaaacaatc aacgtttgcg cctagcttcc tgctgaacat caaaggcaag
     2641 aaaacatctg ttgtcaaaga cagcatcctt gaacaaggac aattaacagt taacaaataa
     2701 aaacgcaaaa gaaaatgccg atatcctatt ggcattgacg gtctccagta aaggtggata
     2761 cggcctgaat gatatctttg aagcgcagaa gattgaatgg catgaatgac agtaaaggtg
     2821 gatacggatc cgaattcgag ctccgtcgac aagcttgcgg ccgcactcga gcaccaccac
     2881 caccaccact gagatccggc tgctaacaaa gcccgaaagg aagctgagtt ggctgctgcc
     2941 accgctgagc aataactagc ataacccctt ggggcctcta aacgggtctt gaggggtttt
     3001 ttgctgaaag gaggaactat atccggattg gcgaatggga cgcgccctgt agcggcgcat
     3061 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag
     3121 cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc
     3181 aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc
     3241 ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt
     3301 ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa
     3361 caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg
     3421 cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat
     3481 taacgtttac aatttcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
     3541 tatttttcta aatacattca aatatgtatc cgctcatgaa ttaattctta gaaaaactca
     3601 tcgagcatca aatgaaactg caatttattc atatcaggat tatcaatacc atatttttga
     3661 aaaagccgtt tctgtaatga aggagaaaac tcaccgaggc agttccatag gatggcaaga
     3721 tcctggtatc ggtctgcgat tccgactcgt ccaacatcaa tacaacctat taatttcccc
     3781 tcgtcaaaaa taaggttatc aagtgagaaa tcaccatgag tgacgactga atccggtgag
     3841 aatggcaaaa gtttatgcat ttctttccag acttgttcaa caggccagcc attacgctcg
     3901 tcatcaaaat cactcgcatc aaccaaaccg ttattcattc gtgattgcgc ctgagcgaga
     3961 cgaaatacgc gatcgctgtt aaaaggacaa ttacaaacag gaatcgaatg caaccggcgc
     4021 aggaacactg ccagcgcatc aacaatattt tcacctgaat caggatattc ttctaatacc
     4081 tggaatgctg ttttcccggg gatcgcagtg gtgagtaacc atgcatcatc aggagtacgg
     4141 ataaaatgct tgatggtcgg aagaggcata aattccgtca gccagtttag tctgaccatc
     4201 tcatctgtaa catcattggc aacgctacct ttgccatgtt tcagaaacaa ctctggcgca
     4261 tcgggcttcc catacaatcg atagattgtc gcacctgatt gcccgacatt atcgcgagcc
     4321 catttatacc catataaatc agcatccatg ttggaattta atcgcggcct agagcaagac
     4381 gtttcccgtt gaatatggct cataacaccc cttgtattac tgtttatgta agcagacagt
     4441 tttattgttc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
     4501 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     4561 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     4621 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt
     4681 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     4741 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     4801 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     4861 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     4921 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
     4981 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
     5041 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
     5101 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
     5161 ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc
     5221 ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag
     5281 cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc
     5341 acaccgcata tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag
     5401 tatacactcc gctatcgcta cgtgactggg tcatggctgc gccccgacac ccgccaacac
     5461 ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga
     5521 ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc
     5581 agctgcggta aagctcatca gcgtggtcgt gaagcgattc acagatgtct gcctgttcat
     5641 ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg ataaagcggg
     5701 ccatgttaag ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa gggggatttc
     5761 tgttcatggg ggtaatgata ccgatgaaac gagagaggat gctcacgata cgggttactg
     5821 atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa acaactggcg gtatggatgc
     5881 ggcgggacca gagaaaaatc actcagggtc aatgccagcg cttcgttaat acagatgtag
     5941 gtgttccaca gggtagccag cagcatcctg cgatgcagat ccggaacata atggtgcagg
     6001 gcgctgactt ccgcgtttcc agactttacg aaacacggaa accgaagacc attcatgttg
     6061 ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg cgtatcggtg
     6121 attcattctg ctaaccagta aggcaacccc gccagcctag ccgggtcctc aacgacagga
     6181 gcacgatcat gcgcacccgt ggggccgcca tgccggcgat aatggcctgc ttctcgccga
     6241 aacgtttggt ggcgggacca gtgacgaagg cttgagcgag ggcgtgcaag attccgaata
     6301 ccgcaagcga caggccgatc atcgtcgcgc tccagcgaaa gcggtcctcg ccgaaaatga
     6361 cccagagcgc tgccggcacc tgtcctacga gttgcatgat aaagaagaca gtcataagtg
     6421 cggcgacgat agtcatgccc cgcgcccacc ggaaggagct gactgggttg aaggctctca
     6481 agggcatcgg tcgagatccc ggtgcctaat gagtgagcta acttacatta attgcgttgc
     6541 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
     6601 aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg tggtttttct tttcaccagt
     6661 gagacgggca acagctgatt gcccttcacc gcctggccct gagagagttg cagcaagcgg
     6721 tccacgctgg tttgccccag caggcgaaaa tcctgtttga tggtggttaa cggcgggata
     6781 taacatgagc tgtcttcggt atcgtcgtat cccactaccg agatatccgc accaacgcgc
     6841 agcccggact cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc
     6901 atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa accggacatg
     6961 gcactccagt cgccttcccg ttccgctatc ggctgaattt gattgcgagt gagatattta
     7021 tgccagccag ccagacgcag acgcgccgag acagaactta atgggcccgc taacagcgcg
     7081 atttgctggt gacccaatgc gaccagatgc tccacgccca gtcgcgtacc gtcttcatgg
     7141 gagaaaataa tactgttgat gggtgtctgg tcagagacat caagaaataa cgccggaaca
     7201 ttagtgcagg cagcttccac agcaatggca tcctggtcat ccagcggata gttaatgatc
     7261 agcccactga cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc ttcgacgccg
     7321 cttcgttcta ccatcgacac caccacgctg gcacccagtt gatcggcgcg agatttaatc
     7381 gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac gccaatcagc
     7441 aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc
     7501 atcgccgctt ccactttttc ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg
     7561 cgggaaacgg tctgataaga gacaccggca tactctgcga catcgtataa cgttactggt
     7621 ttcacattca ccaccctgaa ttgactctct tccgggcgct atcatgccat accgcgaaag
     7681 gttttgcgcc attcgatggt gtccgggatc tcgacgctct cccttatgcg actcctgcat
     7741 taggaagcag cccagtagta ggttgaggcc gttgagcacc gccgccgcaa ggaatggtgc
     7801 atgcaaggag atggcgccca acagtccccc ggccacgggg cctgccacca tacccacgcc
     7861 gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct tccccatcgg tgatgtcggc
     7921 gatataggcg ccagcaaccg cacctgtggc gccggtgatg ccggccacga tgcgtccggc
     7981 gtagaggatc gagatctcga tcccgcgaaa t
//
