LOCUS       40924_41185        6259 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Mutation screening vector pSPRX, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6259)
  AUTHORS   Matin MM, Fowler GJS., Dalton A, Hornby DP.
  TITLE     The MINU (Methyltransferase Inactivation Utilisation) test- A novel 
            method for the screening of truncating mutations
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6259)
  AUTHORS   Matin MM, Hornby DP.
  TITLE     Direct Submission
  JOURNAL   Submitted (22-OCT-2001) Molecular Biology, Krebs Institute, Firth 
            Court, Western Bank, Sheffield S10 2TN, UK
REFERENCE   3  (bases 1 to 6259)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 6259)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (22-OCT-2001) Molecular Biology, Krebs Institute, Firth Court, 
            Western Bank, Sheffield S10 2TN, UK"
COMMENT     SGRef: number: 3; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..6259
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        183..211
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and
                     lac UV5 promoters"
     protein_bind    219..235
                     /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)."
     CDS             258..911
                     /codon_start=1
                     /label=GST
                     /note="glutathione S-transferase from Schistosoma
                     japonicum"
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     CDS             918..935
                     /codon_start=1
                     /label=thrombin site
                     /note="thrombin recognition and cleavage site"
                     /translation="LVPRGS"
     promoter        2562..2666
                     /label=AmpR promoter
     CDS             2667..3524
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      3698..4286
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     promoter        4530..4607
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the
                     promoter boosts expression of the lacI gene about 10-fold."
     CDS             4608..5687
                     /codon_start=1
                     /label=lacI
                     /note="lac repressor"
                     /translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     protein_bind    5703..5724
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     promoter        5739..5769
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5777..5793
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /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)."
     CDS             5813..5986
                     /codon_start=1
                     /label=lacZ-alpha
                     /note="LacZ-alpha fragment of beta-galactosidase"
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGE"
ORIGIN
        1 acgttatcga ctgcacggtg caccaatgct tctggcgtca ggcagccatc ggaagctgtg
       61 gtatggctgt gcaggtcgta aatcactgca taattcgtgt cgctcaaggc gcactcccgt
      121 tctggataat gttttttgcg ccgacatcat aacggttctg gcaaatattc tgaaatgagc
      181 tgttgacaat taatcatcgg ctcgtataat gtgtggaatt gtgagcggat aacaatttca
      241 cacaggaaac agtattcatg tcccctatac taggttattg gaaaattaag ggccttgtgc
      301 aacccactcg acttcttttg gaatatcttg aagaaaaata tgaagagcat ttgtatgagc
      361 gcgatgaagg tgataaatgg cgaaacaaaa agtttgaatt gggtttggag tttcccaatc
      421 ttccttatta tattgatggt gatgttaaat taacacagtc tatggccatc atacgttata
      481 tagctgacaa gcacaacatg ttgggtggtt gtccaaaaga gcgtgcagag atttcaatgc
      541 ttgaaggagc ggttttggat attagatacg gtgtttcgag aattgcatat agtaaagact
      601 ttgaaactct caaagttgat tttcttagca agctacctga aatgctgaaa atgttcgaag
      661 atcgtttatg tcataaaaca tatttaaatg gtgatcatgt aacccatcct gacttcatgt
      721 tgtatgacgc tcttgatgtt gttttataca tggacccaat gtgcctggat gcgttcccaa
      781 aattagtttg ttttaaaaaa cgtattgaag ctatcccaca aattgataag tacttgaaat
      841 ccagcaagta tatagcatgg cctttgcagg gctggcaagc cacgtttggt ggtggcgacc
      901 atcctccaaa atcggatctg gttccgcgtg gatccttggg taaactacgt gtaatgagtc
      961 tttttagtgg gatcggtgga tttgaagctg cactaagaaa cattggggtt ggttatgagc
     1021 tggttggttt tagtgagatt gataaatatg ccgtcaaatc tttttgtgca attcacaacg
     1081 ttgatgagca attaaatttt ggagatgtaa gcaagattga taagaaaaaa ctacctgaat
     1141 ttgatatctt agttggagga tctccttgtc aaagctttag tgtagccggc catcgaaagg
     1201 gatttgaaga tacaagaggt accttgtttt ttcaatacgt tgagactctt aaggaaaagc
     1261 aaccaaagtt ttttgttttt gaaaatgtta aagggttgat caaccatgat aaaggaaata
     1321 cattaaatgt tatggctgaa gctttcagtg aagttgggta cagaattgac ctagagctgc
     1381 ttaattcaaa attctttaat gttccacaaa atagggagcg actttacata attggaatta
     1441 gagaagattt aattaaaaat gaagaatggt ctttggattt taaaagaaag gatatacttc
     1501 aaaaagggaa acagagattg gtagaattag atattaaaag ctttaatttt agatggacag
     1561 ctcaatcggc tgctacgaag aggctaaaag atttattaga agaatacgtt gatgaaaagt
     1621 actacttgaa tgaagataaa acaaacagtt tgatcaaaga gttgtctaca agtcgactta
     1681 atgaaaatct tactgttgag caagtaggta acattaatcc ctctggtaat ggaatgaatg
     1741 gaaatgttta taattcatct ggattaagcc ccacaattac cactaataaa ggagagggac
     1801 tgaaaattgc agttgagtac tctagatctc tgcagctcga gcccggggct agccatatgg
     1861 aattcagagg cctgaatagg aaccaaaaac aaaatgcagt tgttgaggta aggccagtat
     1921 taaccccaga aaggggggag aagcgacaaa atggaagaag atttaaagat gacggtgaac
     1981 cagcatttac agtaaacaca attgacagac acggggtagc ggttggagag tatccaaaat
     2041 acagaattag aagattaaca ccgttagagt gctttaggct acaggctttt gatgacgaag
     2101 attttgaaaa agcttttgct gcgggaataa gtaactcaca attatataag caagccggta
     2161 attcaattac tgtaactgtg cttgagtcaa tattcaagga attaatacat acatacgtta
     2221 ataaagaatc tgaataactg gagctcaagc ttattcatcg tgactgactg acgatctgcc
     2281 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
     2341 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
     2401 ttggcgggtg tcggggcgca gccatgaccc agtcacgtag cgatagcgga gtgtataatt
     2461 cttgaagacg aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa
     2521 tggtttctta gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
     2581 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
     2641 ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc
     2701 ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa
     2761 aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg
     2821 gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag
     2881 ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc
     2941 gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta
     3001 cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg
     3061 cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca
     3121 acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac
     3181 caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat
     3241 taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg
     3301 ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata
     3361 aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta
     3421 agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa
     3481 atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag
     3541 tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg
     3601 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     3661 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     3721 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     3781 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     3841 ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     3901 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     3961 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     4021 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     4081 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     4141 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     4201 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     4261 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     4321 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
     4381 accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca
     4441 gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc cttacgcatc
     4501 tgtgcggtat ttcacaccgc ataaattccg acaccatcga atggtgcaaa acctttcgcg
     4561 gtatggcatg atagcgcccg gaagagagtc aattcagggt ggtgaatgtg aaaccagtaa
     4621 cgttatacga tgtcgcagag tatgccggtg tctcttatca gaccgtttcc cgcgtggtga
     4681 accaggccag ccacgtttct gcgaaaacgc gggaaaaagt ggaagcggcg atggcggagc
     4741 tgaattacat tcccaaccgc gtggcacaac aactggcggg caaacagtcg ttgctgattg
     4801 gcgttgccac ctccagtctg gccctgcacg cgccgtcgca aattgtcgcg gcgattaaat
     4861 ctcgcgccga tcaactgggt gccagcgtgg tggtgtcgat ggtagaacga agcggcgtcg
     4921 aagcctgtaa agcggcggtg cacaatcttc tcgcgcaacg cgtcagtggg ctgatcatta
     4981 actatccgct ggatgaccag gatgccattg ctgtggaagc tgcctgcact aatgttccgg
     5041 cgttatttct tgatgtctct gaccagacac ccatcaacag tattattttc tcccatgaag
     5101 acggtacgcg actgggcgtg gagcatctgg tcgcattggg tcaccagcaa atcgcgctgt
     5161 tagcgggccc attaagttct gtctcggcgc gtctgcgtct ggctggctgg cataaatatc
     5221 tcactcgcaa tcaaattcag ccgatagcgg aacgggaagg cgactggagt gccatgtccg
     5281 gttttcaaca aaccatgcaa atgctgaatg agggcatcgt tcccactgcg atgctggttg
     5341 ccaacgatca gatggcgctg ggcgcaatgc gcgccattac cgagtccggg ctgcgcgttg
     5401 gtgcggatat ctcggtagtg ggatacgacg ataccgaaga cagctcatgt tatatcccgc
     5461 cgttaaccac catcaaacag gattttcgcc tgctggggca aaccagcgtg gaccgcttgc
     5521 tgcaactctc tcagggccag gcggtgaagg gcaatcagct gttgcccgtc tcactggtga
     5581 aaagaaaaac caccctggcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt
     5641 cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca
     5701 attaatgtga gttagctcac tcattaggca ccccaggctt tacactttat gcttccggct
     5761 cgtatgttgt gtggaattgt gagcggataa caatttcaca caggaaacag ctatgaccat
     5821 gattacggat tcactggccg tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac
     5881 ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata gcgaagaggc
     5941 ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatggc gctttgcctg
     6001 gtttccggca ccagaagcgg tgccggaaag ctggctggag tgcgatcttc ctgaggccga
     6061 tactgtcgtc gtcccctcaa actggcagat gcacggttac gatgcgccca tctacaccaa
     6121 cgtaacctat cccattacgg tcaatccgcc gtttgttccc acggagaatc cgacgggttg
     6181 ttactcgctc acatttaatg ttgatgaaag ctggctacag gaaggccaga cgcgaattat
     6241 ttttgatggc gttggaatt
//
