LOCUS       Exported                6259 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Mutation screening vector pSPRX, complete sequence.
ACCESSION   AF438205
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  JOURNAL   Unpublished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)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..6259
                     /organism="Mutation screening vector pSPRX"
                     /mol_type="genomic DNA"
                     /db_xref="taxon:177209"
     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
                     /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             258..2237
                     /codon_start=1
                     /product="GST-M.SPRX methyltransferase fusion protein"
                     /label=GST-M.SPRX methyltransferase fusion protein
                     /protein_id="AAM21516.1"
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDLV
                     PRGSLGKLRVMSLFSGIGGFEAALRNIGVGYELVGFSEIDKYAVKSFCAIHNVDEQLNF
                     GDVSKIDKKKLPEFDILVGGSPCQSFSVAGHRKGFEDTRGTLFFQYVETLKEKQPKFFV
                     FENVKGLINHDKGNTLNVMAEAFSEVGYRIDLELLNSKFFNVPQNRERLYIIGIREDLI
                     KNEEWSLDFKRKDILQKGKQRLVELDIKSFNFRWTAQSAATKRLKDLLEEYVDEKYYLN
                     EDKTNSLIKELSTSRLNENLTVEQVGNINPSGNGMNGNVYNSSGLSPTITTNKGEGLKI
                     AVEYSRSLQLEPGASHMEFRGLNRNQKQNAVVEVRPVLTPERGEKRQNGRRFKDDGEPA
                     FTVNTIDRHGVAVGEYPKYRIRRLTPLECFRLQAFDDEDFEKAFAAGISNSQLYKQAGN
                     SITVTVLESIFKELIHTYVNKESE"
     CDS             258..911
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     CDS             918..935
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     promoter        2562..2666
                     /gene="bla"
                     /label=AmpR promoter
     CDS             2667..3527
                     /codon_start=1
                     /product="beta lactamase"
                     /label=beta lactamase
                     /protein_id="AAM21517.1"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      3698..4286
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     promoter        4530..4607
                     /gene="lacI (mutant)"
                     /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..5690
                     /codon_start=1
                     /gene="laqIq"
                     /product="lac repressor"
                     /label=laqIq
                     /protein_id="AAM21518.1"
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     gene            4608..5690
                     /gene="laqIq"
                     /label=laqIq
     protein_bind    5703..5724
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /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)."
     primer_bind     5801..5817
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             5813..81
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGITLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(5833..5849)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"