LOCUS       sequence-457146-        5818 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express N-terminal HRV 3C protease cleavable GST fused to Bacillus 
            cereus PC-PLC in E.coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5818)
  AUTHORS   Murakami C, Mizuno S, Kado S, Sakane F
  TITLE     Development of a liquid chromatography-mass spectrometry based 
            enzyme activity assay for phosphatidylcholine-specific phospholipase
            C.
  JOURNAL   Anal Biochem. 2017 Jun 1;526:43-49. doi: 10.1016/j.ab.2017.03.010. 
            Epub 2017 Mar 14.
  PUBMED    28315318
REFERENCE   2  (bases 1 to 5818)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Anal 
            Biochem."; date: "2017-06-1"; volume: "526:43-49. doi"; pages: " 
            10.1016/j.ab.2017.03.010. Epub 2017 Mar 14"
COMMENT     Sequence Label: pGEX-GST-BcPC-PLC
FEATURES             Location/Qualifiers
     source          1..5818
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      75..663
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     564..583
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     817..834
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        907..984
                     /gene="lacI"
                     /label=lacI promoter
     CDS             985..2067
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(1004..1023)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(2116..2121,2122..2139,2140..2146)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2154..2170
                     /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     2178..2194
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2178..2194
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             2190..2717
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(2210..2227)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2210..2226)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2219..2241)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     promoter        join(2819..2824,2825..2840,2841..2847)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    2855..2871
                     /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             2894..3547
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     3505..3527
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             3554..3577
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     primer_bind     complement(4504..4526)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4671..4689)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4757..4861
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4862..4930,4931..5722)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(5080..5099)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
       61 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
      121 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
      181 aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag
      241 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
      301 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
      361 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
      421 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
      481 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
      541 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
      601 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
      661 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
      721 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga
      781 gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg
      841 gaagagcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata
      901 aattccgaca ccatcgaatg gcgcaaaacc tttcgcggta tggcatgata gcgcccggaa
      961 gagagtcaat tcagggtggt gaatgtgaaa ccagtaacgt tatacgatgt cgcagagtat
     1021 gccggtgtct cttatcagac cgtttcccgc gtggtgaacc aggccagcca cgtttctgcg
     1081 aaaacgcggg aaaaagtgga agcggcgatg gcggagctga attacattcc caaccgcgtg
     1141 gcacaacaac tggcgggcaa acagtcgttg ctgattggcg ttgccacctc cagtctggcc
     1201 ctgcacgcgc cgtcgcaaat tgtcgcggcg attaaatctc gcgccgatca actgggtgcc
     1261 agcgtggtgg tgtcgatggt agaacgaagc ggcgtcgaag cctgtaaagc ggcggtgcac
     1321 aatcttctcg cgcaacgcgt cagtgggctg atcattaact atccgctgga tgaccaggat
     1381 gccattgctg tggaagctgc ctgcactaat gttccggcgt tatttcttga tgtctctgac
     1441 cagacaccca tcaacagtat tattttctcc catgaagacg gtacgcgact gggcgtggag
     1501 catctggtcg cattgggtca ccagcaaatc gcgctgttag cgggcccatt aagttctgtc
     1561 tcggcgcgtc tgcgtctggc tggctggcat aaatatctca ctcgcaatca aattcagccg
     1621 atagcggaac gggaaggcga ctggagtgcc atgtccggtt ttcaacaaac catgcaaatg
     1681 ctgaatgagg gcatcgttcc cactgcgatg ctggttgcca acgatcagat ggcgctgggc
     1741 gcaatgcgcg ccattaccga gtccgggctg cgcgttggtg cggatatctc ggtagtggga
     1801 tacgacgata ccgaagacag ctcatgttat atcccgccgt taaccaccat caaacaggat
     1861 tttcgcctgc tggggcaaac cagcgtggac cgcttgctgc aactctctca gggccaggcg
     1921 gtgaagggca atcagctgtt gcccgtctca ctggtgaaaa gaaaaaccac gctagcgccc
     1981 aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag
     2041 gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtaagtt agctcactca
     2101 ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag
     2161 cggataacaa tttcacacag gaaacagcta tgaccatgat tacggattca ctggccgtcg
     2221 ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac
     2281 atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac
     2341 agttgcgcag cctgaatggc gaatggcgct ttgcctggtt tccggcacca gaagcggtgc
     2401 cggaaagctg gctggagtgc gatcttcctg aggccgatac tgtcgtcgtc ccctcaaact
     2461 ggcagatgca cggttacgat gcgcccatct acaccaacgt gacctatccc attacggtca
     2521 atccgccgtt tgttcccacg gagaatccga cgggttgtta ctcgctcaca tttaatgttg
     2581 atgaaagctg gctacaggaa ggccagacgc gaattatttt tgatggcgtt ggaattagct
     2641 tatcgactgc acggtgcacc aatgcttctg gcgtcaggca gccatcggaa gctgtggtat
     2701 ggctgtgcag gtcgtaaatc actgcataat tcgtgtcgct caaggcgcac tcccgttctg
     2761 gataatgttt tttgcgccga catcataacg gttctggcaa atattctgaa atgagctgtt
     2821 gacaattaat catcggctcg tataatgtgt ggaattgtga gcggataaca atttcacaca
     2881 ggaaacagta ttcatgtccc ctatactagg ttattggaaa attaagggcc ttgtgcaacc
     2941 cactcgactt cttttggaat atcttgaaga aaaatatgaa gagcatttgt atgagcgcga
     3001 tgaaggtgat aaatggcgaa acaaaaagtt tgaattgggt ttggagtttc ccaatcttcc
     3061 ttattatatt gatggtgatg ttaaattaac acagtctatg gccatcatac gttatatagc
     3121 tgacaagcac aacatgttgg gtggttgtcc aaaagagcgt gcagagattt caatgcttga
     3181 aggagcggtt ttggatatta gatacggtgt ttcgagaatt gcatatagta aagactttga
     3241 aactctcaaa gttgattttc ttagcaagct acctgaaatg ctgaaaatgt tcgaagatcg
     3301 tttatgtcat aaaacatatt taaatggtga tcatgtaacc catcctgact tcatgttgta
     3361 tgacgctctt gatgttgttt tatacatgga cccaatgtgc ctggatgcgt tcccaaaatt
     3421 agtttgtttt aaaaaacgta ttgaagctat cccacaaatt gataagtact tgaaatccag
     3481 caagtatata gcatggcctt tgcagggctg gcaagccacg tttggtggtg gcgaccatcc
     3541 tccaaaatcg gatctggaag ttctgttcca ggggcccctg ggatccatga aaaaaaaagt
     3601 acttgcttta gcggcagcta ttacattagt tgctccatta caaagtgttg catttgctca
     3661 tgaaaatgat gggggacaga gatttggagt tattccgcgc tggtctgctg aagataaaca
     3721 taaagaaggc gtgaattctc atttatggat tgtaaaccgt gcaattgata ttatgtctcg
     3781 taatacaaca cttgtaaaac aagatcgagt tgcactatta aatgaatggc gtactgagtt
     3841 agagaacggt atttatgctg ctgactatga aaatccttat tatgataata gcacatttgc
     3901 ttcacatttc tatgaccctg acaatgggaa aacttatatt ccgtatgcaa agcaagcaaa
     3961 ggaaacagga gctaaatatt ttaaattagc tggtgagtct tataaaaata aagatatgaa
     4021 acaagcgttc ttctatttag gattatctct tcattattta ggggatgtaa accaaccgat
     4081 gcatgcggca aactttacga atctatcgta tccacaagga ttccattcta aatatgaaaa
     4141 ctttgtagat acgataaaag ataattataa agtaacggat ggaaatggat attggaactg
     4201 gaaaggtacg aatccagaag attggattca tggagcggca gtagttgcga aacaagatta
     4261 cgctggcatt gtaaatgata atacgaaaga ttggttcgtg agagctgctg tatcacaaga
     4321 atatgcagat aaatggcgcg ctgaagttac accaatgaca ggtaagcgtt taatggatgc
     4381 acaacgtgtt actgctggat atattcagct ttggtttgat acgtacggaa atcgttaact
     4441 cgagcggccg catcgtgact gactgacgat ctgcctcgcg cgtttcggtg atgacggtga
     4501 aaacctctga cacatgcagc tcccggagac ggtcacagct tgtctgtaag cggatgccgg
     4561 gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg gcgcagccat
     4621 gacccagtca cgtagcgata gcggagtgta taattcttga agacgaaagg gcctcgtgat
     4681 acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac
     4741 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     4801 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
     4861 tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc
     4921 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc
     4981 acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc
     5041 cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc
     5101 ccgtgttgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt
     5161 ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt
     5221 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat
     5281 cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct
     5341 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat
     5401 gcctgcagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc
     5461 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg
     5521 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc
     5581 tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta
     5641 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc
     5701 ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga
     5761 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctc
//
