LOCUS       sequence-353873-        7108 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express human prolyl oligopeptidase (tagged) in bacterial cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7108)
  AUTHORS   Retzl B, Hellinger R, Muratspahic E, Pinto MEF, Bolzani VS, Gruber 
            CW
  TITLE     Discovery of a Beetroot Protease Inhibitor to Identify and Classify 
            Plant-Derived Cystine Knot Peptides.
  JOURNAL   J Nat Prod. 2020 Nov 25;83(11):3305-3314. doi: 
            10.1021/acs.jnatprod.0c00648. Epub 2020 Oct 29.
  PUBMED    33118348
REFERENCE   2  (bases 1 to 7108)
  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"; doi: 
            "10.1021/acs.jnatprod.0c00648"; journalName: "J Nat Prod"; date: 
            "2020-11-25- 25"; volume: "83"; issue: "11"; pages: "3305-3314"
COMMENT     Sequence Label: pGEX6P1-hPREP
FEATURES             Location/Qualifiers
     source          1..7108
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(823..828,829..846,847..853)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    861..877
                     /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     885..901
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     885..901
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             897..1424
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(917..934)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(917..933)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(926..948)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     promoter        join(1526..1531,1532..1547,1548..1554)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    1562..1578
                     /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             1601..2254
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     2212..2234
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             2261..2284
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     primer_bind     complement(4501..4523)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4668..4686)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4754..4858
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4859..4927,4928..5719)
                     /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(5077..5096)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5890..6478
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6379..6398
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6632..6649
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        6722..6799
                     /gene="lacI"
                     /label=lacI promoter
     CDS             join(6800..7108,1..774)
                     /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(6819..6838)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 gtcgaagcct gtaaagcggc ggtgcacaat cttctcgcgc aacgcgtcag tgggctgatc
       61 attaactatc cgctggatga ccaggatgcc attgctgtgg aagctgcctg cactaatgtt
      121 ccggcgttat ttcttgatgt ctctgaccag acacccatca acagtattat tttctcccat
      181 gaagacggta cgcgactggg cgtggagcat ctggtcgcat tgggtcacca gcaaatcgcg
      241 ctgttagcgg gcccattaag ttctgtctcg gcgcgtctgc gtctggctgg ctggcataaa
      301 tatctcactc gcaatcaaat tcagccgata gcggaacggg aaggcgactg gagtgccatg
      361 tccggttttc aacaaaccat gcaaatgctg aatgagggca tcgttcccac tgcgatgctg
      421 gttgccaacg atcagatggc gctgggcgca atgcgcgcca ttaccgagtc cgggctgcgc
      481 gttggtgcgg atatctcggt agtgggatac gacgataccg aagacagctc atgttatatc
      541 ccgccgttaa ccaccatcaa acaggatttt cgcctgctgg ggcaaaccag cgtggaccgc
      601 ttgctgcaac tctctcaggg ccaggcggtg aagggcaatc agctgttgcc cgtctcactg
      661 gtgaaaagaa aaaccacgct agcgcccaat acgcaaaccg cctctccccg cgcgttggcc
      721 gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa
      781 cgcaattaat gtaagttagc tcactcatta ggcaccccag gctttacact ttatgcttcc
      841 ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa acagctatga
      901 ccatgattac ggattcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg
      961 ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt aatagcgaag
     1021 aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tggcgctttg
     1081 cctggtttcc ggcaccagaa gcggtgccgg aaagctggct ggagtgcgat cttcctgagg
     1141 ccgatactgt cgtcgtcccc tcaaactggc agatgcacgg ttacgatgcg cccatctaca
     1201 ccaacgtgac ctatcccatt acggtcaatc cgccgtttgt tcccacggag aatccgacgg
     1261 gttgttactc gctcacattt aatgttgatg aaagctggct acaggaaggc cagacgcgaa
     1321 ttatttttga tggcgttgga attagcttat cgactgcacg gtgcaccaat gcttctggcg
     1381 tcaggcagcc atcggaagct gtggtatggc tgtgcaggtc gtaaatcact gcataattcg
     1441 tgtcgctcaa ggcgcactcc cgttctggat aatgtttttt gcgccgacat cataacggtt
     1501 ctggcaaata ttctgaaatg agctgttgac aattaatcat cggctcgtat aatgtgtgga
     1561 attgtgagcg gataacaatt tcacacagga aacagtattc atgtccccta tactaggtta
     1621 ttggaaaatt aagggccttg tgcaacccac tcgacttctt ttggaatatc ttgaagaaaa
     1681 atatgaagag catttgtatg agcgcgatga aggtgataaa tggcgaaaca aaaagtttga
     1741 attgggtttg gagtttccca atcttcctta ttatattgat ggtgatgtta aattaacaca
     1801 gtctatggcc atcatacgtt atatagctga caagcacaac atgttgggtg gttgtccaaa
     1861 agagcgtgca gagatttcaa tgcttgaagg agcggttttg gatattagat acggtgtttc
     1921 gagaattgca tatagtaaag actttgaaac tctcaaagtt gattttctta gcaagctacc
     1981 tgaaatgctg aaaatgttcg aagatcgttt atgtcataaa acatatttaa atggtgatca
     2041 tgtaacccat cctgacttca tgttgtatga cgctcttgat gttgttttat acatggaccc
     2101 aatgtgcctg gatgcgttcc caaaattagt ttgttttaaa aaacgtattg aagctatccc
     2161 acaaattgat aagtacttga aatccagcaa gtatatagca tggcctttgc agggctggca
     2221 agccacgttt ggtggtggcg accatcctcc aaaatcggat ctggaagttc tgttccaggg
     2281 gcccctggga tccccggaat tcatgctgtc ccttcagtac cccgacgtgt accgcgacga
     2341 gaccgccgta caggattatc atggtcataa aatttgtgac ccttacgcct ggcttgaaga
     2401 ccccgacagt gaacagacta aggcctttgt ggaggcccag aataagatta ctgtgccatt
     2461 tcttgagcag tgtcccatca gaggtttata caaagagaga atgactgaac tatatgatta
     2521 tcccaagtat agttgccact tcaagaaagg aaaacggtat ttttattttt acaatacagg
     2581 tttgcagaac cagcgagtat tatatgtaca ggattcctta gagggtgagg ccagagtgtt
     2641 cctggacccc aacatactgt ctgacgatgg cacagtggca ctccgaggtt atgcgttcag
     2701 cgaagatggt gaatattttg cctatggtct gagtgccagt ggctcagact gggtgacaat
     2761 caagttcatg aaagttgatg gtgccaaaga gcttccagat gtgcttgaaa gagtcaagtt
     2821 cagctgtatg gcctggaccc atgatgggaa gggaatgttc tacaactcat accctcaaca
     2881 ggatggaaaa agtgatggca cagagacatc taccaatctc caccaaaagc tctactacca
     2941 tgtcttggga accgatcagt cagaagatat tttgtgtgct gagtttcctg atgaacctaa
     3001 atggatgggt ggagctgagt tatctgatga tggccgctat gtcttgttat caataaggga
     3061 aggatgtgat ccagtaaacc gactctggta ctgtgaccta cagcaggaat ccagtggcat
     3121 cgcgggaatc ctgaagtggg taaaactgat tgacaacttt gaaggggaat atgactacgt
     3181 gaccaatgag gggacggtgt tcacattcaa gacgaatcgc cagtctccca actatcgcgt
     3241 gatcaacatt gacttcaggg atcctgaaga gtctaagtgg aaagtacttg ttcctgagca
     3301 tgagaaagat gtcttagaat ggatagcttg tgtcaggtcc aacttcttgg tcttatgcta
     3361 cctccatgac gtcaagaaca ttctgcagct ccatgacctg actactggtg ctctccttaa
     3421 gaccttcccg ctcgatgtcg gcagcattgt agggtacagc ggtcagaaga aggacactga
     3481 aatcttctat cagtttactt cctttttatc tccaggtatc atttatcact gtgatcttac
     3541 caaagaggag ctggagccaa gagttttccg agaggtgacc gtaaaaggaa ttgatgcttc
     3601 tgattaccag acagtccaga ttttctaccc tagcaaggat ggtacgaaga ttccaatgtt
     3661 cattgtgcat aaaaaaggca taaaattgga tggctctcat ccagctttct tatatggcta
     3721 tggcggcttc aacatatcca tcacacccaa ctacagtgtt tccaggctta tttttgtgag
     3781 acacatgggt ggtatcctgg cagtggccaa catcagagga ggtggcgaat atggagagac
     3841 gtggcataaa ggtggtatct tggccaacaa acaaaactgc tttgatgact ttcagtgtgc
     3901 tgctgagtat ctgatcaagg aaggttacac atctcccaag aggctgacta ttaatggagg
     3961 ttcaaatgga ggcctcttag tggctgcttg tgcaaatcag agacctgacc tctttggttg
     4021 tgttattgcc caagttggag taatggacat gctgaagttt cataaatata ccatcggcca
     4081 tgcttggacc actgattatg ggtgctcgga cagcaaacaa cactttgaat ggcttgtcaa
     4141 atactctcca ttgcataatg tgaagttacc agaagcagat gacatccagt acccgtccat
     4201 gctgctcctc actgctgacc atgatgaccg cgtggtcccg cttcactccc tgaagttcat
     4261 tgccaccctt cagtacatcg tgggccgcag caggaagcaa agcaaccccc tgcttatcca
     4321 cgtggacacc aaggcgggcc acggggcggg gaagcccaca gccaaagtga tagaggaagt
     4381 ctcagacatg tttgcgttca tcgcgcggtg cctgaacgtc gactggattc cataactcga
     4441 gcggccgcat cgtgactgac tgacgatctg cctcgcgcgt ttcggtgatg acggtgaaaa
     4501 cctctgacac atgcagctcc cggagacggt cacagcttgt ctgtaagcgg atgccgggag
     4561 cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg tgtcggggcg cagccatgac
     4621 ccagtcacgt agcgatagcg gagtgtataa ttcttgaaga cgaaagggcc tcgtgatacg
     4681 cctattttta taggttaatg tcatgataat aatggtttct tagacgtcag gtggcacttt
     4741 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
     4801 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat
     4861 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt
     4921 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg
     4981 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga
     5041 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg
     5101 tgttgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt
     5161 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg
     5221 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg
     5281 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga
     5341 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc
     5401 tgcagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc
     5461 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc
     5521 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg
     5581 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac
     5641 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc
     5701 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt
     5761 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
     5821 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     5881 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     5941 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     6001 aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg
     6061 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     6121 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     6181 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     6241 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     6301 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     6361 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     6421 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     6481 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
     6541 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
     6601 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
     6661 gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcataaattc
     6721 cgacaccatc gaatggcgca aaacctttcg cggtatggca tgatagcgcc cggaagagag
     6781 tcaattcagg gtggtgaatg tgaaaccagt aacgttatac gatgtcgcag agtatgccgg
     6841 tgtctcttat cagaccgttt cccgcgtggt gaaccaggcc agccacgttt ctgcgaaaac
     6901 gcgggaaaaa gtggaagcgg cgatggcgga gctgaattac attcccaacc gcgtggcaca
     6961 acaactggcg ggcaaacagt cgttgctgat tggcgttgcc acctccagtc tggccctgca
     7021 cgcgccgtcg caaattgtcg cggcgattaa atctcgcgcc gatcaactgg gtgccagcgt
     7081 ggtggtgtcg atggtagaac gaagcggc
//
