LOCUS       sequence-192474-        6229 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express D661A mutant of prolyl oligopeptidase from Galerina 
            marginata.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6229)
  AUTHORS   Czekster CM, Naismith JH
  TITLE     Kinetic Landscape of a Peptide Bond-Forming Prolyl Oligopeptidase.
  JOURNAL   Biochemistry. 2017 Apr 18;56(15):2086-2095. doi: 
            10.1021/acs.biochem.7b00012. Epub 2017 Apr 7.
  PUBMED    28332820
REFERENCE   2  (bases 1 to 6229)
  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.biochem.7b00012"; journalName: "Biochemistry"; date: 
            "2017-04-18- 18"; volume: "56"; issue: "15"; pages: "2086-2095"
COMMENT     Sequence Label: pJExpress414_GmPOPB-D661A
FEATURES             Location/Qualifiers
     source          1..6229
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     229..248
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        229..247
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    248..272
                     /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             317..334
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             335..355
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     terminator      2562..2609
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(2832..2851)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     complement(2857..2876)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        2918..3009
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3010..3078,3079..3870)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(3228..3247)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     CDS             3953..5035
                     /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(3972..3991)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     rep_origin      5121..5709
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5610..5629
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5863..5880
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     5978..5995
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5979..5995
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 cctataatta aactattcat ctattattta tgattttttg tatatacaat atttctagtt
       61 tgttaaagag aattaagaaa ataaatctcg aaaataataa agggaaaatc agtttttgat
      121 atcaaaatta tacatgtcaa cgataataca aaatataata caaactataa gatgttatca
      181 gtatttatta tcatttagaa taaattttgt gtcgcccttc cgcgaaatta atacgactca
      241 ctatagggga attgtgagcg gataacaatt cccctctaga aataattttg tttaactttt
      301 aggaggtaaa catatgcacc atcatcatca tcacgagaac ttatactttc aaggcatgag
      361 cagcgttacc tgggcaccgg gcaattaccc gagcacccgc cgttccgatc atgtggacac
      421 gtatcaaagc gcgagcaagg gcgaagtccc ggtgccggat ccgtatcagt ggctggaaga
      481 gagcaccgac gaagtcgaca agtggacgac ggcacaagcg gacctggcac agtcttatct
      541 ggaccaaaac gctgacattc aaaaactggc cgagaaattt cgtgcgagcc gtaattacgc
      601 caagttctct gcaccgactc tgttggacga cggtcactgg tattggttct ataaccgtgg
      661 cttgcagagc caaagcgtct tgtacagatc taaagaacca gccctgccgg acttctctaa
      721 gggcgacgat aacgttggtg acgttttctt tgatccgaat gtgctggcag cggatggtag
      781 cgcgggcatg gtcctgtgca aattctcgcc ggacggtaaa ttctttgcct atgcagttag
      841 ccacctgggt ggcgattact ccacgattta cgtgcgttcc acgagcagcc cgctgagcca
      901 ggcgagcgtc gcgcaaggtg tggacggtcg tctgagcgac gaagtcaaat ggttcaagtt
      961 tagcaccatt atttggacta aagatagcaa gggcttcctg taccagcgct atccggctcg
     1021 cgagcgtcac gagggtaccc gcagcgaccg taatgccatg atgtgttacc acaaagtcgg
     1081 cacgacccaa gaagaagata ttattgtgta tcaggataat gaacatcctg agtggatcta
     1141 tggtgcggat acctccgaag atggcaagta cctgtacctg taccagttta aagataccag
     1201 caagaaaaac ctgttgtggg tcgccgagct ggacgaagat ggcgttaaga gcggtattca
     1261 ctggcgtaaa gttgttaacg aatacgcagc agactacaat atcattacga accacggtag
     1321 cctggtctac attaagacga acctgaatgc accgcaatat aaagttatta ccatcgacct
     1381 gtccaaagac gagccagaga tccgtgactt catcccggaa gaaaaagatg ctaaactggc
     1441 gcaagttaat tgtgcgaacg aagagtattt cgtcgctatt tacaagcgta acgttaaaga
     1501 tgaaatctac ctgtactcga aggctggtgt tcagctgacc cgtctggcgc cggatttcgt
     1561 gggtgcggca agcattgcga atcgtcaaaa acagacccac ttttttctga ccctgagcgg
     1621 ttttaacacc ccgggtacga tcgctcgtta tgattttacc gcgccggaaa cccagcgctt
     1681 tagcatcctg cgtaccacta aagtcaacga actggatcct gatgacttcg agagcacgca
     1741 agtgtggtac gagagcaagg acggtacgaa aatcccgatg ttcatcgttc gtcacaagtc
     1801 caccaagttt gacggtaccg cggctgcgat ccaatatggt tacggcggct tcgccacctc
     1861 cgcggatcca ttcttcagcc cgatcattct gacgtttctg cagacctacg gtgcgatttt
     1921 tgccgtaccg agcatccgtg gtggcggtga gttcggtgag gaatggcata agggcggccg
     1981 ccgcgaaacc aaagtgaaca cgtttgacga tttcattgcg gccgcccaat ttctggtgaa
     2041 gaacaaatat gcggctccgg gtaaagttgc aattaatggt gcgagcaacg gcggcctgct
     2101 ggtgatgggc tctatcgtcc gtgcgcctga gggtacgttc ggtgcggcag tgccagaggg
     2161 cggtgtggcg gatctgctta agttccacaa atttacgggt ggccaggcgt ggatcagcga
     2221 gtatggtaat ccgtctatcc ctgaagaatt tgattacatc tatccgctga gcccggttca
     2281 caatgtccgc accgacaaag ttatgccggc aaccctgatt accgtgaata tcggtgcggg
     2341 ccgtgtggtt ccgatgcaca gcttcaagtt catcgcgact ttgcagcata acgttccgca
     2401 gaatccgcat ccgctgctga ttaagattga taaatcgtgg ttgggtcacg gcatgggtaa
     2461 gccgaccgac aaaaatgtga aagacgccgc cgacaagtgg ggtttcatcg cacgcgccct
     2521 gggtctggaa ctgaaaacgg tcgagtgatg aaagcttccc cctagcataa ccccttgggg
     2581 cctctaaacg ggtcttgagg ggttttttgc ccctgagacg cgtcaatcga gttcgtacct
     2641 aagggcgaca ccccataatt agcccgggcg aaaggcccag tctttcgact gagcctttcg
     2701 ttttatttga tgcctggcag ttccctactc tcgcatgggg agtccccaca ctaccatcgg
     2761 cgctacggcg tttcacttct gagttcggca tggggtcagg tgggaccacc gcgctactgc
     2821 cgccaggcaa acaaggggtg ttatgagcca tattcaggta taaatgggct cgcgataatg
     2881 ttcagaattg gttaattggt tgtaacactg acccctattt gtttattttt ctaaatacat
     2941 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa
     3001 aggaagaata tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt
     3061 tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag
     3121 ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt
     3181 tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg
     3241 gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag
     3301 aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta
     3361 agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg
     3421 acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta
     3481 actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac
     3541 accacgatgc ctgtagcgat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt
     3601 actctagctt cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca
     3661 cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatccgg agccggtgag
     3721 cgtggttctc gcggtatcat cgcagcgctg gggccagatg gtaagccctc ccgtatcgta
     3781 gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag
     3841 ataggtgcct cactgattaa gcattggtaa gcggcgcgcc atcgaatggc gcaaaacctt
     3901 tcgcggtatg gcatgatagc gcccggaaga gagtcaattc agggtggtga atatgaaacc
     3961 agtaacgtta tacgatgtcg cagagtatgc cggtgtctct tatcagaccg tttcccgcgt
     4021 ggtgaaccag gccagccacg tttctgcgaa aacgcgggaa aaagtggaag cggcgatggc
     4081 ggagctgaat tacattccca accgcgtggc acaacaactg gcgggcaaac agtcgttgct
     4141 gattggcgtt gccacctcca gtctggccct gcacgcgccg tcgcaaattg tcgcggcgat
     4201 taaatctcgc gccgatcaac tgggtgccag cgtggtggtg tcgatggtag aacgaagcgg
     4261 cgtcgaagcc tgtaaagcgg cggtgcacaa tcttctcgcg caacgcgtca gtgggctgat
     4321 cattaactat ccgctggatg accaggatgc cattgctgtg gaagctgcct gcactaatgt
     4381 tccggcgtta tttcttgatg tctctgacca gacacccatc aacagtatta ttttctccca
     4441 tgaggacggt acgcgactgg gcgtggagca tctggtcgca ttgggtcacc agcaaatcgc
     4501 gctgttagcg ggcccattaa gttctgtctc ggcgcgtctg cgtctggctg gctggcataa
     4561 atatctcact cgcaatcaaa ttcagccgat agcggaacgg gaaggcgact ggagtgccat
     4621 gtccggtttt caacaaacca tgcaaatgct gaatgagggc atcgttccca ctgcgatgct
     4681 ggttgccaac gatcagatgg cgctgggcgc aatgcgcgcc attaccgagt ccgggctgcg
     4741 cgttggtgcg gatatctcgg tagtgggata cgacgatacc gaagatagct catgttatat
     4801 cccgccgtta accaccatca aacaggattt tcgcctgctg gggcaaacca gcgtggaccg
     4861 cttgctgcaa ctctctcagg gccaggcggt gaagggcaat cagctgttgc cagtctcact
     4921 ggtgaaaaga aaaaccaccc tggcgcccaa tacgcaaacc gcctctcccc gcgcgttggc
     4981 cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgactcat
     5041 gaccaaaatc ccttaacgtg agttacgcgc gcgtcgttcc actgagcgtc agaccccgta
     5101 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     5161 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     5221 tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag
     5281 ccgtagttag cccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     5341 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     5401 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     5461 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     5521 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     5581 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     5641 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     5701 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
     5761 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt
     5821 gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag
     5881 gaagcggaag gcgagagtag ggaactgcca ggcatcaaac taagcagaag gcccctgacg
     5941 gatggccttt ttgcgtttct acaaactctt tctgtgttgt aaaacgacgg ccagtcttaa
     6001 gctcgggccc cctgggcggt tctgataacg agtaatcgtt aatccgcaaa taacgtaaaa
     6061 acccgcttcg gcgggttttt ttatgggggg agtttaggga aagagcattt gtcagaatat
     6121 ttaagggcgc ctgtcacttt gcttgatata tgagaattat ttaaccttat aaatgagaaa
     6181 aaagcaacgc actttaaata agatacgttg ctttttcgat tgatgaaca
//
