LOCUS       sequence-192471-        6229 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express R663A 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-R663A
FEATURES             Location/Qualifiers
     source          1..6229
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      566..1154
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1055..1074
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1308..1325
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     1423..1440
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1424..1440
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1903..1922
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1903..1921
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1922..1946
                     /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             1991..2008
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2009..2029
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     terminator      4236..4283
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(4506..4525)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     complement(4531..4550)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        4592..4683
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4684..4752,4753..5544)
                     /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(4902..4921)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     CDS             join(5627..6229,1..480)
                     /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(5646..5665)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 cataaatatc tcactcgcaa tcaaattcag ccgatagcgg aacgggaagg cgactggagt
       61 gccatgtccg gttttcaaca aaccatgcaa atgctgaatg agggcatcgt tcccactgcg
      121 atgctggttg ccaacgatca gatggcgctg ggcgcaatgc gcgccattac cgagtccggg
      181 ctgcgcgttg gtgcggatat ctcggtagtg ggatacgacg ataccgaaga tagctcatgt
      241 tatatcccgc cgttaaccac catcaaacag gattttcgcc tgctggggca aaccagcgtg
      301 gaccgcttgc tgcaactctc tcagggccag gcggtgaagg gcaatcagct gttgccagtc
      361 tcactggtga aaagaaaaac caccctggcg cccaatacgc aaaccgcctc tccccgcgcg
      421 ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga
      481 ctcatgacca aaatccctta acgtgagtta cgcgcgcgtc gttccactga gcgtcagacc
      541 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
      601 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
      661 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag
      721 tgtagccgta gttagcccac cacttcaaga actctgtagc accgcctaca tacctcgctc
      781 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
      841 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
      901 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
      961 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     1021 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     1081 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     1141 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     1201 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
     1261 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga
     1321 gcgaggaagc ggaaggcgag agtagggaac tgccaggcat caaactaagc agaaggcccc
     1381 tgacggatgg cctttttgcg tttctacaaa ctctttctgt gttgtaaaac gacggccagt
     1441 cttaagctcg ggccccctgg gcggttctga taacgagtaa tcgttaatcc gcaaataacg
     1501 taaaaacccg cttcggcggg tttttttatg gggggagttt agggaaagag catttgtcag
     1561 aatatttaag ggcgcctgtc actttgcttg atatatgaga attatttaac cttataaatg
     1621 agaaaaaagc aacgcacttt aaataagata cgttgctttt tcgattgatg aacacctata
     1681 attaaactat tcatctatta tttatgattt tttgtatata caatatttct agtttgttaa
     1741 agagaattaa gaaaataaat ctcgaaaata ataaagggaa aatcagtttt tgatatcaaa
     1801 attatacatg tcaacgataa tacaaaatat aatacaaact ataagatgtt atcagtattt
     1861 attatcattt agaataaatt ttgtgtcgcc cttccgcgaa attaatacga ctcactatag
     1921 gggaattgtg agcggataac aattcccctc tagaaataat tttgtttaac ttttaggagg
     1981 taaacatatg caccatcatc atcatcacga gaacttatac tttcaaggca tgagcagcgt
     2041 tacctgggca ccgggcaatt acccgagcac ccgccgttcc gatcatgtgg acacgtatca
     2101 aagcgcgagc aagggcgaag tcccggtgcc ggatccgtat cagtggctgg aagagagcac
     2161 cgacgaagtc gacaagtgga cgacggcaca agcggacctg gcacagtctt atctggacca
     2221 aaacgctgac attcaaaaac tggccgagaa atttcgtgcg agccgtaatt acgccaagtt
     2281 ctctgcaccg actctgttgg acgacggtca ctggtattgg ttctataacc gtggcttgca
     2341 gagccaaagc gtcttgtaca gatctaaaga accagccctg ccggacttct ctaagggcga
     2401 cgataacgtt ggtgacgttt tctttgatcc gaatgtgctg gcagcggatg gtagcgcggg
     2461 catggtcctg tgcaaattct cgccggacgg taaattcttt gcctatgcag ttagccacct
     2521 gggtggcgat tactccacga tttacgtgcg ttccacgagc agcccgctga gccaggcgag
     2581 cgtcgcgcaa ggtgtggacg gtcgtctgag cgacgaagtc aaatggttca agtttagcac
     2641 cattatttgg actaaagata gcaagggctt cctgtaccag cgctatccgg ctcgcgagcg
     2701 tcacgagggt acccgcagcg accgtaatgc catgatgtgt taccacaaag tcggcacgac
     2761 ccaagaagaa gatattattg tgtatcagga taatgaacat cctgagtgga tctatggtgc
     2821 ggatacctcc gaagatggca agtacctgta cctgtaccag tttaaagata ccagcaagaa
     2881 aaacctgttg tgggtcgccg agctggacga agatggcgtt aagagcggta ttcactggcg
     2941 taaagttgtt aacgaatacg cagcagacta caatatcatt acgaaccacg gtagcctggt
     3001 ctacattaag acgaacctga atgcaccgca atataaagtt attaccatcg acctgtccaa
     3061 agacgagcca gagatccgtg acttcatccc ggaagaaaaa gatgctaaac tggcgcaagt
     3121 taattgtgcg aacgaagagt atttcgtcgc tatttacaag cgtaacgtta aagatgaaat
     3181 ctacctgtac tcgaaggctg gtgttcagct gacccgtctg gcgccggatt tcgtgggtgc
     3241 ggcaagcatt gcgaatcgtc aaaaacagac ccactttttt ctgaccctga gcggttttaa
     3301 caccccgggt acgatcgctc gttatgattt taccgcgccg gaaacccagc gctttagcat
     3361 cctgcgtacc actaaagtca acgaactgga tcctgatgac ttcgagagca cgcaagtgtg
     3421 gtacgagagc aaggacggta cgaaaatccc gatgttcatc gttcgtcaca agtccaccaa
     3481 gtttgacggt accgcggctg cgatccaata tggttacggc ggcttcgcca cctccgcgga
     3541 tccattcttc agcccgatca ttctgacgtt tctgcagacc tacggtgcga tttttgccgt
     3601 accgagcatc cgtggtggcg gtgagttcgg tgaggaatgg cataagggcg gccgccgcga
     3661 aaccaaagtg aacacgtttg acgatttcat tgcggccgcc caatttctgg tgaagaacaa
     3721 atatgcggct ccgggtaaag ttgcaattaa tggtgcgagc aacggcggcc tgctggtgat
     3781 gggctctatc gtccgtgcgc ctgagggtac gttcggtgcg gcagtgccag agggcggtgt
     3841 ggcggatctg cttaagttcc acaaatttac gggtggccag gcgtggatca gcgagtatgg
     3901 taatccgtct atccctgaag aatttgatta catctatccg ctgagcccgg ttcacaatgt
     3961 ccgcaccgac aaagttatgc cggcaaccct gattaccgtg aatatcggtg atggcgcggt
     4021 ggttccgatg cacagcttca agttcatcgc gactttgcag cataacgttc cgcagaatcc
     4081 gcatccgctg ctgattaaga ttgataaatc gtggttgggt cacggcatgg gtaagccgac
     4141 cgacaaaaat gtgaaagacg ccgccgacaa gtggggtttc atcgcacgcg ccctgggtct
     4201 ggaactgaaa acggtcgagt gatgaaagct tccccctagc ataacccctt ggggcctcta
     4261 aacgggtctt gaggggtttt ttgcccctga gacgcgtcaa tcgagttcgt acctaagggc
     4321 gacaccccat aattagcccg ggcgaaaggc ccagtctttc gactgagcct ttcgttttat
     4381 ttgatgcctg gcagttccct actctcgcat ggggagtccc cacactacca tcggcgctac
     4441 ggcgtttcac ttctgagttc ggcatggggt caggtgggac caccgcgcta ctgccgccag
     4501 gcaaacaagg ggtgttatga gccatattca ggtataaatg ggctcgcgat aatgttcaga
     4561 attggttaat tggttgtaac actgacccct atttgtttat ttttctaaat acattcaaat
     4621 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag
     4681 aatatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt
     4741 cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt
     4801 gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagttttcgc
     4861 cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta
     4921 tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac
     4981 ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa
     5041 ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg
     5101 atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc
     5161 cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg
     5221 atgcctgtag cgatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta
     5281 gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg
     5341 cgctcggccc ttccggctgg ctggtttatt gctgataaat ccggagccgg tgagcgtggt
     5401 tctcgcggta tcatcgcagc gctggggcca gatggtaagc cctcccgtat cgtagttatc
     5461 tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt
     5521 gcctcactga ttaagcattg gtaagcggcg cgccatcgaa tggcgcaaaa cctttcgcgg
     5581 tatggcatga tagcgcccgg aagagagtca attcagggtg gtgaatatga aaccagtaac
     5641 gttatacgat gtcgcagagt atgccggtgt ctcttatcag accgtttccc gcgtggtgaa
     5701 ccaggccagc cacgtttctg cgaaaacgcg ggaaaaagtg gaagcggcga tggcggagct
     5761 gaattacatt cccaaccgcg tggcacaaca actggcgggc aaacagtcgt tgctgattgg
     5821 cgttgccacc tccagtctgg ccctgcacgc gccgtcgcaa attgtcgcgg cgattaaatc
     5881 tcgcgccgat caactgggtg ccagcgtggt ggtgtcgatg gtagaacgaa gcggcgtcga
     5941 agcctgtaaa gcggcggtgc acaatcttct cgcgcaacgc gtcagtgggc tgatcattaa
     6001 ctatccgctg gatgaccagg atgccattgc tgtggaagct gcctgcacta atgttccggc
     6061 gttatttctt gatgtctctg accagacacc catcaacagt attattttct cccatgagga
     6121 cggtacgcga ctgggcgtgg agcatctggt cgcattgggt caccagcaaa tcgcgctgtt
     6181 agcgggccca ttaagttctg tctcggcgcg tctgcgtctg gctggctgg
//
