LOCUS       sequence-192477-        6229 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express R663Q 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-R663Q
FEATURES             Location/Qualifiers
     source          1..6229
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      518..1106
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1007..1026
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1260..1277
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     1375..1392
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1376..1392
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1855..1874
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1855..1873
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1874..1898
                     /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             1943..1960
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1961..1981
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     terminator      4188..4235
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(4458..4477)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     complement(4483..4502)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        4544..4635
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4636..4704,4705..5496)
                     /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(4854..4873)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     CDS             join(5579..6229,1..432)
                     /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(5598..5617)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 ggcgactgga gtgccatgtc cggttttcaa caaaccatgc aaatgctgaa tgagggcatc
       61 gttcccactg cgatgctggt tgccaacgat cagatggcgc tgggcgcaat gcgcgccatt
      121 accgagtccg ggctgcgcgt tggtgcggat atctcggtag tgggatacga cgataccgaa
      181 gatagctcat gttatatccc gccgttaacc accatcaaac aggattttcg cctgctgggg
      241 caaaccagcg tggaccgctt gctgcaactc tctcagggcc aggcggtgaa gggcaatcag
      301 ctgttgccag tctcactggt gaaaagaaaa accaccctgg cgcccaatac gcaaaccgcc
      361 tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa
      421 agcgggcagt gactcatgac caaaatccct taacgtgagt tacgcgcgcg tcgttccact
      481 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
      541 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
      601 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
      661 ctgttcttct agtgtagccg tagttagccc accacttcaa gaactctgta gcaccgccta
      721 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
      781 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
      841 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
      901 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
      961 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     1021 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     1081 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     1141 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
     1201 accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca
     1261 gcgagtcagt gagcgaggaa gcggaaggcg agagtaggga actgccaggc atcaaactaa
     1321 gcagaaggcc cctgacggat ggcctttttg cgtttctaca aactctttct gtgttgtaaa
     1381 acgacggcca gtcttaagct cgggccccct gggcggttct gataacgagt aatcgttaat
     1441 ccgcaaataa cgtaaaaacc cgcttcggcg ggttttttta tggggggagt ttagggaaag
     1501 agcatttgtc agaatattta agggcgcctg tcactttgct tgatatatga gaattattta
     1561 accttataaa tgagaaaaaa gcaacgcact ttaaataaga tacgttgctt tttcgattga
     1621 tgaacaccta taattaaact attcatctat tatttatgat tttttgtata tacaatattt
     1681 ctagtttgtt aaagagaatt aagaaaataa atctcgaaaa taataaaggg aaaatcagtt
     1741 tttgatatca aaattataca tgtcaacgat aatacaaaat ataatacaaa ctataagatg
     1801 ttatcagtat ttattatcat ttagaataaa ttttgtgtcg cccttccgcg aaattaatac
     1861 gactcactat aggggaattg tgagcggata acaattcccc tctagaaata attttgttta
     1921 acttttagga ggtaaacata tgcaccatca tcatcatcac gagaacttat actttcaagg
     1981 catgagcagc gttacctggg caccgggcaa ttacccgagc acccgccgtt ccgatcatgt
     2041 ggacacgtat caaagcgcga gcaagggcga agtcccggtg ccggatccgt atcagtggct
     2101 ggaagagagc accgacgaag tcgacaagtg gacgacggca caagcggacc tggcacagtc
     2161 ttatctggac caaaacgctg acattcaaaa actggccgag aaatttcgtg cgagccgtaa
     2221 ttacgccaag ttctctgcac cgactctgtt ggacgacggt cactggtatt ggttctataa
     2281 ccgtggcttg cagagccaaa gcgtcttgta cagatctaaa gaaccagccc tgccggactt
     2341 ctctaagggc gacgataacg ttggtgacgt tttctttgat ccgaatgtgc tggcagcgga
     2401 tggtagcgcg ggcatggtcc tgtgcaaatt ctcgccggac ggtaaattct ttgcctatgc
     2461 agttagccac ctgggtggcg attactccac gatttacgtg cgttccacga gcagcccgct
     2521 gagccaggcg agcgtcgcgc aaggtgtgga cggtcgtctg agcgacgaag tcaaatggtt
     2581 caagtttagc accattattt ggactaaaga tagcaagggc ttcctgtacc agcgctatcc
     2641 ggctcgcgag cgtcacgagg gtacccgcag cgaccgtaat gccatgatgt gttaccacaa
     2701 agtcggcacg acccaagaag aagatattat tgtgtatcag gataatgaac atcctgagtg
     2761 gatctatggt gcggatacct ccgaagatgg caagtacctg tacctgtacc agtttaaaga
     2821 taccagcaag aaaaacctgt tgtgggtcgc cgagctggac gaagatggcg ttaagagcgg
     2881 tattcactgg cgtaaagttg ttaacgaata cgcagcagac tacaatatca ttacgaacca
     2941 cggtagcctg gtctacatta agacgaacct gaatgcaccg caatataaag ttattaccat
     3001 cgacctgtcc aaagacgagc cagagatccg tgacttcatc ccggaagaaa aagatgctaa
     3061 actggcgcaa gttaattgtg cgaacgaaga gtatttcgtc gctatttaca agcgtaacgt
     3121 taaagatgaa atctacctgt actcgaaggc tggtgttcag ctgacccgtc tggcgccgga
     3181 tttcgtgggt gcggcaagca ttgcgaatcg tcaaaaacag acccactttt ttctgaccct
     3241 gagcggtttt aacaccccgg gtacgatcgc tcgttatgat tttaccgcgc cggaaaccca
     3301 gcgctttagc atcctgcgta ccactaaagt caacgaactg gatcctgatg acttcgagag
     3361 cacgcaagtg tggtacgaga gcaaggacgg tacgaaaatc ccgatgttca tcgttcgtca
     3421 caagtccacc aagtttgacg gtaccgcggc tgcgatccaa tatggttacg gcggcttcgc
     3481 cacctccgcg gatccattct tcagcccgat cattctgacg tttctgcaga cctacggtgc
     3541 gatttttgcc gtaccgagca tccgtggtgg cggtgagttc ggtgaggaat ggcataaggg
     3601 cggccgccgc gaaaccaaag tgaacacgtt tgacgatttc attgcggccg cccaatttct
     3661 ggtgaagaac aaatatgcgg ctccgggtaa agttgcaatt aatggtgcga gcaacggcgg
     3721 cctgctggtg atgggctcta tcgtccgtgc gcctgagggt acgttcggtg cggcagtgcc
     3781 agagggcggt gtggcggatc tgcttaagtt ccacaaattt acgggtggcc aggcgtggat
     3841 cagcgagtat ggtaatccgt ctatccctga agaatttgat tacatctatc cgctgagccc
     3901 ggttcacaat gtccgcaccg acaaagttat gccggcaacc ctgattaccg tgaatatcgg
     3961 tgatggccag gtggttccga tgcacagctt caagttcatc gcgactttgc agcataacgt
     4021 tccgcagaat ccgcatccgc tgctgattaa gattgataaa tcgtggttgg gtcacggcat
     4081 gggtaagccg accgacaaaa atgtgaaaga cgccgccgac aagtggggtt tcatcgcacg
     4141 cgccctgggt ctggaactga aaacggtcga gtgatgaaag cttcccccta gcataacccc
     4201 ttggggcctc taaacgggtc ttgaggggtt ttttgcccct gagacgcgtc aatcgagttc
     4261 gtacctaagg gcgacacccc ataattagcc cgggcgaaag gcccagtctt tcgactgagc
     4321 ctttcgtttt atttgatgcc tggcagttcc ctactctcgc atggggagtc cccacactac
     4381 catcggcgct acggcgtttc acttctgagt tcggcatggg gtcaggtggg accaccgcgc
     4441 tactgccgcc aggcaaacaa ggggtgttat gagccatatt caggtataaa tgggctcgcg
     4501 ataatgttca gaattggtta attggttgta acactgaccc ctatttgttt atttttctaa
     4561 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat
     4621 tgaaaaagga agaatatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg
     4681 gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa
     4741 gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt
     4801 gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt
     4861 ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat
     4921 tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg
     4981 acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta
     5041 cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat
     5101 catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag
     5161 cgtgacacca cgatgcctgt agcgatggca acaacgttgc gcaaactatt aactggcgaa
     5221 ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca
     5281 ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atccggagcc
     5341 ggtgagcgtg gttctcgcgg tatcatcgca gcgctggggc cagatggtaa gccctcccgt
     5401 atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc
     5461 gctgagatag gtgcctcact gattaagcat tggtaagcgg cgcgccatcg aatggcgcaa
     5521 aacctttcgc ggtatggcat gatagcgccc ggaagagagt caattcaggg tggtgaatat
     5581 gaaaccagta acgttatacg atgtcgcaga gtatgccggt gtctcttatc agaccgtttc
     5641 ccgcgtggtg aaccaggcca gccacgtttc tgcgaaaacg cgggaaaaag tggaagcggc
     5701 gatggcggag ctgaattaca ttcccaaccg cgtggcacaa caactggcgg gcaaacagtc
     5761 gttgctgatt ggcgttgcca cctccagtct ggccctgcac gcgccgtcgc aaattgtcgc
     5821 ggcgattaaa tctcgcgccg atcaactggg tgccagcgtg gtggtgtcga tggtagaacg
     5881 aagcggcgtc gaagcctgta aagcggcggt gcacaatctt ctcgcgcaac gcgtcagtgg
     5941 gctgatcatt aactatccgc tggatgacca ggatgccatt gctgtggaag ctgcctgcac
     6001 taatgttccg gcgttatttc ttgatgtctc tgaccagaca cccatcaaca gtattatttt
     6061 ctcccatgag gacggtacgc gactgggcgt ggagcatctg gtcgcattgg gtcaccagca
     6121 aatcgcgctg ttagcgggcc cattaagttc tgtctcggcg cgtctgcgtc tggctggctg
     6181 gcataaatat ctcactcgca atcaaattca gccgatagcg gaacgggaa
//
