LOCUS       sequence-200434-        6229 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express R663K 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-R663K
FEATURES             Location/Qualifiers
     source          1..6229
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      568..1156
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1057..1076
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1310..1327
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     1425..1442
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1426..1442
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1905..1924
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1905..1923
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1924..1948
                     /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             1993..2010
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2011..2031
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     terminator      4238..4285
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(4508..4527)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     complement(4533..4552)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        4594..4685
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4686..4754,4755..5546)
                     /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(4904..4923)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     CDS             join(5629..6229,1..482)
                     /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(5648..5667)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 ggcataaata tctcactcgc aatcaaattc agccgatagc ggaacgggaa ggcgactgga
       61 gtgccatgtc cggttttcaa caaaccatgc aaatgctgaa tgagggcatc gttcccactg
      121 cgatgctggt tgccaacgat cagatggcgc tgggcgcaat gcgcgccatt accgagtccg
      181 ggctgcgcgt tggtgcggat atctcggtag tgggatacga cgataccgaa gatagctcat
      241 gttatatccc gccgttaacc accatcaaac aggattttcg cctgctgggg caaaccagcg
      301 tggaccgctt gctgcaactc tctcagggcc aggcggtgaa gggcaatcag ctgttgccag
      361 tctcactggt gaaaagaaaa accaccctgg cgcccaatac gcaaaccgcc tctccccgcg
      421 cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt
      481 gactcatgac caaaatccct taacgtgagt tacgcgcgcg tcgttccact gagcgtcaga
      541 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
      601 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
      661 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct
      721 agtgtagccg tagttagccc accacttcaa gaactctgta gcaccgccta catacctcgc
      781 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
      841 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
      901 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
      961 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     1021 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     1081 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     1141 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     1201 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac
     1261 cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt
     1321 gagcgaggaa gcggaaggcg agagtaggga actgccaggc atcaaactaa gcagaaggcc
     1381 cctgacggat ggcctttttg cgtttctaca aactctttct gtgttgtaaa acgacggcca
     1441 gtcttaagct cgggccccct gggcggttct gataacgagt aatcgttaat ccgcaaataa
     1501 cgtaaaaacc cgcttcggcg ggttttttta tggggggagt ttagggaaag agcatttgtc
     1561 agaatattta agggcgcctg tcactttgct tgatatatga gaattattta accttataaa
     1621 tgagaaaaaa gcaacgcact ttaaataaga tacgttgctt tttcgattga tgaacaccta
     1681 taattaaact attcatctat tatttatgat tttttgtata tacaatattt ctagtttgtt
     1741 aaagagaatt aagaaaataa atctcgaaaa taataaaggg aaaatcagtt tttgatatca
     1801 aaattataca tgtcaacgat aatacaaaat ataatacaaa ctataagatg ttatcagtat
     1861 ttattatcat ttagaataaa ttttgtgtcg cccttccgcg aaattaatac gactcactat
     1921 aggggaattg tgagcggata acaattcccc tctagaaata attttgttta acttttagga
     1981 ggtaaacata tgcaccatca tcatcatcac gagaacttat actttcaagg catgagcagc
     2041 gttacctggg caccgggcaa ttacccgagc acccgccgtt ccgatcatgt ggacacgtat
     2101 caaagcgcga gcaagggcga agtcccggtg ccggatccgt atcagtggct ggaagagagc
     2161 accgacgaag tcgacaagtg gacgacggca caagcggacc tggcacagtc ttatctggac
     2221 caaaacgctg acattcaaaa actggccgag aaatttcgtg cgagccgtaa ttacgccaag
     2281 ttctctgcac cgactctgtt ggacgacggt cactggtatt ggttctataa ccgtggcttg
     2341 cagagccaaa gcgtcttgta cagatctaaa gaaccagccc tgccggactt ctctaagggc
     2401 gacgataacg ttggtgacgt tttctttgat ccgaatgtgc tggcagcgga tggtagcgcg
     2461 ggcatggtcc tgtgcaaatt ctcgccggac ggtaaattct ttgcctatgc agttagccac
     2521 ctgggtggcg attactccac gatttacgtg cgttccacga gcagcccgct gagccaggcg
     2581 agcgtcgcgc aaggtgtgga cggtcgtctg agcgacgaag tcaaatggtt caagtttagc
     2641 accattattt ggactaaaga tagcaagggc ttcctgtacc agcgctatcc ggctcgcgag
     2701 cgtcacgagg gtacccgcag cgaccgtaat gccatgatgt gttaccacaa agtcggcacg
     2761 acccaagaag aagatattat tgtgtatcag gataatgaac atcctgagtg gatctatggt
     2821 gcggatacct ccgaagatgg caagtacctg tacctgtacc agtttaaaga taccagcaag
     2881 aaaaacctgt tgtgggtcgc cgagctggac gaagatggcg ttaagagcgg tattcactgg
     2941 cgtaaagttg ttaacgaata cgcagcagac tacaatatca ttacgaacca cggtagcctg
     3001 gtctacatta agacgaacct gaatgcaccg caatataaag ttattaccat cgacctgtcc
     3061 aaagacgagc cagagatccg tgacttcatc ccggaagaaa aagatgctaa actggcgcaa
     3121 gttaattgtg cgaacgaaga gtatttcgtc gctatttaca agcgtaacgt taaagatgaa
     3181 atctacctgt actcgaaggc tggtgttcag ctgacccgtc tggcgccgga tttcgtgggt
     3241 gcggcaagca ttgcgaatcg tcaaaaacag acccactttt ttctgaccct gagcggtttt
     3301 aacaccccgg gtacgatcgc tcgttatgat tttaccgcgc cggaaaccca gcgctttagc
     3361 atcctgcgta ccactaaagt caacgaactg gatcctgatg acttcgagag cacgcaagtg
     3421 tggtacgaga gcaaggacgg tacgaaaatc ccgatgttca tcgttcgtca caagtccacc
     3481 aagtttgacg gtaccgcggc tgcgatccaa tatggttacg gcggcttcgc cacctccgcg
     3541 gatccattct tcagcccgat cattctgacg tttctgcaga cctacggtgc gatttttgcc
     3601 gtaccgagca tccgtggtgg cggtgagttc ggtgaggaat ggcataaggg cggccgccgc
     3661 gaaaccaaag tgaacacgtt tgacgatttc attgcggccg cccaatttct ggtgaagaac
     3721 aaatatgcgg ctccgggtaa agttgcaatt aatggtgcga gcaacggcgg cctgctggtg
     3781 atgggctcta tcgtccgtgc gcctgagggt acgttcggtg cggcagtgcc agagggcggt
     3841 gtggcggatc tgcttaagtt ccacaaattt acgggtggcc aggcgtggat cagcgagtat
     3901 ggtaatccgt ctatccctga agaatttgat tacatctatc cgctgagccc ggttcacaat
     3961 gtccgcaccg acaaagttat gccggcaacc ctgattaccg tgaatatcgg tgatggcaaa
     4021 gtggttccga tgcacagctt caagttcatc gcgactttgc agcataacgt tccgcagaat
     4081 ccgcatccgc tgctgattaa gattgataaa tcgtggttgg gtcacggcat gggtaagccg
     4141 accgacaaaa atgtgaaaga cgccgccgac aagtggggtt tcatcgcacg cgccctgggt
     4201 ctggaactga aaacggtcga gtgatgaaag cttcccccta gcataacccc ttggggcctc
     4261 taaacgggtc ttgaggggtt ttttgcccct gagacgcgtc aatcgagttc gtacctaagg
     4321 gcgacacccc ataattagcc cgggcgaaag gcccagtctt tcgactgagc ctttcgtttt
     4381 atttgatgcc tggcagttcc ctactctcgc atggggagtc cccacactac catcggcgct
     4441 acggcgtttc acttctgagt tcggcatggg gtcaggtggg accaccgcgc tactgccgcc
     4501 aggcaaacaa ggggtgttat gagccatatt caggtataaa tgggctcgcg ataatgttca
     4561 gaattggtta attggttgta acactgaccc ctatttgttt atttttctaa atacattcaa
     4621 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
     4681 agaatatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc
     4741 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg
     4801 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
     4861 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat
     4921 tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg
     4981 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag
     5041 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa
     5101 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc
     5161 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca
     5221 cgatgcctgt agcgatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc
     5281 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc
     5341 tgcgctcggc ccttccggct ggctggttta ttgctgataa atccggagcc ggtgagcgtg
     5401 gttctcgcgg tatcatcgca gcgctggggc cagatggtaa gccctcccgt atcgtagtta
     5461 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag
     5521 gtgcctcact gattaagcat tggtaagcgg cgcgccatcg aatggcgcaa aacctttcgc
     5581 ggtatggcat gatagcgccc ggaagagagt caattcaggg tggtgaatat gaaaccagta
     5641 acgttatacg atgtcgcaga gtatgccggt gtctcttatc agaccgtttc ccgcgtggtg
     5701 aaccaggcca gccacgtttc tgcgaaaacg cgggaaaaag tggaagcggc gatggcggag
     5761 ctgaattaca ttcccaaccg cgtggcacaa caactggcgg gcaaacagtc gttgctgatt
     5821 ggcgttgcca cctccagtct ggccctgcac gcgccgtcgc aaattgtcgc ggcgattaaa
     5881 tctcgcgccg atcaactggg tgccagcgtg gtggtgtcga tggtagaacg aagcggcgtc
     5941 gaagcctgta aagcggcggt gcacaatctt ctcgcgcaac gcgtcagtgg gctgatcatt
     6001 aactatccgc tggatgacca ggatgccatt gctgtggaag ctgcctgcac taatgttccg
     6061 gcgttatttc ttgatgtctc tgaccagaca cccatcaaca gtattatttt ctcccatgag
     6121 gacggtacgc gactgggcgt ggagcatctg gtcgcattgg gtcaccagca aatcgcgctg
     6181 ttagcgggcc cattaagttc tgtctcggcg cgtctgcgtc tggctggct
//
