LOCUS       sequence-177749-        6226 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express W695-deletion mutant of prolyl oligopeptidase from Galerina 
            marginata.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6226)
  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 6226)
  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-W695del
FEATURES             Location/Qualifiers
     source          1..6226
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      27..615
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     516..535
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     769..786
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     884..901
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     885..901
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1364..1383
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1364..1382
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1383..1407
                     /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             1452..1469
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1470..1490
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     terminator      3694..3741
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(3964..3983)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     complement(3989..4008)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        4050..4141
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4142..4210,4211..5002)
                     /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(4360..4379)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     CDS             5085..6167
                     /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(5104..5123)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
       61 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
      121 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta
      181 gtgtagccgt agttagccca ccacttcaag aactctgtag caccgcctac atacctcgct
      241 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
      301 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
      361 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
      421 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
      481 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
      541 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
      601 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
      661 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc
      721 gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg
      781 agcgaggaag cggaaggcga gagtagggaa ctgccaggca tcaaactaag cagaaggccc
      841 ctgacggatg gcctttttgc gtttctacaa actctttctg tgttgtaaaa cgacggccag
      901 tcttaagctc gggccccctg ggcggttctg ataacgagta atcgttaatc cgcaaataac
      961 gtaaaaaccc gcttcggcgg gtttttttat ggggggagtt tagggaaaga gcatttgtca
     1021 gaatatttaa gggcgcctgt cactttgctt gatatatgag aattatttaa ccttataaat
     1081 gagaaaaaag caacgcactt taaataagat acgttgcttt ttcgattgat gaacacctat
     1141 aattaaacta ttcatctatt atttatgatt ttttgtatat acaatatttc tagtttgtta
     1201 aagagaatta agaaaataaa tctcgaaaat aataaaggga aaatcagttt ttgatatcaa
     1261 aattatacat gtcaacgata atacaaaata taatacaaac tataagatgt tatcagtatt
     1321 tattatcatt tagaataaat tttgtgtcgc ccttccgcga aattaatacg actcactata
     1381 ggggaattgt gagcggataa caattcccct ctagaaataa ttttgtttaa cttttaggag
     1441 gtaaacatat gcaccatcat catcatcacg agaacttata ctttcaaggc atgagcagcg
     1501 ttacctgggc accgggcaat tacccgagca cccgccgttc cgatcatgtg gacacgtatc
     1561 aaagcgcgag caagggcgaa gtcccggtgc cggatccgta tcagtggctg gaagagagca
     1621 ccgacgaagt cgacaagtgg acgacggcac aagcggacct ggcacagtct tatctggacc
     1681 aaaacgctga cattcaaaaa ctggccgaga aatttcgtgc gagccgtaat tacgccaagt
     1741 tctctgcacc gactctgttg gacgacggtc actggtattg gttctataac cgtggcttgc
     1801 agagccaaag cgtcttgtac agatctaaag aaccagccct gccggacttc tctaagggcg
     1861 acgataacgt tggtgacgtt ttctttgatc cgaatgtgct ggcagcggat ggtagcgcgg
     1921 gcatggtcct gtgcaaattc tcgccggacg gtaaattctt tgcctatgca gttagccacc
     1981 tgggtggcga ttactccacg atttacgtgc gttccacgag cagcccgctg agccaggcga
     2041 gcgtcgcgca aggtgtggac ggtcgtctga gcgacgaagt caaatggttc aagtttagca
     2101 ccattatttg gactaaagat agcaagggct tcctgtacca gcgctatccg gctcgcgagc
     2161 gtcacgaggg tacccgcagc gaccgtaatg ccatgatgtg ttaccacaaa gtcggcacga
     2221 cccaagaaga agatattatt gtgtatcagg ataatgaaca tcctgagtgg atctatggtg
     2281 cggatacctc cgaagatggc aagtacctgt acctgtacca gtttaaagat accagcaaga
     2341 aaaacctgtt gtgggtcgcc gagctggacg aagatggcgt taagagcggt attcactggc
     2401 gtaaagttgt taacgaatac gcagcagact acaatatcat tacgaaccac ggtagcctgg
     2461 tctacattaa gacgaacctg aatgcaccgc aatataaagt tattaccatc gacctgtcca
     2521 aagacgagcc agagatccgt gacttcatcc cggaagaaaa agatgctaaa ctggcgcaag
     2581 ttaattgtgc gaacgaagag tatttcgtcg ctatttacaa gcgtaacgtt aaagatgaaa
     2641 tctacctgta ctcgaaggct ggtgttcagc tgacccgtct ggcgccggat ttcgtgggtg
     2701 cggcaagcat tgcgaatcgt caaaaacaga cccacttttt tctgaccctg agcggtttta
     2761 acaccccggg tacgatcgct cgttatgatt ttaccgcgcc ggaaacccag cgctttagca
     2821 tcctgcgtac cactaaagtc aacgaactgg atcctgatga cttcgagagc acgcaagtgt
     2881 ggtacgagag caaggacggt acgaaaatcc cgatgttcat cgttcgtcac aagtccacca
     2941 agtttgacgg taccgcggct gcgatccaat atggttacgg cggcttcgcc acctccgcgg
     3001 atccattctt cagcccgatc attctgacgt ttctgcagac ctacggtgcg atttttgccg
     3061 taccgagcat ccgtggtggc ggtgagttcg gtgaggaatg gcataagggc ggccgccgcg
     3121 aaaccaaagt gaacacgttt gacgatttca ttgcggccgc ccaatttctg gtgaagaaca
     3181 aatatgcggc tccgggtaaa gttgcaatta atggtgcgag caacggcggc ctgctggtga
     3241 tgggctctat cgtccgtgcg cctgagggta cgttcggtgc ggcagtgcca gagggcggtg
     3301 tggcggatct gcttaagttc cacaaattta cgggtggcca ggcgtggatc agcgagtatg
     3361 gtaatccgtc tatccctgaa gaatttgatt acatctatcc gctgagcccg gttcacaatg
     3421 tccgcaccga caaagttatg ccggcaaccc tgattaccgt gaatatcggt gatggccgtg
     3481 tggttccgat gcacagcttc aagttcatcg cgactttgca gcataacgtt ccgcagaatc
     3541 cgcatccgct gctgattaag attgataaat cgttgggtca cggcatgggt aagccgaccg
     3601 acaaaaatgt gaaagacgcc gccgacaagt ggggtttcat cgcacgcgcc ctgggtctgg
     3661 aactgaaaac ggtcgagtga tgaaagcttc cccctagcat aaccccttgg ggcctctaaa
     3721 cgggtcttga ggggtttttt gcccctgaga cgcgtcaatc gagttcgtac ctaagggcga
     3781 caccccctaa ttagcccggg cgaaaggccc agtctttcga ctgagccttt cgttttattt
     3841 gatgcctggc agttccctac tctcgcatgg ggagtcccca cactaccatc ggcgctacgg
     3901 cgtttcactt ctgagttcgg catggggtca ggtgggacca ccgcgctact gccgccaggc
     3961 aaacaagggg tgttatgagc catattcagg tataaatggg ctcgcgataa tgttcagaat
     4021 tggttaattg gttgtaacac tgacccctat ttgtttattt ttctaaatac attcaaatat
     4081 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagaa
     4141 tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc
     4201 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc
     4261 acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc
     4321 cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc
     4381 ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt
     4441 ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt
     4501 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat
     4561 cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct
     4621 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat
     4681 gcctgtagcg atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc
     4741 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg
     4801 ctcggccctt ccggctggct ggtttattgc tgataaatcc ggagccggtg agcgtggttc
     4861 tcgcggtatc atcgcagcgc tggggccaga tggtaagccc tcccgtatcg tagttatcta
     4921 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc
     4981 ctcactgatt aagcattggt aagcggcgcg ccatcgaatg gcgcaaaacc tttcgcggta
     5041 tggcatgata gcgcccggaa gagagtcaat tcagggtggt gaatatgaaa ccagtaacgt
     5101 tatacgatgt cgcagagtat gccggtgtct cttatcagac cgtttcccgc gtggtgaacc
     5161 aggccagcca cgtttctgcg aaaacgcggg aaaaagtgga agcggcgatg gcggagctga
     5221 attacattcc caaccgcgtg gcacaacaac tggcgggcaa acagtcgttg ctgattggcg
     5281 ttgccacctc cagtctggcc ctgcacgcgc cgtcgcaaat tgtcgcggcg attaaatctc
     5341 gcgccgatca actgggtgcc agcgtggtgg tgtcgatggt agaacgaagc ggcgtcgaag
     5401 cctgtaaagc ggcggtgcac aatcttctcg cgcaacgcgt cagtgggctg atcattaact
     5461 atccgctgga tgaccaggat gccattgctg tggaagctgc ctgcactaat gttccggcgt
     5521 tatttcttga tgtctctgac cagacaccca tcaacagtat tattttctcc catgaggacg
     5581 gtacgcgact gggcgtggag catctggtcg cattgggtca ccagcaaatc gcgctgttag
     5641 cgggcccatt aagttctgtc tcggcgcgtc tgcgtctggc tggctggcat aaatatctca
     5701 ctcgcaatca aattcagccg atagcggaac gggaaggcga ctggagtgcc atgtccggtt
     5761 ttcaacaaac catgcaaatg ctgaatgagg gcatcgttcc cactgcgatg ctggttgcca
     5821 acgatcagat ggcgctgggc gcaatgcgcg ccattaccga gtccgggctg cgcgttggtg
     5881 cggatatctc ggtagtggga tacgacgata ccgaagatag ctcatgttat atcccgccgt
     5941 taaccaccat caaacaggat tttcgcctgc tggggcaaac cagcgtggac cgcttgctgc
     6001 aactctctca gggccaggcg gtgaagggca atcagctgtt gccagtctca ctggtgaaaa
     6061 gaaaaaccac cctggcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat
     6121 taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgactc atgaccaaaa
     6181 tcccttaacg tgagttacgc gcgcgtcgtt ccactgagcg tcagac
//
