LOCUS       sequence-192479-        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      432..1020
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     921..940
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1174..1191
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     1289..1306
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1290..1306
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1769..1788
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1769..1787
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1788..1812
                     /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             1857..1874
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1875..1895
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     terminator      4099..4146
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(4369..4388)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     complement(4394..4413)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        4455..4546
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4547..4615,4616..5407)
                     /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(4765..4784)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     CDS             join(5490..6226,1..346)
                     /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(5509..5528)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 cgatcagatg gcgctgggcg caatgcgcgc cattaccgag tccgggctgc gcgttggtgc
       61 ggatatctcg gtagtgggat acgacgatac cgaagatagc tcatgttata tcccgccgtt
      121 aaccaccatc aaacaggatt ttcgcctgct ggggcaaacc agcgtggacc gcttgctgca
      181 actctctcag ggccaggcgg tgaagggcaa tcagctgttg ccagtctcac tggtgaaaag
      241 aaaaaccacc ctggcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt
      301 aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgactca tgaccaaaat
      361 cccttaacgt gagttacgcg cgcgtcgttc cactgagcgt cagaccccgt agaaaagatc
      421 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
      481 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
      541 gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta
      601 gcccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
      661 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
      721 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
      781 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
      841 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
      901 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
      961 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     1021 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
     1081 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct
     1141 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa
     1201 ggcgagagta gggaactgcc aggcatcaaa ctaagcagaa ggcccctgac ggatggcctt
     1261 tttgcgtttc tacaaactct ttctgtgttg taaaacgacg gccagtctta agctcgggcc
     1321 ccctgggcgg ttctgataac gagtaatcgt taatccgcaa ataacgtaaa aacccgcttc
     1381 ggcgggtttt tttatggggg gagtttaggg aaagagcatt tgtcagaata tttaagggcg
     1441 cctgtcactt tgcttgatat atgagaatta tttaacctta taaatgagaa aaaagcaacg
     1501 cactttaaat aagatacgtt gctttttcga ttgatgaaca cctataatta aactattcat
     1561 ctattattta tgattttttg tatatacaat atttctagtt tgttaaagag aattaagaaa
     1621 ataaatctcg aaaataataa agggaaaatc agtttttgat atcaaaatta tacatgtcaa
     1681 cgataataca aaatataata caaactataa gatgttatca gtatttatta tcatttagaa
     1741 taaattttgt gtcgcccttc cgcgaaatta atacgactca ctatagggga attgtgagcg
     1801 gataacaatt cccctctaga aataattttg tttaactttt aggaggtaaa catatgcacc
     1861 atcatcatca tcacgagaac ttatactttc aaggcatgag cagcgttacc tgggcaccgg
     1921 gcaattaccc gagcacccgc cgttccgatc atgtggacac gtatcaaagc gcgagcaagg
     1981 gcgaagtccc ggtgccggat ccgtatcagt ggctggaaga gagcaccgac gaagtcgaca
     2041 agtggacgac ggcacaagcg gacctggcac agtcttatct ggaccaaaac gctgacattc
     2101 aaaaactggc cgagaaattt cgtgcgagcc gtaattacgc caagttctct gcaccgactc
     2161 tgttggacga cggtcactgg tattggttct ataaccgtgg cttgcagagc caaagcgtct
     2221 tgtacagatc taaagaacca gccctgccgg acttctctaa gggcgacgat aacgttggtg
     2281 acgttttctt tgatccgaat gtgctggcag cggatggtag cgcgggcatg gtcctgtgca
     2341 aattctcgcc ggacggtaaa ttctttgcct atgcagttag ccacctgggt ggcgattact
     2401 ccacgattta cgtgcgttcc acgagcagcc cgctgagcca ggcgagcgtc gcgcaaggtg
     2461 tggacggtcg tctgagcgac gaagtcaaat ggttcaagtt tagcaccatt atttggacta
     2521 aagatagcaa gggcttcctg taccagcgct atccggctcg cgagcgtcac gagggtaccc
     2581 gcagcgaccg taatgccatg atgtgttacc acaaagtcgg cacgacccaa gaagaagata
     2641 ttattgtgta tcaggataat gaacatcctg agtggatcta tggtgcggat acctccgaag
     2701 atggcaagta cctgtacctg taccagttta aagataccag caagaaaaac ctgttgtggg
     2761 tcgccgagct ggacgaagat ggcgttaaga gcggtattca ctggcgtaaa gttgttaacg
     2821 aatacgcagc agactacaat atcattacga accacggtag cctggtctac attaagacga
     2881 acctgaatgc accgcaatat aaagttatta ccatcgacct gtccaaagac gagccagaga
     2941 tccgtgactt catcccggaa gaaaaagatg ctaaactggc gcaagttaat tgtgcgaacg
     3001 aagagtattt cgtcgctatt tacaagcgta acgttaaaga tgaaatctac ctgtactcga
     3061 aggctggtgt tcagctgacc cgtctggcgc cggatttcgt gggtgcggca agcattgcga
     3121 atcgtcaaaa acagacccac ttttttctga ccctgagcgg ttttaacacc ccgggtacga
     3181 tcgctcgtta tgattttacc gcgccggaaa cccagcgctt tagcatcctg cgtaccacta
     3241 aagtcaacga actggatcct gatgacttcg agagcacgca agtgtggtac gagagcaagg
     3301 acggtacgaa aatcccgatg ttcatcgttc gtcacaagtc caccaagttt gacggtaccg
     3361 cggctgcgat ccaatatggt tacggcggct tcgccacctc cgcggatcca ttcttcagcc
     3421 cgatcattct gacgtttctg cagacctacg gtgcgatttt tgccgtaccg agcatccgtg
     3481 gtggcggtga gttcggtgag gaatggcata agggcggccg ccgcgaaacc aaagtgaaca
     3541 cgtttgacga tttcattgcg gccgcccaat ttctggtgaa gaacaaatat gcggctccgg
     3601 gtaaagttgc aattaatggt gcgagcaacg gcggcctgct ggtgatgggc tctatcgtcc
     3661 gtgcgcctga gggtacgttc ggtgcggcag tgccagaggg cggtgtggcg gatctgctta
     3721 agttccacaa atttacgggt ggccaggcgt ggatcagcga gtatggtaat ccgtctatcc
     3781 ctgaagaatt tgattacatc tatccgctga gcccggttca caatgtccgc accgacaaag
     3841 ttatgccggc aaccctgatt accgtgaata tcggtgatgg ccgtgtggtt ccgatgcaca
     3901 gcttcaagtt catcgcgact ttgcagcata acgttccgca gaatccgcat ccgctgctga
     3961 ttaagattga taaatcgttg ggtcacggca tgggtaagcc gaccgacaaa aatgtgaaag
     4021 acgccgccga caagtggggt ttcatcgcac gcgccctggg tctggaactg aaaacggtcg
     4081 agtgatgaaa gcttccccct agcataaccc cttggggcct ctaaacgggt cttgaggggt
     4141 tttttgcccc tgagacgcgt caatcgagtt cgtacctaag ggcgacaccc cataattagc
     4201 ccgggcgaaa ggcccagtct ttcgactgag cctttcgttt tatttgatgc ctggcagttc
     4261 cctactctcg catggggagt ccccacacta ccatcggcgc tacggcgttt cacttctgag
     4321 ttcggcatgg ggtcaggtgg gaccaccgcg ctactgccgc caggcaaaca aggggtgtta
     4381 tgagccatat tcaggtataa atgggctcgc gataatgttc agaattggtt aattggttgt
     4441 aacactgacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
     4501 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagaatatga gtattcaaca
     4561 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc
     4621 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat
     4681 cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc
     4741 aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg
     4801 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc
     4861 agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat
     4921 aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga
     4981 gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc
     5041 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcgatggc
     5101 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt
     5161 aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc
     5221 tggctggttt attgctgata aatccggagc cggtgagcgt ggttctcgcg gtatcatcgc
     5281 agcgctgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca
     5341 ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca
     5401 ttggtaagcg gcgcgccatc gaatggcgca aaacctttcg cggtatggca tgatagcgcc
     5461 cggaagagag tcaattcagg gtggtgaata tgaaaccagt aacgttatac gatgtcgcag
     5521 agtatgccgg tgtctcttat cagaccgttt cccgcgtggt gaaccaggcc agccacgttt
     5581 ctgcgaaaac gcgggaaaaa gtggaagcgg cgatggcgga gctgaattac attcccaacc
     5641 gcgtggcaca acaactggcg ggcaaacagt cgttgctgat tggcgttgcc acctccagtc
     5701 tggccctgca cgcgccgtcg caaattgtcg cggcgattaa atctcgcgcc gatcaactgg
     5761 gtgccagcgt ggtggtgtcg atggtagaac gaagcggcgt cgaagcctgt aaagcggcgg
     5821 tgcacaatct tctcgcgcaa cgcgtcagtg ggctgatcat taactatccg ctggatgacc
     5881 aggatgccat tgctgtggaa gctgcctgca ctaatgttcc ggcgttattt cttgatgtct
     5941 ctgaccagac acccatcaac agtattattt tctcccatga ggacggtacg cgactgggcg
     6001 tggagcatct ggtcgcattg ggtcaccagc aaatcgcgct gttagcgggc ccattaagtt
     6061 ctgtctcggc gcgtctgcgt ctggctggct ggcataaata tctcactcgc aatcaaattc
     6121 agccgatagc ggaacgggaa ggcgactgga gtgccatgtc cggttttcaa caaaccatgc
     6181 aaatgctgaa tgagggcatc gttcccactg cgatgctggt tgccaa
//
