LOCUS       sequence-394865-        5731 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression plasmids for production of C247A mutant of 
            asparaginyl endopeptidase 1 from O. affinis (OaAEP1_C247A).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5731)
  AUTHORS   Antonenko A, Singh AK, Mosna K, Krezel A
  TITLE     OaAEP1 Ligase-Assisted Chemoenzymatic Synthesis of Full 
            Cysteine-Rich Metal-Binding Cyanobacterial Metallothionein SmtA.
  JOURNAL   Bioconjug Chem. 2023 Mar 15;34(4):719-27. doi: 
            10.1021/acs.bioconjchem.3c00037.
  PUBMED    36921066
REFERENCE   2  (bases 1 to 5731)
  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.bioconjchem"; journalName: "Bioconjug Chem"; date: 
            "2023-03-15- 15"; volume: "34"; issue: "4"; pages: "719-27"
COMMENT     Sequence Label: OaAEP1_C247A
FEATURES             Location/Qualifiers
     source          1..5731
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /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             100..117
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             124..414
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="SDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLR
                     RLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     CDS             2285..2329
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(2367..2385)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2381..2428
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(2658..3473)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(2658..3473)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     3381..3400
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(3474..3565)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(3581..4330)
                     /direction=LEFT
                     /label=RSF ori
                     /note="Plasmids containing the RSF 1030 origin of 
                     replication can be propagated in E. coli cells that contain
                     additional plasmids with compatible origins."
     primer_bind     complement(3591..3608)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(3831..3850)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(4526..5608)
                     /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     5570..5589
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5609..5686)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctgta gaaataattt
       61 tgtttaactt taataaggag atataccatg ggcagcagcc atcatcatca tcatcacagc
      121 actagtgact cagaagtcaa tcaagaagct aagccagagg tcaagccaga agtcaagcct
      181 gagactcaca tcaatttaaa ggtgtccgat ggatcttcag aaatcttctt taagatcaaa
      241 aagaccactc ctttaagaag gctgatggaa gcgttcgcta aaagacaggg taaggaaatg
      301 gactccttaa gattcttgta cgacggtatt agaattcaag ctgatcagac ccctgaagat
      361 ttggacatgg aggataacga tattattgag gctcacagag aacagattgg tggatccgct
      421 cgcgatggtg attatctgca tctgccgagc gaagtgagcc gtttctttcg cccgcaggaa
      481 acaaacgatg atcatggtga agatagcgtg ggtacccgct gggcggtgct gattgcgggc
      541 tctaaaggct atgcgaacta tcgccatcag gctggcgtgt gccatgcgta tcagattctg
      601 aaacgcggcg gcctgaaaga tgaaaacatt gttgtgttca tgtatgatga tattgcgtat
      661 aacgaaagca atccacgtcc gggcgtgatt attaacagcc cgcatggtag cgatgtttat
      721 gctggcgtgc cgaaagatta tacaggcgaa gaagtgaacg cgaaaaactt tctggcggcg
      781 attctgggca acaaaagcgc gattaccggc ggcagcggca aagtggtgga tagcggtccg
      841 aacgaccata tttttattta ttataccgat catggcgctg cgggcgtgat tggcatgccg
      901 agcaaaccgt atctgtatgc agatgaactg aacgatgcgc tgaaaaagaa acatgcgagc
      961 ggtacatata aaagcctggt gttttatctg gaagcgtgcg aaagcggcag catgtttgaa
     1021 ggtattttgc cggaagattt gaacatttat gcgctgacca gcaccaacac caccgaaagc
     1081 agctgggcgt attattgtcc ggcgcaggaa aacccgccac cgccggaata taacgtgtgc
     1141 ctgggcgatc tgtttagcgt ggcgtggctg gaagatagcg atgtgcagaa ttcctggtat
     1201 gaaaccctga accagcagta tcatcatgtg gataaacgca ttagccatgc gagccatgcg
     1261 acccagtatg gcaacctgaa attgggcgaa gaaggcctgt ttgtgtatat gggcagcaac
     1321 ccggcgaacg ataactatac ctctctggat ggcaacgcgc tgaccccgtc atcgattgtg
     1381 gtgaaccagc gcgatgcgga tctgctgcat ctgtgggaaa aattccgcaa agcgccggaa
     1441 ggcagcgcgc gcaaagaaga agcgcagacc cagattttta aagcgatgag ccatcgcgtg
     1501 catattgata gcagcattaa actgattggc aaactgctgt ttggcattga aaaatgcacc
     1561 gaaagcagct gggcgtatta ttgtccggcg caggaaaacc cgccaccgcc ggaatataac
     1621 gtgtgcctgg gcgatctgtt tagcgtggcg tggctggaag atagcgatgt gcagaattcc
     1681 tggtatgaaa ccctgaacca gcagtatcat catgtggata aacgcattag ccatgcgagc
     1741 catgcgaccc agtatggcaa cctgaaattg ggcgaagaag gcctgtttgt gtatatgggc
     1801 agcaacccgg cgaacgataa ctatacctct ctggatggca acgcgctgac cccgtcatcg
     1861 attgtggtga accagcgcga tgcggatctg ctgcatctgt gggaaaaatt ccgcaaagcg
     1921 ccggaaggca gcgcgcgcaa agaagaagcg cagacccaga tttttaaagc gatgagccat
     1981 cgcgtgcata ttgatagcag cattaaactg attggcaaac tgctgtttgg cattgaaaaa
     2041 tgcaccgaaa ttctgaacgc ggttcgtccg gcaggtcaac cgctggtgga tgattgggcg
     2101 tgcctgcgta gcctggtggg cacctttgaa acccattgcg gcagcctgag cgaatatggc
     2161 atgcgccata cccgcaccat tgcgaacatt tgcaacgcgg gcattagcga agaacagatg
     2221 gcggaagcgg cgagccaggc gtgcgcgagc attccgtaag cttgcggccg cactcgagtc
     2281 tggtaaagaa accgctgctg cgaaatttga acgccagcac atggactcgt ctactagcgc
     2341 agcttaatta acctaggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc
     2401 ctctaaacgg gtcttgaggg gttttttgct gaaacctcag gcatttgaga agcacacggt
     2461 cacactgctt ccggtagtca ataaaccggt aaaccagcaa tagacataag cggctattta
     2521 acgaccctgc cctgaaccga cgacaagctg acgaccgggt ctccgcaagt ggcacttttc
     2581 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc
     2641 cgctcatgaa ttaattctta gaaaaactca tcgagcatca aatgaaactg caatttattc
     2701 atatcaggat tatcaatacc atatttttga aaaagccgtt tctgtaatga aggagaaaac
     2761 tcaccgaggc agttccatag gatggcaaga tcctggtatc ggtctgcgat tccgactcgt
     2821 ccaacatcaa tacaacctat taatttcccc tcgtcaaaaa taaggttatc aagtgagaaa
     2881 tcaccatgag tgacgactga atccggtgag aatggcaaaa gtttatgcat ttctttccag
     2941 acttgttcaa caggccagcc attacgctcg tcatcaaaat cactcgcatc aaccaaaccg
     3001 ttattcattc gtgattgcgc ctgagcgaga cgaaatacgc ggtcgctgtt aaaaggacaa
     3061 ttacaaacag gaatcgaatg caaccggcgc aggaacactg ccagcgcatc aacaatattt
     3121 tcacctgaat caggatattc ttctaatacc tggaatgctg ttttcccggg gatcgcagtg
     3181 gtgagtaacc atgcatcatc aggagtacgg ataaaatgct tgatggtcgg aagaggcata
     3241 aattccgtca gccagtttag tctgaccatc tcatctgtaa catcattggc aacgctacct
     3301 ttgccatgtt tcagaaacaa ctctggcgca tcgggcttcc catacaatcg atagattgtc
     3361 gcacctgatt gcccgacatt atcgcgagcc catttatacc catataaatc agcatccatg
     3421 ttggaattta atcgcggcct agagcaagac gtttcccgtt gaatatggct catactcttc
     3481 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     3541 gaatgtattt agaaaaataa acaaataggc atgcagcgct cttccgcttc ctcgctcact
     3601 gactcgctac gctcggtcgt tcgactgcgg cgagcggtgt cagctcactc aaaagcggta
     3661 atacggttat ccacagaatc aggggataaa gccggaaaga acatgtgagc aaaaagcaaa
     3721 gcaccggaag aagccaacgc cgcaggcgtt tttccatagg ctccgccccc ctgacgagca
     3781 tcacaaaaat cgacgctcaa gccagaggtg gcgaaacccg acaggactat aaagatacca
     3841 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     3901 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgttg
     3961 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     4021 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     4081 cgacttatcg ccactggcag cagccattgg taactgattt agaggacttt gtcttgaagt
     4141 tatgcacctg ttaaggctaa actgaaagaa cagattttgg tgagtgcggt cctccaaccc
     4201 acttaccttg gttcaaagag ttggtagctc agcgaacctt gagaaaacca ccgttggtag
     4261 cggtggtttt tctttattta tgagatgatg aatcaatcgg tctatcaagt caacgaacag
     4321 ctattccgtt actctagatt tcagtgcaat ttatctcttc aaatgtagca cctgaagtca
     4381 gccccatacg atataagttg taattctcat gttagtcatg ccccgcgccc accggaagga
     4441 gctgactggg ttgaaggctc tcaagggcat cggtcgagat cccggtgcct aatgagtgag
     4501 ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     4561 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca
     4621 gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc accgcctggc
     4681 cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga aaatcctgtt
     4741 tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg tatcccacta
     4801 ccgagatgtc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg
     4861 ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc agcatttgca
     4921 tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct atcggctgaa
     4981 tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc gagacagaac
     5041 ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc
     5101 ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc tggtcagaga
     5161 catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg gcatcctggt
     5221 catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg
     5281 ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg ctggcaccca
     5341 gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc agggccagac
     5401 tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt gccacgcggt
     5461 tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa
     5521 cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg gcatactctg
     5581 cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc tcttccgggc
     5641 gctatcatgc cataccgcga aaggttttgc gccattcgat ggtgtccggg atctcgacgc
     5701 tctcccttat gcgactcctg cattaggaaa t
//
