LOCUS       40924_6352        5245 bp DNA     circular SYN 13-MAY-2021
DEFINITION  Express asparaginyl endopeptidase 1  from  O. affinis (OaAEP1).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5245)
  AUTHORS   Mikula KM, Tascon I, Tommila JJ, Iwai H
  TITLE     Segmental isotopic labeling of a single-domain globular protein 
            without any refolding step by an asparaginyl endopeptidase.
  JOURNAL   FEBS Lett. 2017 Mar 30. doi: 10.1002/1873-3468.12640.
  PUBMED    28369872
REFERENCE   2  (bases 1 to 5245)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 5245)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "FEBS Lett. 
            2017 Mar 30. doi: 10.1002/1873-3468.12640."
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..5245
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /label=lac operator
                     /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)."
     RBS             59..81
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             100..117
                     /codon_start=1
                     /label=6xHis
                     /note="6xHis affinity tag"
                     /translation="HHHHHH"
     CDS             124..414
                     /codon_start=1
                     /label=SUMO
                     /note="cleavable ubiquitin-like protein tag"
                     /translation="SDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLR
                     RLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     CDS             1799..1843
                     /codon_start=1
                     /label=S-Tag
                     /note="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /translation="KETAAAKFERQHMDS"
     terminator      1895..1942
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(2175..2987)
                     /codon_start=1
                     /label=KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     promoter        complement(2988..3079)
                     /label=AmpR promoter
     rep_origin      complement(3095..3844)
                     /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."
     protein_bind    complement(4006..4027)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             complement(4043..5122)
                     /codon_start=1
                     /label=lacI
                     /note="lac repressor"
                     /translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     promoter        complement(5123..5200)
                     /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 agctggtgct 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 gaaattctga acgcggttcg tccggcaggt caaccgctgg tggatgattg ggcgtgcctg
     1621 cgtagcctgg tgggcacctt tgaaacccat tgcggcagcc tgagcgaata tggcatgcgc
     1681 catacccgca ccattgcgaa catttgcaac gcgggcatta gcgaagaaca gatggcggaa
     1741 gcggcgagcc aggcgtgcgc gagcattccg taagcttgcg gccgcactcg agtctggtaa
     1801 agaaaccgct gctgcgaaat ttgaacgcca gcacatggac tcgtctacta gcgcagctta
     1861 attaacctag gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa
     1921 acgggtcttg aggggttttt tgctgaaacc tcaggcattt gagaagcaca cggtcacact
     1981 gcttccggta gtcaataaac cggtaaacca gcaatagaca taagcggcta tttaacgacc
     2041 ctgccctgaa ccgacgacaa gctgacgacc gggtctccgc aagtggcact tttcggggaa
     2101 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     2161 tgaattaatt cttagaaaaa ctcatcgagc atcaaatgaa actgcaattt attcatatca
     2221 ggattatcaa taccatattt ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg
     2281 aggcagttcc ataggatggc aagatcctgg tatcggtctg cgattccgac tcgtccaaca
     2341 tcaatacaac ctattaattt cccctcgtca aaaataaggt tatcaagtga gaaatcacca
     2401 tgagtgacga ctgaatccgg tgagaatggc aaaagtttat gcatttcttt ccagacttgt
     2461 tcaacaggcc agccattacg ctcgtcatca aaatcactcg catcaaccaa accgttattc
     2521 attcgtgatt gcgcctgagc gagacgaaat acgcggtcgc tgttaaaagg acaattacaa
     2581 acaggaatcg aatgcaaccg gcgcaggaac actgccagcg catcaacaat attttcacct
     2641 gaatcaggat attcttctaa tacctggaat gctgttttcc cggggatcgc agtggtgagt
     2701 aaccatgcat catcaggagt acggataaaa tgcttgatgg tcggaagagg cataaattcc
     2761 gtcagccagt ttagtctgac catctcatct gtaacatcat tggcaacgct acctttgcca
     2821 tgtttcagaa acaactctgg cgcatcgggc ttcccataca atcgatagat tgtcgcacct
     2881 gattgcccga cattatcgcg agcccattta tacccatata aatcagcatc catgttggaa
     2941 tttaatcgcg gcctagagca agacgtttcc cgttgaatat ggctcatact cttccttttt
     3001 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     3061 atttagaaaa ataaacaaat aggcatgcag cgctcttccg cttcctcgct cactgactcg
     3121 ctacgctcgg tcgttcgact gcggcgagcg gtgtcagctc actcaaaagc ggtaatacgg
     3181 ttatccacag aatcagggga taaagccgga aagaacatgt gagcaaaaag caaagcaccg
     3241 gaagaagcca acgccgcagg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     3301 aaatcgacgc tcaagccaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     3361 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     3421 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gttggtatct
     3481 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     3541 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     3601 atcgccactg gcagcagcca ttggtaactg atttagagga ctttgtcttg aagttatgca
     3661 cctgttaagg ctaaactgaa agaacagatt ttggtgagtg cggtcctcca acccacttac
     3721 cttggttcaa agagttggta gctcagcgaa ccttgagaaa accaccgttg gtagcggtgg
     3781 tttttcttta tttatgagat gatgaatcaa tcggtctatc aagtcaacga acagctattc
     3841 cgttactcta gatttcagtg caatttatct cttcaaatgt agcacctgaa gtcagcccca
     3901 tacgatataa gttgtaattc tcatgttagt catgccccgc gcccaccgga aggagctgac
     3961 tgggttgaag gctctcaagg gcatcggtcg agatcccggt gcctaatgag tgagctaact
     4021 tacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct
     4081 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gccagggtgg
     4141 tttttctttt caccagtgag acgggcaaca gctgattgcc cttcaccgcc tggccctgag
     4201 agagttgcag caagcggtcc acgctggttt gccccagcag gcgaaaatcc tgtttgatgg
     4261 tggttaacgg cgggatataa catgagctgt cttcggtatc gtcgtatccc actaccgaga
     4321 tgtccgcacc aacgcgcagc ccggactcgg taatggcgcg cattgcgccc agcgccatct
     4381 gatcgttggc aaccagcatc gcagtgggaa cgatgccctc attcagcatt tgcatggttt
     4441 gttgaaaacc ggacatggca ctccagtcgc cttcccgttc cgctatcggc tgaatttgat
     4501 tgcgagtgag atatttatgc cagccagcca gacgcagacg cgccgagaca gaacttaatg
     4561 ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc acgcccagtc
     4621 gcgtaccgtc ttcatgggag aaaataatac tgttgatggg tgtctggtca gagacatcaa
     4681 gaaataacgc cggaacatta gtgcaggcag cttccacagc aatggcatcc tggtcatcca
     4741 gcggatagtt aatgatcagc ccactgacgc gttgcgcgag aagattgtgc accgccgctt
     4801 tacaggcttc gacgccgctt cgttctacca tcgacaccac cacgctggca cccagttgat
     4861 cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc agactggagg
     4921 tggcaacgcc aatcagcaac gactgtttgc ccgccagttg ttgtgccacg cggttgggaa
     4981 tgtaattcag ctccgccatc gccgcttcca ctttttcccg cgttttcgca gaaacgtggc
     5041 tggcctggtt caccacgcgg gaaacggtct gataagagac accggcatac tctgcgacat
     5101 cgtataacgt tactggtttc acattcacca ccctgaattg actctcttcc gggcgctatc
     5161 atgccatacc gcgaaaggtt ttgcgccatt cgatggtgtc cgggatctcg acgctctccc
     5221 ttatgcgact cctgcattag gaaat
//
