LOCUS Exported 5245 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Express asparaginyl endopeptidase 1 from O. affinis (OaAEP1). ACCESSION . VERSION . KEYWORDS pBHRSF184 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) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5245 /organism="synthetic DNA construct" /mol_type="other DNA" 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 127..411 /codon_start=1 /gene="S. cerevisiae SMT3 (truncated)" /product="cleavable ubiquitin-like protein tag" /label=SUMO /translation="DSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRR LMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIG" CDS 1799..1843 /codon_start=1 /product="affinity and epitope tag derived from pancreatic ribonuclease A" /label=S-Tag /translation="KETAAAKFERQHMDS" primer_bind complement(1881..1899) /label=T7 Term /note="T7 terminator, reverse primer" terminator 1895..1942 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" CDS complement(2172..2987) /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 2895..2914 /label=Kan-R /note="Kanamycin resistance gene, reverse primer" promoter complement(2988..3079) /gene="bla" /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." primer_bind complement(3105..3122) /label=L4440 /note="L4440 vector, forward primer" primer_bind complement(3345..3364) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(4040..5122) /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 5084..5103 /label=LacI-R /note="LacI, reverse primer" promoter complement(5123..5200) /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 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 //