LOCUS Exported 6192 bp ds-DNA circular SYN 09-DEC-2021 DEFINITION Expression of recombinant fusion protein in E. coli. ACCESSION . VERSION . KEYWORDS CoV-2-NSP3-c002 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6192) AUTHORS Sowa ST, Galera-Prat A, Wazir S, Alanen HI, Maksimainen MM, Lehtio L TITLE A molecular toolbox for ADP-ribosyl binding proteins JOURNAL Cell Reports Methods, November 10 2021 REFERENCE 2 (bases 1 to 6192) AUTHORS . TITLE Direct Submission JOURNAL Exported Dec 9, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6192 /organism="synthetic DNA construct" /mol_type="other DNA" CDS 4..21 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" CDS 31..357 /codon_start=1 /gene="trxA" /product="E. coli thioredoxin" /label=TrxA /translation="MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDE IADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFL DANLA" CDS 364..384 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQS" CDS 969..986 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" primer_bind complement(1039..1057) /label=T7 Term /note="T7 terminator, reverse primer" terminator 1053..1100 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" rep_origin 1137..1592 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(1224..1243) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 1434..1455 /label=F1ori-F /note="F1 origin, forward primer" CDS complement(1684..2499) /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 2407..2426 /label=Kan-R /note="Kanamycin resistance gene, reverse primer" primer_bind 2491..2510 /label=pENTR-R /note="pENTR vectors, reverse primer" rep_origin 2621..3209 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 3110..3129 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 3363..3380 /label=L4440 /note="L4440 vector, forward primer" misc_feature 3395..3534 /label=bom /note="basis of mobility region from pBR322" primer_bind complement(3460..3479) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 3620..3642 /label=pGEX 3' /note="pGEX vectors, reverse primer" CDS complement(3636..3827) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" CDS complement(4636..5718) /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 5680..5699 /label=LacI-R /note="LacI, reverse primer" promoter complement(5719..5796) /gene="lacI" /label=lacI promoter primer_bind 6002..6021 /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" primer_bind 6105..6124 /label=T7 /note="T7 promoter, forward primer" promoter 6105..6123 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" protein_bind 6124..6148 /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)." RBS 6163..6185 /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" ORIGIN 1 atgcaccatc atcatcatca ttcttctggt atgagcgata aaattattca cctgactgac 61 gacagttttg acacggatgt actcaaagcg gacggggcga tcctcgtcga tttctgggca 121 gagtggtgcg gtccgtgcaa aatgatcgcc ccgattctgg atgaaatcgc tgacgaatat 181 cagggcaaac tgaccgttgc aaaactgaac atcgatcaaa accctggcac tgcgccgaaa 241 tatggcatcc gtggtatccc gactctgctg ctgttcaaaa acggtgaagt ggcggcaacc 301 aaagtgggcg cactgtctaa aggtcagttg aaagagttcc tcgacgctaa cctggccggt 361 accgagaact tgtacttcca atccatggag gtgaatagtt tctcagggta tttaaagttg 421 actgataatg tttacatcaa gaacgcggat attgttgaag aggccaagaa ggtgaaacct 481 accgtggtgg taaacgctgc aaacgtgtac cttaagcacg gcgggggagt cgcgggagca 541 ctgaataaag ctacgaataa tgcaatgcaa gtggaatcag atgactacat cgcaaccaat 601 ggcccattaa aagtgggtgg tagctgcgtc ctgtctggac ataacctggc aaaacactgt 661 ttacatgtag ttgggcccaa cgtcaataaa ggagaggaca tccaattatt gaaatctgct 721 tatgaaaact tcaatcagca cgaggtgtta cttgcaccgt tgttgtccgc tgggattttt 781 ggcgctgacc ccattcattc tcttcgtgtc tgtgttgata ctgtgcgtac taacgtatac 841 ctggcagttt ttgataagaa tttatatgac aaattggtgt catcttttct tgagtaggac 901 ggtctccagt aaaggtggat acggatccga attcgagctc cgtcgacaag cttgcggccg 961 cactcgagca ccaccaccac caccactgag atccggctgc taacaaagcc cgaaaggaag 1021 ctgagttggc tgctgccacc gctgagcaat aactagcata accccttggg gcctctaaac 1081 gggtcttgag gggttttttg ctgaaaggag gaactatatc cggattggcg aatgggacgc 1141 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac 1201 acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt 1261 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc 1321 tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc 1381 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact 1441 cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg 1501 gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc 1561 gaattttaac aaaatattaa cgcttacaat ttaggtggca cttttcgggg aaatgtgcgc 1621 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgaattaa 1681 ttcttagaaa aactcatcga gcatcaaatg aaactgcaat ttattcatat caggattatc 1741 aataccatat ttttgaaaaa gccgtttctg taatgaagga gaaaactcac cgaggcagtt 1801 ccataggatg gcaagatcct ggtatcggtc tgcgattccg actcgtccaa catcaataca 1861 acctattaat ttcccctcgt caaaaataag gttatcaagt gagaaatcac catgagtgac 1921 gactgaatcc ggtgagaatg gcaaaagttt atgcatttct ttccagactt gttcaacagg 1981 ccagccatta cgctcgtcat caaaatcact cgcatcaacc aaaccgttat tcattcgtga 2041 ttgcgcctga gcgagacgaa atacgcgatc gctgttaaaa ggacaattac aaacaggaat 2101 cgaatgcaac cggcgcagga acactgccag cgcatcaaca atattttcac ctgaatcagg 2161 atattcttct aatacctgga atgctgtttt cccggggatc gcagtggtga gtaaccatgc 2221 atcatcagga gtacggataa aatgcttgat ggtcggaaga ggcataaatt ccgtcagcca 2281 gtttagtctg accatctcat ctgtaacatc attggcaacg ctacctttgc catgtttcag 2341 aaacaactct ggcgcatcgg gcttcccata caatcgatag attgtcgcac ctgattgccc 2401 gacattatcg cgagcccatt tatacccata taaatcagca tccatgttgg aatttaatcg 2461 cggcctagag caagacgttt cccgttgaat atggctcata acaccccttg tattactgtt 2521 tatgtaagca gacagtttta ttgttcatga ccaaaatccc ttaacgtgag ttttcgttcc 2581 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc 2641 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg 2701 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa 2761 atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc 2821 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt 2881 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa 2941 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc 3001 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc 3061 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct 3121 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat 3181 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc 3241 tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg 3301 ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc 3361 gcagcgagtc agtgagcgag gaagcggaag agcgcctgat gcggtatttt ctccttacgc 3421 atctgtgcgg tatttcacac cgcaatggtg cactctcagt acaatctgct ctgatgccgc 3481 atagttaagc cagtatacac tccgctatcg ctacgtgact gggtcatggc tgcgccccga 3541 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac 3601 agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg 3661 aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg 3721 tctgcctgtt catccgcgtc cagctcgttg agtttctcca gaagcgttaa tgtctggctt 3781 ctgataaagc gggccatgtt aagggcggtt ttttcctgtt tggtcactga tgcctccgtg 3841 taagggggat ttctgttcat gggggtaatg ataccgatga aacgagagag gatgctcacg 3901 atacgggtta ctgatgatga acatgcccgg ttactggaac gttgtgaggg taaacaactg 3961 gcggtatgga tgcggcggga ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt 4021 aatacagatg taggtgttcc acagggtagc cagcagcatc ctgcgatgca gatccggaac 4081 ataatggtgc agggcgctga cttccgcgtt tccagacttt acgaaacacg gaaaccgaag 4141 accattcatg ttgttgctca ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc 4201 tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac cccgccagcc tagccgggtc 4261 ctcaacgaca ggagcacgat catgcgcacc cgtggggccg ccatgccggc gataatggcc 4321 tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga aggcttgagc gagggcgtgc 4381 aagattccga ataccgcaag cgacaggccg atcatcgtcg cgctccagcg aaagcggtcc 4441 tcgccgaaaa tgacccagag cgctgccggc acctgtccta cgagttgcat gataaagaag 4501 acagtcataa gtgcggcgac gatagtcatg ccccgcgccc accggaagga gctgactggg 4561 ttgaaggctc tcaagggcat cggtcgagat cccggtgcct aatgagtgag ctaacttaca 4621 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat 4681 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca gggtggtttt 4741 tcttttcacc agtgagacgg gcaacagctg attgcccttc accgcctggc cctgagagag 4801 ttgcagcaag cggtccacgc tggtttgccc cagcaggcga aaatcctgtt tgatggtggt 4861 taacggcggg atataacatg agctgtcttc ggtatcgtcg tatcccacta ccgagatatc 4921 cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc 4981 gttggcaacc agcatcgcag tgggaacgat gccctcattc agcatttgca tggtttgttg 5041 aaaaccggac atggcactcc agtcgccttc ccgttccgct atcggctgaa tttgattgcg 5101 agtgagatat ttatgccagc cagccagacg cagacgcgcc gagacagaac ttaatgggcc 5161 cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt 5221 accgtcttca tgggagaaaa taatactgtt gatgggtgtc tggtcagaga catcaagaaa 5281 taacgccgga acattagtgc aggcagcttc cacagcaatg gcatcctggt catccagcgg 5341 atagttaatg atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg ccgctttaca 5401 ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg ctggcaccca gttgatcggc 5461 gcgagattta atcgccgcga caatttgcga cggcgcgtgc agggccagac tggaggtggc 5521 aacgccaatc agcaacgact gtttgcccgc cagttgttgt gccacgcggt tgggaatgta 5581 attcagctcc gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc 5641 ctggttcacc acgcgggaaa cggtctgata agagacaccg gcatactctg cgacatcgta 5701 taacgttact ggtttcacat tcaccaccct gaattgactc tcttccgggc gctatcatgc 5761 cataccgcga aaggttttgc gccattcgat ggtgtccggg atctcgacgc tctcccttat 5821 gcgactcctg cattaggaag cagcccagta gtaggttgag gccgttgagc accgccgccg 5881 caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc cccggccacg gggcctgcca 5941 ccatacccac gccgaaacaa gcgctcatga gcccgaagtg gcgagcccga tcttccccat 6001 cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt ggcgccggtg atgccggcca 6061 cgatgcgtcc ggcgtagagg atcgagatct cgatcccgcg aaattaatac gactcactat 6121 aggggaattg tgagcggata acaattcccc tctagaaata attttgttta actttaagaa 6181 ggagatatac at //