LOCUS Exported 6721 bp ds-DNA circular SYN 20-JUL-2021 DEFINITION Expression of SARS-COV-2 Nsp12 D218A in E. coli. ACCESSION . VERSION . KEYWORDS pIA1405 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6721) TITLE Nsp9 NMPylation JOURNAL Unpublished REFERENCE 2 (bases 1 to 6721) AUTHORS . TITLE Direct Submission JOURNAL Exported Jul 20, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6721 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 812..831 /label=LacI-R /note="LacI, reverse primer" promoter complement(851..928) /gene="lacI" /label=lacI promoter primer_bind 974..993 /label=T7 /note="T7 promoter, forward primer" promoter 974..992 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" protein_bind 993..1017 /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 1073..1090 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" CDS 1100..1117 /codon_start=1 /product="thrombin recognition and cleavage site" /label=thrombin site /translation="LVPRGS" CDS 1130..1420 /codon_start=1 /gene="S. cerevisiae SMT3 (truncated)" /product="cleavable ubiquitin-like protein tag" /label=SUMO /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIG" CDS 4248..4292 /codon_start=1 /product="affinity and epitope tag derived from pancreatic ribonuclease A" /label=S-Tag /translation="KETAAAKFERQHMDS" primer_bind complement(4330..4348) /label=T7 Term /note="T7 terminator, reverse primer" terminator 4344..4391 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" CDS complement(4621..5436) /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 5344..5363 /label=Kan-R /note="Kanamycin resistance gene, reverse primer" promoter complement(5437..5528) /gene="bla" /label=AmpR promoter rep_origin complement(5544..6293) /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(5554..5571) /label=L4440 /note="L4440 vector, forward primer" primer_bind complement(5794..5813) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(6489..850) /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" ORIGIN 1 ggatataaca tgagctgtct tcggtatcgt cgtatcccac taccgagatg tccgcaccaa 61 cgcgcagccc ggactcggta atggcgcgca ttgcgcccag cgccatctga tcgttggcaa 121 ccagcatcgc agtgggaacg atgccctcat tcagcatttg catggtttgt tgaaaaccgg 181 acatggcact ccagtcgcct tcccgttccg ctatcggctg aatttgattg cgagtgagat 241 atttatgcca gccagccaga cgcagacgcg ccgagacaga acttaatggg cccgctaaca 301 gcgcgatttg ctggtgaccc aatgcgacca gatgctccac gcccagtcgc gtaccgtctt 361 catgggagaa aataatactg ttgatgggtg tctggtcaga gacatcaaga aataacgccg 421 gaacattagt gcaggcagct tccacagcaa tggcatcctg gtcatccagc ggatagttaa 481 tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta caggcttcga 541 cgccgcttcg ttctaccatc gacaccacca cgctggcacc cagttgatcg gcgcgagatt 601 taatcgccgc gacaatttgc gacggcgcgt gcagggccag actggaggtg gcaacgccaa 661 tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg taattcagct 721 ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga aacgtggctg gcctggttca 781 ccacgcggga aacggtctga taagagacac cggcatactc tgcgacatcg tataacgtta 841 ctggtttcac attcaccacc ctgaattgac tctcttccgg gcgctatcat gccataccgc 901 gaaaggtttt gcgccattcg atggtgtccg ggatctcgac gctctccctt atgcgactcc 961 tgcattagga aattaatacg actcactata ggggaattgt gagcggataa caattcccct 1021 gtagaaataa ttttgtttaa ctttaataag gagatatacc atgggcagca gccatcatca 1081 tcatcatcac agcagcggcc tggtgccgcg cggcagccac atggcaagca tgtcggactc 1141 agaagtcaat caagaagcta agccagaggt caagccagaa gtcaagcctg agactcacat 1201 caatttaaag gtgtccgatg gatcttcaga gatcttcttc aagatcaaaa agaccactcc 1261 tttaagaagg ctgatggaag cgttcgctaa aagacagggt aaggaaatgg actccttaag 1321 attcttgtac gacggtatta gaatccaagc tgatcagacc cctgaagatt tggacatgga 1381 ggataacgat attattgagg ctcacagaga acagattggt ggatccgccg acgcgcagtc 1441 ttttctgaac cgcgtatgcg gggtaagcgc agctcgcttg acaccctgtg gtactgggac 1501 gtctacagac gtggtttatc gcgcgttcga catctataac gacaaagtcg ccggtttcgc 1561 taagttcttg aagaccaatt gctgtcgctt tcaagagaaa gatgaagatg ataatcttat 1621 tgactcttat tttgtagtaa agcgtcacac cttcagcaac tatcagcatg aagaaaccat 1681 ctataatctt ttgaaggact gtccagcagt agccaagcac gattttttca aatttcgtat 1741 tgacggtgac atggtccccc acatttcacg ccagcgcctt accaaataca caatggcgga 1801 tctggtttat gctcttcgtc attttgacga aggaaattgc gacaccctta aggagattct 1861 ggtaacttat aattgttgcg acgatgacta ttttaataaa aaggattggt atgattttgt 1921 cgaaaatccg gatattttac gtgtgtacgc aaatctggga gagcgcgttc gccaagcgtt 1981 gttaaagacg gtacagttct gcgacgccat gcgtaatgcc ggtatcgtgg gtgtgctgac 2041 cttagataac caagatctta acggtaattg gtatgcattc ggagatttta ttcagaccac 2101 tcccggctcc ggcgtgccgg tagtggattc ctattatagt ctgctgatgc caatcttgac 2161 gttgacccgt gccttgacgg cggagagcca tgtggacaca gacttaacga agccttacat 2221 taaatgggat ttgcttaaat acgacttcac ggaggagcgt cttaaacttt ttgaccgtta 2281 ctttaagtat tgggaccaga cgtatcaccc caactgcgtc aactgcttag acgatcgctg 2341 catcttacat tgtgcaaact ttaatgtttt attttccaca gtttttccgc cgacttcctt 2401 cgggccactg gtacgcaaga tttttgtcga cggcgtgccg tttgtagtct ctacggggta 2461 ccactttcgt gaacttggtg ttgttcataa ccaggatgtt aatttacatt cgtcacgtct 2521 ttcattcaaa gagttactgg tatacgcggc cgatcccgcc atgcacgcag cgtccgggaa 2581 tttacttctg gataaacgta ctacgtgttt cagtgttgca gcgctgacca acaatgtagc 2641 tttccaaaca gtaaaaccag ggaatttcaa taaggatttt tacgacttcg cagtcagtaa 2701 gggttttttt aaagagggga gctcggttga gttaaaacac ttttttttcg ctcaggatgg 2761 gaacgctgca atctccgatt acgactacta tcgctacaac ttacctacga tgtgcgatat 2821 tcgccaatta ctgttcgtag ttgaagtagt agacaagtat ttcgactgtt acgatggagg 2881 atgtattaat gcgaaccaag taatcgtcaa taatttagat aaatctgcag gttttccctt 2941 taataagtgg ggaaaggcgc gcctgtacta cgattcaatg tcgtatgagg accaggacgc 3001 tcttttcgca tataccaaac gcaatgttat cccgacgatt acgcaaatga atctgaaata 3061 tgccatttcc gctaaaaatc gcgctcgcac ggtcgcggga gtatcgattt gttctactat 3121 gactaaccgc caattccatc aaaagttgct taaatctatc gcagcaacgc gtggggcaac 3181 ggtggtcatc gggacctcga agttttatgg aggttggcac aacatgctga aaaccgtgta 3241 tagtgacgtg gaaaacccgc atctgatggg gtgggattat ccgaaatgcg atcgcgcaat 3301 gcctaatatg cttcgcatca tggcctcttt agtgctagca cgtaagcata cgacatgttg 3361 cagcttgtcc catcgctttt accgtttagc taatgagtgc gcacaagtat tatcggagat 3421 ggtaatgtgc ggcgggagtt tatacgtgaa gccaggtggt actagttctg gggatgcaac 3481 cacagcgtat gctaattcgg ttttcaatat ctgtcaggcc gttaccgcca acgtaaacgc 3541 acttctgtca acggatggta ataaaattgc tgataagtac gtacgcaacc tgcaacatcg 3601 tctgtatgag tgcctttatc gcaaccgcga tgtcgacacc gactttgtaa acgagttcta 3661 tgcatatttg cgtaagcatt tctctatgat gatcctttca gacgatgccg ttgtgtgttt 3721 caacagtacc tatgcctcgc aaggcctagt agcctccatc aaaaacttta agtccgttct 3781 ttactatcag aacaacgtct ttatgtcaga ggccaagtgc tggaccgaga cggacttaac 3841 taaaggccca cacgagttct gctcacaaca tactatgctt gtaaagcaag gtgatgacta 3901 cgtctatctt ccttatcctg acccaagccg catcctggga gctggttgtt tcgttgacga 3961 tatcgtgaag accgatggca ccctgatgat tgagcgtttc gtctctctgg cgattgacgc 4021 gtatccgtta actaagcacc ccaatcaaga atacgcggac gtttttcact tatacttaca 4081 gtatatccgt aagttacacg atgagctgac cggtcacatg cttgatatgt acagcgttat 4141 gctgacgaat gacaacactt cgcgttattg ggagcctgag ttttatgaag cgatgtatac 4201 acctcacaca gttctgcagt aagcttgcgg ccgcactcga gtctggtaaa gaaaccgctg 4261 ctgcgaaatt tgaacgccag cacatggact cgtctactag cgcagcttaa ttaacctagg 4321 ctgctgccac cgctgagcaa taactagcat aaccccttgg ggcctctaaa cgggtcttga 4381 ggggtttttt gctgaaacct caggcatttg agaagcacac ggtcacactg cttccggtag 4441 tcaataaacc ggtaaaccag caatagacat aagcggctat ttaacgaccc tgccctgaac 4501 cgacgacaag ctgacgaccg ggtctccgca agtggcactt ttcggggaaa tgtgcgcgga 4561 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gaattaattc 4621 ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta ttcatatcag gattatcaat 4681 accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga ggcagttcca 4741 taggatggca agatcctggt atcggtctgc gattccgact cgtccaacat caatacaacc 4801 tattaatttc ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat gagtgacgac 4861 tgaatccggt gagaatggca aaagtttatg catttctttc cagacttgtt caacaggcca 4921 gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa ccgttattca ttcgtgattg 4981 cgcctgagcg agacgaaata cgcggtcgct gttaaaagga caattacaaa caggaatcga 5041 atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata ttttcacctg aatcaggata 5101 ttcttctaat acctggaatg ctgttttccc ggggatcgca gtggtgagta accatgcatc 5161 atcaggagta cggataaaat gcttgatggt cggaagaggc ataaattccg tcagccagtt 5221 tagtctgacc atctcatctg taacatcatt ggcaacgcta cctttgccat gtttcagaaa 5281 caactctggc gcatcgggct tcccatacaa tcgatagatt gtcgcacctg attgcccgac 5341 attatcgcga gcccatttat acccatataa atcagcatcc atgttggaat ttaatcgcgg 5401 cctagagcaa gacgtttccc gttgaatatg gctcatactc ttcctttttc aatattattg 5461 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa 5521 taaacaaata ggcatgcagc gctcttccgc ttcctcgctc actgactcgc tacgctcggt 5581 cgttcgactg cggcgagcgg tgtcagctca ctcaaaagcg gtaatacggt tatccacaga 5641 atcaggggat aaagccggaa agaacatgtg agcaaaaagc aaagcaccgg aagaagccaa 5701 cgccgcaggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct 5761 caagccagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa 5821 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc 5881 tcccttcggg aagcgtggcg ctttctcata gctcacgctg ttggtatctc agttcggtgt 5941 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg 6001 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg 6061 cagcagccat tggtaactga tttagaggac tttgtcttga agttatgcac ctgttaaggc 6121 taaactgaaa gaacagattt tggtgagtgc ggtcctccaa cccacttacc ttggttcaaa 6181 gagttggtag ctcagcgaac cttgagaaaa ccaccgttgg tagcggtggt ttttctttat 6241 ttatgagatg atgaatcaat cggtctatca agtcaacgaa cagctattcc gttactctag 6301 atttcagtgc aatttatctc ttcaaatgta gcacctgaag tcagccccat acgatataag 6361 ttgtaattct catgttagtc atgccccgcg cccaccggaa ggagctgact gggttgaagg 6421 ctctcaaggg catcggtcga gatcccggtg cctaatgagt gagctaactt acattaattg 6481 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa 6541 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ccagggtggt ttttcttttc 6601 accagtgaga cgggcaacag ctgattgccc ttcaccgcct ggccctgaga gagttgcagc 6661 aagcggtcca cgctggtttg ccccagcagg cgaaaatcct gtttgatggt ggttaacggc 6721 g //