LOCUS Exported 7760 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Expresses inactive Mpro mutant and a near-infrared fluorescent protein mIFP fused to H2B. ACCESSION . VERSION . KEYWORDS pcDNA3 H2B-mIFP T2A Mpro (C145A) SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7760) TITLE FlipGFP-based activity reporter of SARS-CoV-2 main protease Mpro JOURNAL Unpublished REFERENCE 2 (bases 1 to 7760) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7760 /organism="synthetic DNA construct" /mol_type="other DNA" enhancer 1..380 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 381..584 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 535..555 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" primer_bind 629..648 /label=T7 /note="T7 promoter, forward primer" promoter 629..647 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" CDS 695..1072 /codon_start=1 /gene="HIST1H2BJ" /product="human histone H2B" /label=H2B /translation="MPEPAKSAPAPKKGSKKAVTKAQKKGGKKRKRSRKESYSIYVYKV LKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLP GELAKHAVSEGTKAITKYTSAK" CDS 1142..2101 /codon_start=1 /product="naturally monomeric infrared fluorescent protein derived from a truncated bacteriophytochrome (Yu et al., 2015)" /label=mIFP /translation="MSVPLTTSAFGHAFLANCEREQIHLAGSIQPHGILLAVKEPDNVV IQASINAAEFLNTNSVVGRPLRDLGGDLPLQILPHLNGPLHLAPMTLRCTVGSPPRRVD CTIHRPSNGGLIVELEPATKTTNIAPALDGAFHRITSSSSLMGLCDETATIIREITGYD RVMVVRFDEEGNGEILSERRRADLEAFLGNRYPASTIPQIARRLYEHNRVRLLVDVNYT PVPLQPRISPLNGRDLDMSLSCLRSMSPIHQKYMQDMGVGATLVCSLMVSGRLWGLIAC HHYEPRFVPFHIRAAGEALAETCAIRIATLESFAQSQSK" CDS 2108..2161 /codon_start=1 /product="2A peptide from Thosea asigna virus capsid protein" /label=T2A /note="Eukaryotic ribosomes fail to insert a peptide bond between the Gly and Pro residues, yielding separate polypeptides." /translation="EGRGSLLTCGDVEENPGP" primer_bind complement(3117..3134) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 3123..3347 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" rep_origin 3393..3821 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(3480..3499) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 3690..3711 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(3830..3850) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 3835..4164 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 4015..4150 /label=SV40 ori /note="SV40 origin of replication" primer_bind 4077..4096 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" CDS 4231..5025 /codon_start=1 /gene="aph(3')-II (or nptII)" /product="aminoglycoside phosphotransferase from Tn5" /label=NeoR/KanR /note="confers resistance to neomycin, kanamycin, and G418 (Geneticin(R))" /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF" primer_bind complement(4285..4304) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 4895..4914 /label=Neo-F /note="Neomycin resistance gene, forward primer" polyA_signal 5199..5320 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(5236..5255) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 5290..5309 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(5369..5385) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(5369..5385) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(5382..5404) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 5393..5409 /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)." promoter complement(5417..5447) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 5462..5483 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(5600..5617) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(5771..6359) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(5851..5870) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(6530..7390) /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind 7153..7172 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(7391..7495) /gene="bla" /label=AmpR promoter primer_bind complement(7570..7589) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" ORIGIN 1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc 61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca 121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga 181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc 241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct 301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat 361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc 421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt 481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa 541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg ctaactagag 601 aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag acccaagctg 661 gctagcgttt aaacttaagc ttggtaccgc caccatgcca gagccagcga agtctgctcc 721 cgccccgaaa aagggctcca agaaggcggt gactaaggcg cagaagaaag gcggcaagaa 781 gcgcaagcgc agccgcaagg agagctattc catctatgtg tacaaggttc tgaagcaggt 841 ccaccctgac accggcattt cgtccaaggc catgggcatc atgaattcgt ttgtgaacga 901 cattttcgag cgcatcgcag gtgaggcttc ccgcctggcg cattacaaca agcgctcgac 961 catcacctcc agggagatcc agacggccgt gcgcctgctg ctgcctgggg agttggccaa 1021 gcacgccgtg tccgagggta ctaaggccat caccaagtac accagcgcta agtctggtgg 1081 cgggagtggt ggcgggtccc tcgagtccgg actcagatct ggcagcggtg gaggcgatat 1141 catgtcggta ccgctgacta cctcagcatt cggccacgcg tttctggcta actgtgaacg 1201 cgagcagatc cacctggcgg gctccattca gccgcacggt atcctgctgg ctgtgaaaga 1261 gccggacaac gtggtgatcc aggcttctat taacgctgcg gagttcctga acaccaactc 1321 tgttgttggc cgtccgctgc gtgacctggg cggcgatctg cctttgcaga tcctgccgca 1381 cctgaacggc ccgctgcacc tggctccgat gaccctgcgt tgtaccgtgg gttctccgcc 1441 gcgtcgtgtg gactgtacca ttcatcgtcc gtctaacggc ggcctgatcg tagaactgga 1501 accagcaacc aagaccacta acattgcgcc ggctctggac ggtgcgtttc atcgtatcac 1561 ttcttcatcc tccctgatgg gcctgtgtga cgaaaccgcg actattatcc gtgagattac 1621 tggctacgac cgtgtgatgg tagtacgttt cgatgaagag ggtaatggcg aaattctgtc 1681 cgaacgtcgt cgtgcggacc tggaagcgtt cctgggtaac cgctacccgg cgtctactat 1741 tccgcagatc gctcgtcgcc tgtacgaaca taaccgtgtt cgcctgctgg tagatgtgaa 1801 ctatactccg gttccgctac agccgcgcat cagcccgctg aacggtcgtg atctggatat 1861 gtccctgtct tgcctgcgct ctatgtcccc gatccaccag aaatacatgc aggacatggg 1921 cgttggcgcg accctggttt gctctctgat ggtgtctggt cgtctgtggg gtctgatcgc 1981 ttgccaccac tacgaaccgc gcttcgttcc gttccacatt cgcgctgctg gcgaagcgct 2041 ggcggaaact tgtgcgatcc gcatcgcgac gctggagagc tttgcacagt ctcagtccaa 2101 aggatccgag ggcagaggaa gtctgctaac atgcggtgac gtcgaggaga atcctggccc 2161 aaccgggatg tctggtttta ggaaaatggc gttccccagc ggtaaagttg aaggatgtat 2221 ggtccaagta acctgtggta ccactaccct taatgggctt tggttggacg acgtagtcta 2281 ctgcccccga cacgtaatct gcactagtga ggatatgctt aatcccaatt acgaagacct 2341 tttgattcgg aaatccaatc acaacttcct ggtccaagcg ggcaacgtcc aactcagggt 2401 tattggacat agtatgcaga attgcgtact gaagctcaaa gtcgatactg caaaccccaa 2461 gacgcccaag tataaattcg tccgaatcca accaggccaa acattttccg tattggcttg 2521 ctataatgga agccccagcg gtgtctacca atgtgcaatg agaccaaact ttacgataaa 2581 gggttcattt ctgaacggct ctgccggttc cgttggtttt aacatcgact atgactgtgt 2641 atccttttgc tacatgcacc atatggaact ccctaccggt gtccacgccg gtacagatct 2701 ggaaggaaat ttctacggtc cgttcgttga ccggcaaacc gcgcaagcgg ctggaaccga 2761 cacaacgatt acagtgaatg tgctcgcgtg gctgtacgca gcagtcataa acggagacag 2821 gtggtttctg aaccgattta cgacgactct caatgacttc aaccttgttg cgatgaagta 2881 caattacgag ccactcaccc aggaccatgt tgatatcctg ggtcccctca gtgcccagac 2941 agggatcgca gttctcgata tgtgcgcgtc actgaaggag cttctccaaa atggaatgaa 3001 tgggcggacc atacttggtt ccgcactcct cgaagatgaa tttactccat ttgacgtggt 3061 cagacaatgc agtggggtca ctttccagta agggcccgtt taaacccgct gatcagcctc 3121 gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac 3181 cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg 3241 tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga 3301 ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatggctt ctgaggcgga 3361 aagaaccagc tggggctcta gggggtatcc ccacgcgccc tgtagcggcg cattaagcgc 3421 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc 3481 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct 3541 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa 3601 acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc 3661 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact 3721 caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg 3781 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat taattctgtg gaatgtgtgt 3841 cagttagggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat 3901 ctcaattagt cagcaaccag gtgtggaaag tccccaggct ccccagcagg cagaagtatg 3961 caaagcatgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg 4021 cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt 4081 tatgcagagg ccgaggccgc ctctgcctct gagctattcc agaagtagtg aggaggcttt 4141 tttggaggcc taggcttttg caaaaagctc ccgggagctt gtatatccat tttcggatct 4201 gatcaagaga caggatgagg atcgtttcgc atgattgaac aagatggatt gcacgcaggt 4261 tctccggccg cttgggtgga gaggctattc ggctatgact gggcacaaca gacaatcggc 4321 tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct ttttgtcaag 4381 accgacctgt ccggtgccct gaatgaactg caggacgagg cagcgcggct atcgtggctg 4441 gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc gggaagggac 4501 tggctgctat tgggcgaagt gccggggcag gatctcctgt catctcacct tgctcctgcc 4561 gagaaagtat ccatcatggc tgatgcaatg cggcggctgc atacgcttga tccggctacc 4621 tgcccattcg accaccaagc gaaacatcgc atcgagcgag cacgtactcg gatggaagcc 4681 ggtcttgtcg atcaggatga tctggacgaa gagcatcagg ggctcgcgcc agccgaactg 4741 ttcgccaggc tcaaggcgcg catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat 4801 gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt ctggattcat cgactgtggc 4861 cggctgggtg tggcggaccg ctatcaggac atagcgttgg ctacccgtga tattgctgaa 4921 gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat 4981 tcgcagcgca tcgccttcta tcgccttctt gacgagttct tctgagcggg actctggggt 5041 tcgaaatgac cgaccaagcg acgcccaacc tgccatcacg agatttcgat tccaccgccg 5101 ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg atgatcctcc 5161 agcgcgggga tctcatgctg gagttcttcg cccaccccaa cttgtttatt gcagcttata 5221 atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc 5281 attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgt ataccgtcga 5341 cctctagcta gagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc 5401 cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct 5461 aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa 5521 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta 5581 ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc 5641 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg 5701 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt 5761 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa 5821 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct 5881 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc 5941 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg 6001 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct 6061 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag 6121 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga 6181 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga 6241 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg 6301 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag 6361 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag 6421 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat 6481 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct 6541 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac 6601 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa 6661 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg 6721 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt 6781 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca 6841 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt 6901 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct 6961 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg 7021 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg 7081 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg 7141 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa 7201 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt 7261 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt 7321 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt 7381 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca 7441 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat 7501 ttccccgaaa agtgccacct gacgtcgacg gatcgggaga tctcccgatc ccctatggtg 7561 cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatctgc tccctgcttg 7621 tgtgttggag gtcgctgagt agtgcgcgag caaaatttaa gctacaacaa ggcaaggctt 7681 gaccgacaat tgcatgaaga atctgcttag ggttaggcgt tttgcgctgc ttcgcgatgt 7741 acgggccaga tatacgcgtt //