LOCUS Exported 7509 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION mammalian expression and localization. ACCESSION . VERSION . KEYWORDS EGFP-Nsp12 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7509) AUTHORS Miserey-Lenkei S, Trajkovic K, D'Ambrosio JM, Patel AJ, Copic A, Mathur P, Schauer K, Goud b, Albanese V, Gautier R, Subra M, Kovacs D, Barelli H, Antonny B TITLE A comprehensive library of fluorescent constructs of SARS-CoV-2 proteins and their initial characterization in different cell types JOURNAL bioRxiv 423586 REFERENCE 2 (bases 1 to 7509) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7509 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 1..204 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 155..175 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" regulatory 243..252 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 249..965 /codon_start=1 /product="enhanced GFP" /label=EGFP /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" CDS 249..965 /codon_start=1 /product="the original enhanced GFP (Yang et al., 1996)" /label=EGFP /note="mammalian codon-optimized" /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" primer_bind complement(294..315) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(555..574) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 902..923 /label=EGFP-C /note="EGFP, forward primer" polyA_signal 3933..4054 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(3970..3989) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 4024..4043 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" rep_origin complement(4061..4516) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(4198..4219) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 4410..4429 /label=F1ori-R /note="F1 origin, reverse primer" promoter 4543..4647 /gene="bla" /label=AmpR promoter promoter 4649..5006 /label=SV40 promoter /note="SV40 enhancer and early promoter" primer_bind complement(4672..4692) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" rep_origin 4857..4992 /label=SV40 ori /note="SV40 origin of replication" primer_bind 4919..4938 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" CDS 5041..5835 /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 GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF" primer_bind complement(5095..5114) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 5705..5724 /label=Neo-F /note="Neomycin resistance gene, forward primer" primer_bind complement(6023..6042) /label=TK-pA-R /note="Thymidine kinase polyA, reverse primer" polyA_signal 6067..6114 /label=HSV TK poly(A) signal /note="herpes simplex virus thymidine kinase polyadenylation signal (Cole and Stacy, 1985)" rep_origin 6443..7031 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 6932..6951 /label=pBR322ori-F /note="pBR322 origin, forward primer" enhancer 7206..7509 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" ORIGIN 1 gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt 61 ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac 121 tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg 181 tgggaggtct atataagcag agctggttta gtgaaccgtc agatccgcta gcgctaccgg 241 tcgccaccat ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg 301 agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg 361 ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct 421 ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc taccccgacc 481 acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca 541 ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg 601 acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc 661 tggggcacaa gctggagtac aactacaaca gccacaacgt ctatatcatg gccgacaagc 721 agaagaacgg catcaaggtg aacttcaaga tccgccacaa catcgaggac ggcagcgtgc 781 agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg 841 acaaccacta cctgagcacc cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc 901 acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg gacgagctgt 961 acaagtccgg actcagatct cgagctcaag cttcgaattc tatgtcagca gacgcacaaa 1021 gttttctgaa ccgggtatgc ggagtcagtg cggcgaggct tacgccctgt ggtacgggaa 1081 catcaacgga cgttgtttac cgcgccttcg acatatacaa tgataaagtc gccggcttcg 1141 caaagttcct taaaacgaat tgctgtaggt tccaagaaaa ggacgaggat gataatttga 1201 tagactccta cttcgtggtc aaacgacaca cgttttcaaa ttatcagcac gaggaaacga 1261 tatataatct tcttaaagac tgccctgcag tggcaaaaca cgacttcttt aagttccgca 1321 ttgacgggga tatggtcccc catatctccc gacagagatt gacgaaatat acaatggcgg 1381 atctggtgta cgccctgaga cacttcgatg agggcaattg tgatacactc aaggagattc 1441 tcgtgaccta caattgttgc gatgatgact atttcaataa gaaagactgg tacgacttcg 1501 tcgaaaaccc tgatatattg agggtgtacg caaacctggg tgaacgagta agacaggcac 1561 ttctgaaaac cgtccaattc tgcgacgcta tgagaaacgc gggcattgtt ggtgtgctta 1621 cgctggataa ccaagacctc aatggtaatt ggtacgattt tggggacttc atccaaacca 1681 ctcctggctc aggcgttccc gtggtagaca gctactattc tttgttgatg ccaattctta 1741 cactcacaag ggccctgacc gctgagtcac acgtcgatac ggacttgacc aagccctaca 1801 tcaagtggga tcttctgaag tacgacttta ccgaggaacg attgaagctt tttgatcgct 1861 actttaaata ctgggatcag acgtaccacc caaactgcgt gaattgcctg gatgaccgat 1921 gtatactcca ttgcgcgaac tttaacgttt tgttctctac agtatttcct ccgacgagct 1981 tcggaccgct tgtgagaaag atattcgtcg acggcgtccc atttgttgtc agcactggtt 2041 accactttcg cgagttgggg gtggtccaca atcaggatgt caatttgcat tcctccaggc 2101 tttcttttaa agagttgctt gtttatgccg ctgaccccgc gatgcatgca gcctctggga 2161 atcttttgct ggacaaaagg acgacgtgct ttagcgttgc tgcccttaca aataacgtag 2221 catttcaaac cgtaaaaccc ggtaatttta ataaagattt ttatgatttt gcagtctcta 2281 agggtttttt caaggagggg agtagtgtag agcttaaaca tttttttttc gctcaggatg 2341 gtaacgctgc gatcagcgac tacgactact atcgatacaa tctcccgacg atgtgtgata 2401 tcaggcagtt gttgttcgtt gtagaagtgg ttgataagta ttttgactgt tatgacggcg 2461 gctgtatcaa tgccaaccag gtcattgtga ataacctcga taaatctgcc gggttcccct 2521 ttaacaagtg ggggaaggcg cgactgtact acgacagcat gtcttacgaa gatcaggacg 2581 cactctttgc atacacaaag aggaatgtga taccgactat aactcaaatg aaccttaagt 2641 atgcaatcag cgcgaaaaat cgggctagga ctgttgccgg agttagcata tgtagtacca 2701 tgacaaaccg acagttccac cagaagttgc tgaaatcaat tgctgcgacc cggggtgcga 2761 ctgtagtaat cgggacatca aagttctacg gcgggtggca taacatgctc aagacggtct 2821 actcagacgt tgagaatccg catcttatgg gttgggatta tccaaagtgt gacagggcga 2881 tgcccaacat gttgcggata atggcctcct tggtacttgc acggaaacat acaacctgct 2941 gtagtttgtc acaccgattt taccgactgg caaacgaatg tgctcaggtt ctcagtgaaa 3001 tggtaatgtg tggcggctcc ctctacgtta agcccggtgg cacaagctct ggagacgcaa 3061 ctactgccta tgcaaacagt gtattcaaca tctgccaggc tgtcacggcc aatgttaacg 3121 cattgctttc cactgacgga aataagattg cagacaaata cgtgagaaat ttgcaacata 3181 gactctacga atgcctctat cgcaatcgcg atgttgatac ggacttcgtt aatgagttct 3241 atgcttactt gcgaaagcac ttctccatga tgattctgtc agacgacgct gtagtatgct 3301 ttaattcaac gtatgcctcc cagggtctgg tcgcgtccat aaaaaatttt aaaagcgtcc 3361 tgtattacca aaataacgtt ttcatgagcg aagcgaaatg ttggaccgag actgatctta 3421 ctaagggccc tcatgagttc tgctcacaac acactatgtt ggtcaagcag ggcgacgatt 3481 acgtgtatct tccttatccc gatcctagta ggattcttgg agcaggttgc ttcgtggatg 3541 atatcgtcaa aacggacggc accctcatga tagagaggtt cgtctctttg gctatagacg 3601 cgtatccgct tactaaacat cctaaccaag aatacgcgga tgtattccat ctttatctgc 3661 agtatatcag aaagttgcat gatgagctga ccggtcacat gttggacatg tatagcgtga 3721 tgcttaccaa tgacaatact tcacggtact gggaacctga attttacgag gcgatgtata 3781 cgcctcacac cgtcctgcag taaggatcca ccggatctag ataactgatc ataatcagcc 3841 ataccacatt tgtagaggtt ttacttgctt taaaaaacct cccacacctc cccctgaacc 3901 tgaaacataa aatgaatgca attgttgttg ttaacttgtt tattgcagct tataatggtt 3961 acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta 4021 gttgtggttt gtccaaactc atcaatgtat cttaacgcgt aaattgtaag cgttaatatt 4081 ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa 4141 atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca 4201 gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc 4261 gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg 4321 aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg 4381 ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg 4441 gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg 4501 ccgctacagg gcgcgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt 4561 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg 4621 cttcaataat attgaaaaag gaagagtcct gaggcggaaa gaaccagctg tggaatgtgt 4681 gtcagttagg gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc 4741 atctcaatta gtcagcaacc aggtgtggaa agtccccagg ctccccagca ggcagaagta 4801 tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc 4861 cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta 4921 tttatgcaga ggccgaggcc gcctcggcct ctgagctatt ccagaagtag tgaggaggct 4981 tttttggagg cctaggcttt tgcaaagatc gatcaagaga caggatgagg atcgtttcgc 5041 atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc 5101 ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca 5161 gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg 5221 caagacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg 5281 ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag 5341 gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg 5401 cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc 5461 atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa 5521 gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgag catgcccgac 5581 ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat 5641 ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac 5701 atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc 5761 ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt 5821 gacgagttct tctgagcggg actctggggt tcgaaatgac cgaccaagcg acgcccaacc 5881 tgccatcacg agatttcgat tccaccgccg ccttctatga aaggttgggc ttcggaatcg 5941 ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg 6001 cccaccctag ggggaggcta actgaaacac ggaaggagac aataccggaa ggaacccgcg 6061 ctatgacggc aataaaaaga cagaataaaa cgcacggtgt tgggtcgttt gttcataaac 6121 gcggggttcg gtcccagggc tggcactctg tcgatacccc accgagaccc cattggggcc 6181 aatacgcccg cgtttcttcc ttttccccac cccacccccc aagttcgggt gaaggcccag 6241 ggctcgcagc caacgtcggg gcggcaggcc ctgccatagc ctcaggttac tcatatatac 6301 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg 6361 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg 6421 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc 6481 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc 6541 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt 6601 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc 6661 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact 6721 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac 6781 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag 6841 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg 6901 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg 6961 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga 7021 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt 7081 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcca 7141 tgcattagtt attaatagta atcaattacg gggtcattag ttcatagccc atatatggag 7201 ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc 7261 ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac tttccattga 7321 cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat 7381 atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc 7441 cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct 7501 attaccatg //