LOCUS Exported 6224 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION mammalian expression and localization. ACCESSION . VERSION . KEYWORDS Nsp4-EGFP SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6224) 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 6224) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6224 /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" misc_feature 1740..1792 /label=MCS /note="multiple cloning site" regulatory 1800..1809 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 1806..2525 /codon_start=1 /product="the original enhanced GFP (Yang et al., 1996)" /label=EGFP /note="mammalian codon-optimized" /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" CDS 1806..2522 /codon_start=1 /product="enhanced GFP" /label=EGFP /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" primer_bind complement(1851..1872) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(2112..2131) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 2459..2480 /label=EGFP-C /note="EGFP, forward primer" polyA_signal 2648..2769 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(2685..2704) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 2739..2758 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" rep_origin complement(2776..3231) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(2913..2934) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 3125..3144 /label=F1ori-R /note="F1 origin, reverse primer" promoter 3258..3362 /gene="bla" /label=AmpR promoter promoter 3364..3721 /label=SV40 promoter /note="SV40 enhancer and early promoter" primer_bind complement(3387..3407) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" rep_origin 3572..3707 /label=SV40 ori /note="SV40 origin of replication" primer_bind 3634..3653 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" CDS 3756..4550 /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(3810..3829) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 4420..4439 /label=Neo-F /note="Neomycin resistance gene, forward primer" primer_bind complement(4738..4757) /label=TK-pA-R /note="Thymidine kinase polyA, reverse primer" polyA_signal 4782..4829 /label=HSV TK poly(A) signal /note="herpes simplex virus thymidine kinase polyadenylation signal (Cole and Stacy, 1985)" rep_origin 5158..5746 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 5647..5666 /label=pBR322ori-F /note="pBR322 origin, forward primer" enhancer 5921..6224 /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 gccaccatga 241 agattgtaaa caattggctt aaacaactta ttaaagtaac cctggtattc ttgtttgtcg 301 ccgcaatctt ttacttgata acaccggtcc acgtgatgtc taaacacaca gactttagta 361 gtgagataat cggatataaa gctatcgatg gcggcgttac ccgcgacatc gcttcaaccg 421 acacatgttt cgctaataag cacgccgatt ttgacacttg gtttagtcaa agaggaggaa 481 gctacacaaa cgacaaggct tgtccgctta tcgcggctgt aataacaagg gaagttgggt 541 ttgtcgtgcc tggtttgcca ggtactatac tcagaaccac aaacggtgac tttctgcact 601 tcttgccaag ggtttttagc gcggtaggca atatctgcta tacgccaagc aagctgattg 661 aatacaccga ctttgcgacg agcgcatgtg tcctcgctgc agagtgtacg atatttaagg 721 atgcgagcgg aaagcccgta ccttactgct atgatactaa tgtccttgag ggatcagttg 781 cgtatgagag tcttaggccg gatacacgat atgtgctcat ggatggttcc ataatccagt 841 tcccaaatac ctacctcgaa gggtccgttc gggtcgtcac cacatttgat agcgaatatt 901 gcagacacgg aacatgcgaa cggagtgaag caggggtctg cgtctccaca agtgggcggt 961 gggtcctcaa taacgattat taccggagcc tgcccggcgt gttttgcggg gtcgatgcgg 1021 ttaacttgtt gacgaatatg tttacgcctc tcatacaacc cattggcgca ctggatatat 1081 ctgcaagtat agtcgctgga ggcattgtgg caatcgtggt tacgtgtttg gcgtattatt 1141 ttatgaggtt ccgccgggct ttcggggaat attcccacgt agtcgccttc aacacactct 1201 tgtttctcat gtctttcact gtactgtgtt tgactcctgt ctactctttc ttgccgggtg 1261 tatactcagt tatttacttg tacctcacgt tttatttgac aaacgatgtt tcatttctcg 1321 cgcatatcca atggatggtg atgtttacgc cattggtgcc tttctggatc acgatcgctt 1381 atatcatttg catcagcacc aaacacttct attggttctt tagtaactac ttgaagcgca 1441 gggttgtttt taacggtgtc agcttctcaa cgtttgaaga ggcagcactg tgtactttcc 1501 ttctgaataa ggaaatgtat ctcaagctgc ggtccgatgt cctgcttccg cttacccagt 1561 ataatcgcta tctcgccctg tataataagt acaagtactt ctcaggcgcg atggatacaa 1621 ccagttatag agaggctgcc tgctgccatc ttgccaaagc tttgaatgac ttcagcaata 1681 gtggctcaga cgttctctac caaccaccgc aaacttccat aacctcagcc gtactccagc 1741 tcgagctcaa gcttcgaatt ctgcagtcga cggtaccgcg ggcccgggat ccaccggtcg 1801 ccaccatggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc 1861 tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca 1921 cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc 1981 ccaccctcgt gaccaccctg acctacggcg tgcagtgctt cagccgctac cccgaccaca 2041 tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca 2101 tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca 2161 ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg 2221 ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcatggcc gacaagcaga 2281 agaacggcat caaggtgaac ttcaagatcc gccacaacat cgaggacggc agcgtgcagc 2341 tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca 2401 accactacct gagcacccag tccgccctga gcaaagaccc caacgagaag cgcgatcaca 2461 tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtaca 2521 agtaaagcgg ccgcgactct agatcataat cagccatacc acatttgtag aggttttact 2581 tgctttaaaa aacctcccac acctccccct gaacctgaaa cataaaatga atgcaattgt 2641 tgttgttaac ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa 2701 tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa 2761 tgtatcttaa ggcgtaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt 2821 gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa 2881 aagaatagac cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa 2941 agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac 3001 gtgaaccatc accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga 3061 accctaaagg gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa 3121 aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc 3181 tgcgcgtaac caccacaccc gccgcgctta atgcgccgct acagggcgcg tcaggtggca 3241 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata 3301 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga 3361 gtcctgaggc ggaaagaacc agctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc 3421 aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg 3481 tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc 3541 agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc 3601 ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc 3661 ggcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa 3721 agatcgatca agagacagga tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg 3781 caggttctcc ggccgcttgg gtggagaggc tattcggcta tgactgggca caacagacaa 3841 tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg gttctttttg 3901 tcaagaccga cctgtccggt gccctgaatg aactgcaaga cgaggcagcg cggctatcgt 3961 ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa 4021 gggactggct gctattgggc gaagtgccgg ggcaggatct cctgtcatct caccttgctc 4081 ctgccgagaa agtatccatc atggctgatg caatgcggcg gctgcatacg cttgatccgg 4141 ctacctgccc attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg 4201 aagccggtct tgtcgatcag gatgatctgg acgaagagca tcaggggctc gcgccagccg 4261 aactgttcgc caggctcaag gcgagcatgc ccgacggcga ggatctcgtc gtgacccatg 4321 gcgatgcctg cttgccgaat atcatggtgg aaaatggccg cttttctgga ttcatcgact 4381 gtggccggct gggtgtggcg gaccgctatc aggacatagc gttggctacc cgtgatattg 4441 ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt gctttacggt atcgccgctc 4501 ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga gcgggactct 4561 ggggttcgaa atgaccgacc aagcgacgcc caacctgcca tcacgagatt tcgattccac 4621 cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat 4681 cctccagcgc ggggatctca tgctggagtt cttcgcccac cctaggggga ggctaactga 4741 aacacggaag gagacaatac cggaaggaac ccgcgctatg acggcaataa aaagacagaa 4801 taaaacgcac ggtgttgggt cgtttgttca taaacgcggg gttcggtccc agggctggca 4861 ctctgtcgat accccaccga gaccccattg gggccaatac gcccgcgttt cttccttttc 4921 cccaccccac cccccaagtt cgggtgaagg cccagggctc gcagccaacg tcggggcggc 4981 aggccctgcc atagcctcag gttactcata tatactttag attgatttaa aacttcattt 5041 ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta 5101 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg 5161 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc 5221 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag 5281 cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa 5341 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc 5401 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc 5461 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta 5521 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag 5581 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct 5641 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga 5701 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc 5761 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt 5821 atcccctgat tctgtggata accgtattac cgccatgcat tagttattaa tagtaatcaa 5881 ttacggggtc attagttcat agcccatata tggagttccg cgttacataa cttacggtaa 5941 atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg 6001 ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt 6061 aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg 6121 tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc 6181 ctacttggca gtacatctac gtattagtca tcgctattac catg //