LOCUS Exported 6242 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION mammalian expression and localization. ACCESSION . VERSION . KEYWORDS Nsp14D-EGFP SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6242) 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 6242) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6242 /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 1758..1810 /label=MCS /note="multiple cloning site" regulatory 1818..1827 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 1824..2543 /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 1824..2540 /codon_start=1 /product="enhanced GFP" /label=EGFP /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" primer_bind complement(1869..1890) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(2130..2149) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 2477..2498 /label=EGFP-C /note="EGFP, forward primer" polyA_signal 2666..2787 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(2703..2722) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 2757..2776 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" rep_origin complement(2794..3249) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(2931..2952) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 3143..3162 /label=F1ori-R /note="F1 origin, reverse primer" promoter 3276..3380 /gene="bla" /label=AmpR promoter promoter 3382..3739 /label=SV40 promoter /note="SV40 enhancer and early promoter" primer_bind complement(3405..3425) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" rep_origin 3590..3725 /label=SV40 ori /note="SV40 origin of replication" primer_bind 3652..3671 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" CDS 3774..4568 /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(3828..3847) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 4438..4457 /label=Neo-F /note="Neomycin resistance gene, forward primer" primer_bind complement(4756..4775) /label=TK-pA-R /note="Thymidine kinase polyA, reverse primer" polyA_signal 4800..4847 /label=HSV TK poly(A) signal /note="herpes simplex virus thymidine kinase polyadenylation signal (Cole and Stacy, 1985)" rep_origin 5176..5764 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 5665..5684 /label=pBR322ori-F /note="pBR322 origin, forward primer" enhancer 5939..6242 /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 gccaccatgc 241 aagcacctac ccacctttcc gtagatacga agttcaaaac ggaaggattg tgtgtggata 301 taccagggat accaaaggat atgacgtacc gaaggctgat ttccatgatg ggttttaaga 361 tgaattacca agttaatggc taccccaaca tgttcatcac cagggaggag gcaattagac 421 acgtaagagc ctggataggc ttcgatgttg aaggttgcca tgcaacccgg gaagccgtag 481 gcacaaacct tccgttgcag cttggctttt ccacgggcgt caacctcgtt gccgtaccga 541 ctggctatgt tgacacgccg aacaacaccg atttctctcg cgtaagtgct aagcctcctc 601 cgggagatca attcaagcat cttatacctc tcatgtacaa gggactgccg tggaatgtag 661 ttcgcattaa gatcgtgcag atgctctccg atactttgaa aaatctgagc gacagggtgg 721 tcttcgttct gtgggcacat ggcttcgaac ttacttctat gaaatacttt gtaaaaatcg 781 ggcccgagcg gacatgttgt ttgtgcgacc gacgagctac ttgttttagc acagcatctg 841 acacctacgc atgctggcac cacagtatag gcttcgatta cgtctacaat ccctttatga 901 tagacgtaca acaatggggc tttacgggta acttgcagag taatcacgat ctttactgcc 961 aagttcatgg gaacgcacac gtggcctcct gcgatgcgat aatgacgagg tgcttggccg 1021 tgcacgagtg ctttgttaag cgggtcgatt ggactataga gtatcccata atcggtgacg 1081 aacttaaaat taatgctgca tgcaggaaag tgcagcacat ggtagtaaaa gcagcgctcc 1141 tcgcggataa gtttcctgtg ctccacgata tcggcaaccc taaggccatt aaatgcgtcc 1201 cccaagctga tgtagagtgg aaattctatg acgctcagcc atgtagtgac aaagcataca 1261 agattgaaga attgttttat tcatatgcga cacactccga taagttcact gacggagttt 1321 gtctcttttg gaactgtaat gttgataggt atccggcgaa tagcatagtg tgtcgatttg 1381 acacgcgcgt tctctctaat ttgaacctcc ctggctgcga cggggggtct ttgtacgtaa 1441 acaagcatgc atttcatacc ccagcttttg acaaatccgc tttcgtaaat ctgaaacaac 1501 ttcccttctt ctattattca gatagccctt gcgaatctca cggtaagcaa gtggttagtg 1561 acattgacta tgtaccactt aaaagtgcaa cgtgcattac acgatgcaat ttggggggcg 1621 cggtttgccg acaccatgcc aacgaataca gattgtacct ggatgcatat aacatgatga 1681 taagtgcagg gtttagtctt tgggtgtata agcagttcga tacgtataat ctctggaaca 1741 catttacccg attgcagctc gagctcaagc ttcgaattct gcagtcgacg gtaccgcggg 1801 cccgggatcc accggtcgcc accatggtga gcaagggcga ggagctgttc accggggtgg 1861 tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc gtgtccggcg 1921 agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca 1981 agctgcccgt gccctggccc accctcgtga ccaccctgac ctacggcgtg cagtgcttca 2041 gccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct 2101 acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg 2161 tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg 2221 aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac aacgtctata 2281 tcatggccga caagcagaag aacggcatca aggtgaactt caagatccgc cacaacatcg 2341 aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc 2401 ccgtgctgct gcccgacaac cactacctga gcacccagtc cgccctgagc aaagacccca 2461 acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg atcactctcg 2521 gcatggacga gctgtacaag taaagcggcc gcgactctag atcataatca gccataccac 2581 atttgtagag gttttacttg ctttaaaaaa cctcccacac ctccccctga acctgaaaca 2641 taaaatgaat gcaattgttg ttgttaactt gtttattgca gcttataatg gttacaaata 2701 aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg 2761 tttgtccaaa ctcatcaatg tatcttaagg cgtaaattgt aagcgttaat attttgttaa 2821 aattcgcgtt aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca 2881 aaatccctta taaatcaaaa gaatagaccg agatagggtt gagtgttgtt ccagtttgga 2941 acaagagtcc actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc 3001 agggcgatgg cccactacgt gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc 3061 gtaaagcact aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc 3121 cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg 3181 caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac 3241 agggcgcgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt 3301 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat 3361 aatattgaaa aaggaagagt cctgaggcgg aaagaaccag ctgtggaatg tgtgtcagtt 3421 agggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa 3481 ttagtcagca accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag 3541 catgcatctc aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct 3601 aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc 3661 agaggccgag gccgcctcgg cctctgagct attccagaag tagtgaggag gcttttttgg 3721 aggcctaggc ttttgcaaag atcgatcaag agacaggatg aggatcgttt cgcatgattg 3781 aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg 3841 actgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg 3901 ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaagacg 3961 aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg 4021 ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc 4081 tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc 4141 tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc 4201 gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc 4261 aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gagcatgccc gacggcgagg 4321 atctcgtcgt gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct 4381 tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt 4441 tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc 4501 tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt 4561 tcttctgagc gggactctgg ggttcgaaat gaccgaccaa gcgacgccca acctgccatc 4621 acgagatttc gattccaccg ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg 4681 ggacgccggc tggatgatcc tccagcgcgg ggatctcatg ctggagttct tcgcccaccc 4741 tagggggagg ctaactgaaa cacggaagga gacaataccg gaaggaaccc gcgctatgac 4801 ggcaataaaa agacagaata aaacgcacgg tgttgggtcg tttgttcata aacgcggggt 4861 tcggtcccag ggctggcact ctgtcgatac cccaccgaga ccccattggg gccaatacgc 4921 ccgcgtttct tccttttccc caccccaccc cccaagttcg ggtgaaggcc cagggctcgc 4981 agccaacgtc ggggcggcag gccctgccat agcctcaggt tactcatata tactttagat 5041 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct 5101 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa 5161 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa 5221 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc 5281 gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta 5341 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct 5401 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg 5461 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag 5521 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc 5581 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg 5641 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt 5701 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg 5761 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca 5821 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg ccatgcatta 5881 gttattaata gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg 5941 ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga 6001 cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat 6061 gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa 6121 gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca 6181 tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc gctattacca 6241 tg //