LOCUS Exported 6089 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION mammalian expression and localization. ACCESSION . VERSION . KEYWORDS Nsp4 deltaC-EGFP SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6089) 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 6089) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6089 /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 1605..1657 /label=MCS /note="multiple cloning site" regulatory 1665..1674 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 1671..2390 /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 1671..2387 /codon_start=1 /product="enhanced GFP" /label=EGFP /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" primer_bind complement(1716..1737) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(1977..1996) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 2324..2345 /label=EGFP-C /note="EGFP, forward primer" polyA_signal 2513..2634 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(2550..2569) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 2604..2623 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" rep_origin complement(2641..3096) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(2778..2799) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 2990..3009 /label=F1ori-R /note="F1 origin, reverse primer" promoter 3123..3227 /gene="bla" /label=AmpR promoter promoter 3229..3586 /label=SV40 promoter /note="SV40 enhancer and early promoter" primer_bind complement(3252..3272) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" rep_origin 3437..3572 /label=SV40 ori /note="SV40 origin of replication" primer_bind 3499..3518 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" CDS 3621..4415 /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(3675..3694) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 4285..4304 /label=Neo-F /note="Neomycin resistance gene, forward primer" primer_bind complement(4603..4622) /label=TK-pA-R /note="Thymidine kinase polyA, reverse primer" polyA_signal 4647..4694 /label=HSV TK poly(A) signal /note="herpes simplex virus thymidine kinase polyadenylation signal (Cole and Stacy, 1985)" rep_origin 5023..5611 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 5512..5531 /label=pBR322ori-F /note="pBR322 origin, forward primer" enhancer 5786..6089 /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 ctcactcgag ctcaagcttc 1621 gaattctgca gtcgacggta ccgcgggccc gggatccacc ggtcgccacc atggtgagca 1681 agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa 1741 acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac ggcaagctga 1801 ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca 1861 ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag cagcacgact 1921 tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg 1981 acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca 2041 tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt 2101 acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac ggcatcaagg 2161 tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc gaccactacc 2221 agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagca 2281 cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt 2341 tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa agcggccgcg 2401 actctagatc ataatcagcc ataccacatt tgtagaggtt ttacttgctt taaaaaacct 2461 cccacacctc cccctgaacc tgaaacataa aatgaatgca attgttgttg ttaacttgtt 2521 tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc 2581 atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttaaggcgt 2641 aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat 2701 tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagaccgaga 2761 tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca 2821 acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccct 2881 aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc 2941 cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag 3001 cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca 3061 cacccgccgc gcttaatgcg ccgctacagg gcgcgtcagg tggcactttt cggggaaatg 3121 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga 3181 gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtcct gaggcggaaa 3241 gaaccagctg tggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccagcagg 3301 cagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg 3361 ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc 3421 gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca 3481 tggctgacta atttttttta tttatgcaga ggccgaggcc gcctcggcct ctgagctatt 3541 ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaagatc gatcaagaga 3601 caggatgagg atcgtttcgc atgattgaac aagatggatt gcacgcaggt tctccggccg 3661 cttgggtgga gaggctattc ggctatgact gggcacaaca gacaatcggc tgctctgatg 3721 ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct ttttgtcaag accgacctgt 3781 ccggtgccct gaatgaactg caagacgagg cagcgcggct atcgtggctg gccacgacgg 3841 gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc gggaagggac tggctgctat 3901 tgggcgaagt gccggggcag gatctcctgt catctcacct tgctcctgcc gagaaagtat 3961 ccatcatggc tgatgcaatg cggcggctgc atacgcttga tccggctacc tgcccattcg 4021 accaccaagc gaaacatcgc atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg 4081 atcaggatga tctggacgaa gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc 4141 tcaaggcgag catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc 4201 cgaatatcat ggtggaaaat ggccgctttt ctggattcat cgactgtggc cggctgggtg 4261 tggcggaccg ctatcaggac atagcgttgg ctacccgtga tattgctgaa gagcttggcg 4321 gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca 4381 tcgccttcta tcgccttctt gacgagttct tctgagcggg actctggggt tcgaaatgac 4441 cgaccaagcg acgcccaacc tgccatcacg agatttcgat tccaccgccg ccttctatga 4501 aaggttgggc ttcggaatcg ttttccggga cgccggctgg atgatcctcc agcgcgggga 4561 tctcatgctg gagttcttcg cccaccctag ggggaggcta actgaaacac ggaaggagac 4621 aataccggaa ggaacccgcg ctatgacggc aataaaaaga cagaataaaa cgcacggtgt 4681 tgggtcgttt gttcataaac gcggggttcg gtcccagggc tggcactctg tcgatacccc 4741 accgagaccc cattggggcc aatacgcccg cgtttcttcc ttttccccac cccacccccc 4801 aagttcgggt gaaggcccag ggctcgcagc caacgtcggg gcggcaggcc ctgccatagc 4861 ctcaggttac tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat 4921 ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt 4981 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct 5041 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc 5101 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc 5161 aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc 5221 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc 5281 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg 5341 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata 5401 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta 5461 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc 5521 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg 5581 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt 5641 cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt 5701 ggataaccgt attaccgcca tgcattagtt attaatagta atcaattacg gggtcattag 5761 ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct 5821 gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc 5881 caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg 5941 cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat 6001 ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca 6061 tctacgtatt agtcatcgct attaccatg //