LOCUS Exported 6658 bp ds-DNA circular SYN 17-NOV-2021 DEFINITION Transient mammalian expression of codon optimized SARS-CoV-2 Nucleocapsid (G204R). Used for generating RNA packaging virus-like particles.. ACCESSION . VERSION . KEYWORDS CoV2-N-G204R SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6658) AUTHORS Syed AM, Taha TY, Tabata T, Chen IP, Ciling A, Khalid MM, Sreekumar B, Chen PY, Hayashi JM, Soczek KM, Ott M, Doudna JA TITLE Rapid assessment of SARS-CoV-2 evolved variants using virus-like particles. JOURNAL Science. 2021 Nov 4:eabl6184. doi: 10.1126/science.abl6184. PUBMED 34735219 REFERENCE 2 (bases 1 to 6658) AUTHORS . TITLE Direct Submission JOURNAL Exported Nov 17, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6658 /organism="synthetic DNA construct" /mol_type="other DNA" enhancer 144..523 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 524..727 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 678..698 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" primer_bind 772..791 /label=T7 /note="T7 promoter, forward primer" promoter 772..790 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(2158..2175) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 2164..2388 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" rep_origin 2434..2862 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(2521..2540) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 2731..2752 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(2871..2891) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 2876..3205 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 3056..3191 /label=SV40 ori /note="SV40 origin of replication" primer_bind 3118..3137 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" CDS 3272..4066 /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(3326..3345) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 3936..3955 /label=Neo-F /note="Neomycin resistance gene, forward primer" polyA_signal 4240..4361 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(4277..4296) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 4331..4350 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(4410..4426) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4410..4426) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(4423..4445) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 4434..4450 /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(4458..4488) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 4503..4524 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(4641..4658) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(4812..5400) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(4892..4911) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(5571..6431) /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 6194..6213 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(6432..6536) /gene="bla" /label=AmpR promoter primer_bind complement(6611..6630) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" ORIGIN 1 ttgtgtgttg gaggtcgctg agtagtgcgc gagcaaaatt taagctacaa caaggcaagg 61 cttgaccgac aattgcatga agaatctgct tagggttagg cgttttgcgc tgcttcgcga 121 tgtacgggcc agatatacgc gttgacattg attattgact agttattaat agtaatcaat 181 tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa 241 tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt 301 tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta 361 aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt 421 caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc 481 tacttggcag tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca 541 gtacatcaat gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat 601 tgacgtcaat gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa 661 caactccgcc ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag 721 cagagctctc tggctaacta gagaacccac tgcttactgg cttatcgaaa ttaatacgac 781 tcactatagg gagacccaag ctggctagcc ttccatttca ggtgtcgtga ggatctattt 841 ccggtgaatt cgccgccacc atgagcgaca acggccccca aaaccaacgg aatgcccctc 901 ggataacatt tggtggccct agtgattcca caggaagtaa ccaaaacgga gaacggtctg 961 gagcccgctc taaacagagg cgaccacagg gattgccaaa taacacagcg tcatggttta 1021 cagccctgac tcaacatgga aaggaggact tgaaatttcc acgaggacag ggtgtaccaa 1081 taaacacaaa cagtagtcca gacgatcaga tcgggtatta tagaagggcc acgagacgca 1141 tcagaggtgg agatggtaaa atgaaagatc tcagtccgcg ctggtacttt tattatttgg 1201 gaaccggccc agaagcgggc ctcccatatg gagccaataa ggacgggatt atatgggtgg 1261 ctacagaggg agccctcaat acgccaaaag atcacatcgg aacaagaaac cctgctaata 1321 atgccgcaat cgtgctgcag ttgcctcagg ggacgacgct tcctaaaggc ttttacgcag 1381 aaggatcacg cggcggcagc caagcatcct caaggtctag ttccagaagt cgaaacagct 1441 ccagaaactc cacaccaggg tccagtaggc gaacaagtcc ggcgcggatg gcgggcaacg 1501 gcggagacgc cgcactcgct ctgttgctcc tggaccggct caaccaactt gaatccaaga 1561 tgagtggtaa gggacagcag caacaaggtc aaaccgtaac caagaaaagc gctgcagaag 1621 cttctaaaaa acctcgacaa aaacggaccg ctacgaaggc atataacgtt actcaagcct 1681 tcgggagacg agggccggag caaacccagg ggaattttgg agatcaggag ctcatccggc 1741 aagggacaga ttacaaacat tggcctcaaa tcgctcagtt cgcgccgtcc gcaagtgctt 1801 ttttcggaat gtctcgcatc ggtatggaag taacaccatc cgggacatgg ctcacttata 1861 ccggcgccat caagctggac gacaaagatc caaatttcaa agatcaggtg attctcttga 1921 ataaacacat agacgcgtat aagacgtttc cacccaccga acccaagaag gacaaaaaga 1981 aaaaagctga tgaaacacaa gcgttgcccc agcgacagaa gaagcagcaa accgtcaccc 2041 tcctgccagc cgctgatctc gatgacttct ccaagcaact tcaacagtcc atgagctcag 2101 ccgattctac tcaggcgtaa ctcgagtcta gagggcccgt ttaaacccgc tgatcagcct 2161 cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga 2221 ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt 2281 gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg 2341 attgggaaga caatagcagg catgctgggg atgcggtggg ctctatggct tctgaggcgg 2401 aaagaaccag ctggggctct agggggtatc cccacgcgcc ctgtagcggc gcattaagcg 2461 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg 2521 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc 2581 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa 2641 aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc 2701 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac 2761 tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt 2821 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg 2881 tcagttaggg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca 2941 tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag gcagaagtat 3001 gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc 3061 gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat 3121 ttatgcagag gccgaggccg cctctgcctc tgagctattc cagaagtagt gaggaggctt 3181 ttttggaggc ctaggctttt gcaaaaagct cccgggagct tgtatatcca ttttcggatc 3241 tgatcaagag acaggatgag gatcgtttcg catgattgaa caagatggat tgcacgcagg 3301 ttctccggcc gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg 3361 ctgctctgat gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa 3421 gaccgacctg tccggtgccc tgaatgaact gcaggacgag gcagcgcggc tatcgtggct 3481 ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga 3541 ctggctgcta ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc 3601 cgagaaagta tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac 3661 ctgcccattc gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc 3721 cggtcttgtc gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact 3781 gttcgccagg ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga cccatggcga 3841 tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg 3901 ccggctgggt gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga 3961 agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga 4021 ttcgcagcgc atcgccttct atcgccttct tgacgagttc ttctgagcgg gactctgggg 4081 ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac gagatttcga ttccaccgcc 4141 gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc 4201 cagcgcgggg atctcatgct ggagttcttc gcccacccca acttgtttat tgcagcttat 4261 aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg 4321 cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg tataccgtcg 4381 acctctagct agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat 4441 ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc 4501 taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga 4561 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt 4621 attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg 4681 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac 4741 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg 4801 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca 4861 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc 4921 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc 4981 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag 5041 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc 5101 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca 5161 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg 5221 aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg 5281 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct 5341 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa 5401 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa 5461 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa 5521 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc 5581 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga 5641 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca 5701 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc 5761 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat 5821 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc 5881 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt 5941 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc 6001 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg 6061 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt 6121 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg 6181 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga 6241 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg 6301 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg 6361 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt 6421 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc 6481 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca 6541 tttccccgaa aagtgccacc tgacgtcgac ggatcgggag atctcccgat cccctatggt 6601 gcactctcag tacaatctgc tctgatgccg catagttaag ccagtatctg ctccctgc //