LOCUS       sequence-348185-        6658 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Transient mammalian expression of codon optimized SARS-CoV-2 
            Nucleocapsid (S235F). Used for generating RNA packaging virus-like 
            particles..
ACCESSION   .
VERSION     .
KEYWORDS    .
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 Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Science. 
            2021 Nov 4:eabl6184. doi: 10.1126/science.abl6184."
COMMENT     Sequence Label: CoV2-N-S235F
FEATURES             Location/Qualifiers
     source          1..6658
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        125..504
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        505..708
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     659..679
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     753..772
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        753..771
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2139..2156)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2145..2369
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2415..2843
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2502..2521)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2712..2733
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2852..2872)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2857..3186
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3037..3172
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3099..3118
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3253..4047
                     /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(3307..3326)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3917..3936
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4221..4342
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4258..4277)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4312..4331
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4391..4407)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4391..4407)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4404..4426)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4415..4431
                     /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(join(4439..4445,4446..4463,4464..4469))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4484..4505
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4622..4639)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4793..5381)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4873..4892)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5552..6343,6344..6412))
                     /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     6175..6194
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6413..6517)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6592..6611)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gagtagtgcg cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg
       61 aagaatctgc ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg
      121 cgttgacatt gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat
      181 agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg
      241 cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata
      301 gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta
      361 catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc
      421 gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca gtacatctac
      481 gtattagtca tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga
      541 tagcggtttg actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg
      601 ttttggcacc aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg
      661 caaatgggcg gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact
      721 agagaaccca ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa
      781 gctggctagc cttccatttc aggtgtcgtg aggatctatt tccggtgaat tcgccgccac
      841 catgagcgac aacggccccc aaaaccaacg gaatgcccct cggataacat ttggtggccc
      901 tagtgattcc acaggaagta accaaaacgg agaacggtct ggagcccgct ctaaacagag
      961 gcgaccacag ggattgccaa ataacacagc gtcatggttt acagccctga ctcaacatgg
     1021 aaaggaggac ttgaaatttc cacgaggaca gggtgtacca ataaacacaa acagtagtcc
     1081 agacgatcag atcgggtatt atagaagggc cacgagacgc atcagaggtg gagatggtaa
     1141 aatgaaagat ctcagtccgc gctggtactt ttattatttg ggaaccggcc cagaagcggg
     1201 cctcccatat ggagccaata aggacgggat tatatgggtg gctacagagg gagccctcaa
     1261 tacgccaaaa gatcacatcg gaacaagaaa ccctgctaat aatgccgcaa tcgtgctgca
     1321 gttgcctcag gggacgacgc ttcctaaagg cttttacgca gaaggatcac gcggcggcag
     1381 ccaagcatcc tcaaggtcta gttccagaag tcgaaacagc tccagaaact ccacaccagg
     1441 gtccagtagg ggcacaagtc cggcgcggat ggcgggcaac ggcggagacg ccgcactcgc
     1501 tctgttgctc ctggaccggc tcaaccaact tgaatccaag atgttcggta agggacagca
     1561 gcaacaaggt caaaccgtaa ccaagaaaag cgctgcagaa gcttctaaaa aacctcgaca
     1621 aaaacggacc gctacgaagg catataacgt tactcaagcc ttcgggagac gagggccgga
     1681 gcaaacccag gggaattttg gagatcagga gctcatccgg caagggacag attacaaaca
     1741 ttggcctcaa atcgctcagt tcgcgccgtc cgcaagtgct tttttcggaa tgtctcgcat
     1801 cggtatggaa gtaacaccat ccgggacatg gctcacttat accggcgcca tcaagctgga
     1861 cgacaaagat ccaaatttca aagatcaggt gattctcttg aataaacaca tagacgcgta
     1921 taagacgttt ccacccaccg aacccaagaa ggacaaaaag aaaaaagctg atgaaacaca
     1981 agcgttgccc cagcgacaga agaagcagca aaccgtcacc ctcctgccag ccgctgatct
     2041 cgatgacttc tccaagcaac ttcaacagtc catgagctca gccgattcta ctcaggcgta
     2101 actcgagtct agagggcccg tttaaacccg ctgatcagcc tcgactgtgc cttctagttg
     2161 ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc
     2221 cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc
     2281 tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag
     2341 gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca gctggggctc
     2401 tagggggtat ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
     2461 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc
     2521 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
     2581 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg
     2641 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
     2701 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta
     2761 ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat
     2821 ttaacaaaaa tttaacgcga attaattctg tggaatgtgt gtcagttagg gtgtggaaag
     2881 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
     2941 aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat
     3001 tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt
     3061 tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc
     3121 gcctctgcct ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt
     3181 tgcaaaaagc tcccgggagc ttgtatatcc attttcggat ctgatcaaga gacaggatga
     3241 ggatcgtttc gcatgattga acaagatgga ttgcacgcag gttctccggc cgcttgggtg
     3301 gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga tgccgccgtg
     3361 ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct gtccggtgcc
     3421 ctgaatgaac tgcaggacga ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct
     3481 tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct attgggcgaa
     3541 gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt atccatcatg
     3601 gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt cgaccaccaa
     3661 gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt cgatcaggat
     3721 gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg
     3781 cgcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt gccgaatatc
     3841 atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg tgtggcggac
     3901 cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg cggcgaatgg
     3961 gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg catcgccttc
     4021 tatcgccttc ttgacgagtt cttctgagcg ggactctggg gttcgaaatg accgaccaag
     4081 cgacgcccaa cctgccatca cgagatttcg attccaccgc cgccttctat gaaaggttgg
     4141 gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc
     4201 tggagttctt cgcccacccc aacttgttta ttgcagctta taatggttac aaataaagca
     4261 atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt
     4321 ccaaactcat caatgtatct tatcatgtct gtataccgtc gacctctagc tagagcttgg
     4381 cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca
     4441 acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca
     4501 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     4561 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt
     4621 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     4681 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     4741 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     4801 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     4861 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     4921 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     4981 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     5041 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     5101 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     5161 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     5221 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     5281 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     5341 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     5401 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     5461 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     5521 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     5581 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     5641 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     5701 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     5761 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     5821 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     5881 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     5941 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     6001 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     6061 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     6121 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     6181 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     6241 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     6301 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     6361 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     6421 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     6481 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
     6541 ctgacgtcga cggatcggga gatctcccga tcccctatgg tgcactctca gtacaatctg
     6601 ctctgatgcc gcatagttaa gccagtatct gctccctgct tgtgtgttgg aggtcgct
//
