LOCUS       sequence-348191-        6658 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Transient mammalian expression of codon optimized SARS-CoV-2 
            Nucleocapsid (R203K). 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-R203K
FEATURES             Location/Qualifiers
     source          1..6658
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        104..483
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        484..687
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     638..658
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     732..751
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        732..750
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2118..2135)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2124..2348
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2394..2822
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2481..2500)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2691..2712
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2831..2851)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2836..3165
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3016..3151
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3078..3097
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3232..4026
                     /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(3286..3305)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3896..3915
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4200..4321
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4237..4256)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4291..4310
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4370..4386)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4370..4386)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4383..4405)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4394..4410
                     /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(4418..4424,4425..4442,4443..4448))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4463..4484
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4601..4618)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4772..5360)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4852..4871)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5531..6322,6323..6391))
                     /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     6154..6173
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6392..6496)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6571..6590)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 taagctacaa caaggcaagg cttgaccgac aattgcatga agaatctgct tagggttagg
       61 cgttttgcgc tgcttcgcga tgtacgggcc agatatacgc gttgacattg attattgact
      121 agttattaat agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc
      181 gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg
      241 acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa
      301 tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca
      361 agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac
      421 atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc
      481 atggtgatgc ggttttggca gtacatcaat gggcgtggat agcggtttga ctcacgggga
      541 tttccaagtc tccaccccat tgacgtcaat gggagtttgt tttggcacca aaatcaacgg
      601 gactttccaa aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg taggcgtgta
      661 cggtgggagg tctatataag cagagctctc tggctaacta gagaacccac tgcttactgg
      721 cttatcgaaa ttaatacgac tcactatagg gagacccaag ctggctagcc ttccatttca
      781 ggtgtcgtga ggatctattt ccggtgaatt cgccgccacc atgagcgaca acggccccca
      841 aaaccaacgg aatgcccctc ggataacatt tggtggccct agtgattcca caggaagtaa
      901 ccaaaacgga gaacggtctg gagcccgctc taaacagagg cgaccacagg gattgccaaa
      961 taacacagcg tcatggttta cagccctgac tcaacatgga aaggaggact tgaaatttcc
     1021 acgaggacag ggtgtaccaa taaacacaaa cagtagtcca gacgatcaga tcgggtatta
     1081 tagaagggcc acgagacgca tcagaggtgg agatggtaaa atgaaagatc tcagtccgcg
     1141 ctggtacttt tattatttgg gaaccggccc agaagcgggc ctcccatatg gagccaataa
     1201 ggacgggatt atatgggtgg ctacagaggg agccctcaat acgccaaaag atcacatcgg
     1261 aacaagaaac cctgctaata atgccgcaat cgtgctgcag ttgcctcagg ggacgacgct
     1321 tcctaaaggc ttttacgcag aaggatcacg cggcggcagc caagcatcct caaggtctag
     1381 ttccagaagt cgaaacagct ccagaaactc cacaccaggg tccagtaagg gcacaagtcc
     1441 ggcgcggatg gcgggcaacg gcggagacgc cgcactcgct ctgttgctcc tggaccggct
     1501 caaccaactt gaatccaaga tgagtggtaa gggacagcag caacaaggtc aaaccgtaac
     1561 caagaaaagc gctgcagaag cttctaaaaa acctcgacaa aaacggaccg ctacgaaggc
     1621 atataacgtt actcaagcct tcgggagacg agggccggag caaacccagg ggaattttgg
     1681 agatcaggag ctcatccggc aagggacaga ttacaaacat tggcctcaaa tcgctcagtt
     1741 cgcgccgtcc gcaagtgctt ttttcggaat gtctcgcatc ggtatggaag taacaccatc
     1801 cgggacatgg ctcacttata ccggcgccat caagctggac gacaaagatc caaatttcaa
     1861 agatcaggtg attctcttga ataaacacat agacgcgtat aagacgtttc cacccaccga
     1921 acccaagaag gacaaaaaga aaaaagctga tgaaacacaa gcgttgcccc agcgacagaa
     1981 gaagcagcaa accgtcaccc tcctgccagc cgctgatctc gatgacttct ccaagcaact
     2041 tcaacagtcc atgagctcag ccgattctac tcaggcgtaa ctcgagtcta gagggcccgt
     2101 ttaaacccgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc
     2161 ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa
     2221 tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg
     2281 gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg
     2341 ctctatggct tctgaggcgg aaagaaccag ctggggctct agggggtatc cccacgcgcc
     2401 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
     2461 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
     2521 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
     2581 acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc
     2641 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt
     2701 gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat
     2761 tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa
     2821 ttaattctgt ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc
     2881 agaagtatgc aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc
     2941 tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg
     3001 cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat
     3061 ggctgactaa ttttttttat ttatgcagag gccgaggccg cctctgcctc tgagctattc
     3121 cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaaaagct cccgggagct
     3181 tgtatatcca ttttcggatc tgatcaagag acaggatgag gatcgtttcg catgattgaa
     3241 caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt cggctatgac
     3301 tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc agcgcagggg
     3361 cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact gcaggacgag
     3421 gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt
     3481 gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca ggatctcctg
     3541 tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat gcggcggctg
     3601 catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg catcgagcga
     3661 gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga agagcatcag
     3721 gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc gcatgcccga cggcgaggat
     3781 ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt
     3841 tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga catagcgttg
     3901 gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt
     3961 tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc
     4021 ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac
     4081 gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc gttttccggg
     4141 acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc gcccacccca
     4201 acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa
     4261 ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt
     4321 atcatgtctg tataccgtcg acctctagct agagcttggc gtaatcatgg tcatagctgt
     4381 ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa
     4441 agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac
     4501 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
     4561 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc
     4621 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
     4681 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
     4741 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     4801 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     4861 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     4921 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     4981 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     5041 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     5101 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     5161 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat
     5221 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     5281 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     5341 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     5401 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
     5461 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
     5521 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     5581 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     5641 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
     5701 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     5761 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     5821 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
     5881 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     5941 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     6001 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     6061 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     6121 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
     6181 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     6241 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     6301 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     6361 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
     6421 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     6481 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtcgac ggatcgggag
     6541 atctcccgat cccctatggt gcactctcag tacaatctgc tctgatgccg catagttaag
     6601 ccagtatctg ctccctgctt gtgtgttgga ggtcgctgag tagtgcgcga gcaaaatt
//
