LOCUS       sequence-348193-        6658 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Transient mammalian expression of codon optimized SARS-CoV-2 
            Nucleocapsid (S202R). 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-S202R
FEATURES             Location/Qualifiers
     source          1..6658
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        128..507
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        508..711
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     662..682
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     756..775
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        756..774
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2142..2159)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2148..2372
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2418..2846
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2505..2524)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2715..2736
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2855..2875)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2860..3189
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3040..3175
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3102..3121
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3256..4050
                     /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(3310..3329)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3920..3939
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4224..4345
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4261..4280)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4315..4334
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4394..4410)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4394..4410)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4407..4429)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4418..4434
                     /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(4442..4448,4449..4466,4467..4472))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4487..4508
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4625..4642)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4796..5384)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4876..4895)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5555..6346,6347..6415))
                     /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     6178..6197
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6416..6520)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6595..6614)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gctgagtagt gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc
       61 atgaagaatc tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat
      121 acgcgttgac attgattatt gactagttat taatagtaat caattacggg gtcattagtt
      181 catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga
      241 ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca
      301 atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca
      361 gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg
      421 cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc
      481 tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt
      541 ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt
      601 ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg
      661 acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc tctctggcta
      721 actagagaac ccactgctta ctggcttatc gaaattaata cgactcacta tagggagacc
      781 caagctggct agccttccat ttcaggtgtc gtgaggatct atttccggtg aattcgccgc
      841 caccatgagc gacaacggcc cccaaaacca acggaatgcc cctcggataa catttggtgg
      901 ccctagtgat tccacaggaa gtaaccaaaa cggagaacgg tctggagccc gctctaaaca
      961 gaggcgacca cagggattgc caaataacac agcgtcatgg tttacagccc tgactcaaca
     1021 tggaaaggag gacttgaaat ttccacgagg acagggtgta ccaataaaca caaacagtag
     1081 tccagacgat cagatcgggt attatagaag ggccacgaga cgcatcagag gtggagatgg
     1141 taaaatgaaa gatctcagtc cgcgctggta cttttattat ttgggaaccg gcccagaagc
     1201 gggcctccca tatggagcca ataaggacgg gattatatgg gtggctacag agggagccct
     1261 caatacgcca aaagatcaca tcggaacaag aaaccctgct aataatgccg caatcgtgct
     1321 gcagttgcct caggggacga cgcttcctaa aggcttttac gcagaaggat cacgcggcgg
     1381 cagccaagca tcctcaaggt ctagttccag aagtcgaaac agctccagaa actccacacc
     1441 agggtcccga aggggcacaa gtccggcgcg gatggcgggc aacggcggag acgccgcact
     1501 cgctctgttg ctcctggacc ggctcaacca acttgaatcc aagatgagtg gtaagggaca
     1561 gcagcaacaa ggtcaaaccg taaccaagaa aagcgctgca gaagcttcta aaaaacctcg
     1621 acaaaaacgg accgctacga aggcatataa cgttactcaa gccttcggga gacgagggcc
     1681 ggagcaaacc caggggaatt ttggagatca ggagctcatc cggcaaggga cagattacaa
     1741 acattggcct caaatcgctc agttcgcgcc gtccgcaagt gcttttttcg gaatgtctcg
     1801 catcggtatg gaagtaacac catccgggac atggctcact tataccggcg ccatcaagct
     1861 ggacgacaaa gatccaaatt tcaaagatca ggtgattctc ttgaataaac acatagacgc
     1921 gtataagacg tttccaccca ccgaacccaa gaaggacaaa aagaaaaaag ctgatgaaac
     1981 acaagcgttg ccccagcgac agaagaagca gcaaaccgtc accctcctgc cagccgctga
     2041 tctcgatgac ttctccaagc aacttcaaca gtccatgagc tcagccgatt ctactcaggc
     2101 gtaactcgag tctagagggc ccgtttaaac ccgctgatca gcctcgactg tgccttctag
     2161 ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac
     2221 tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca
     2281 ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag
     2341 caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa ccagctgggg
     2401 ctctaggggg tatccccacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
     2461 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt
     2521 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
     2581 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga
     2641 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
     2701 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt
     2761 ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct
     2821 gatttaacaa aaatttaacg cgaattaatt ctgtggaatg tgtgtcagtt agggtgtgga
     2881 aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca
     2941 accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
     3001 aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc
     3061 agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag
     3121 gccgcctctg cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc
     3181 ttttgcaaaa agctcccggg agcttgtata tccattttcg gatctgatca agagacagga
     3241 tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg caggttctcc ggccgcttgg
     3301 gtggagaggc tattcggcta tgactgggca caacagacaa tcggctgctc tgatgccgcc
     3361 gtgttccggc tgtcagcgca ggggcgcccg gttctttttg tcaagaccga cctgtccggt
     3421 gccctgaatg aactgcagga cgaggcagcg cggctatcgt ggctggccac gacgggcgtt
     3481 ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa gggactggct gctattgggc
     3541 gaagtgccgg ggcaggatct cctgtcatct caccttgctc ctgccgagaa agtatccatc
     3601 atggctgatg caatgcggcg gctgcatacg cttgatccgg ctacctgccc attcgaccac
     3661 caagcgaaac atcgcatcga gcgagcacgt actcggatgg aagccggtct tgtcgatcag
     3721 gatgatctgg acgaagagca tcaggggctc gcgccagccg aactgttcgc caggctcaag
     3781 gcgcgcatgc ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat
     3841 atcatggtgg aaaatggccg cttttctgga ttcatcgact gtggccggct gggtgtggcg
     3901 gaccgctatc aggacatagc gttggctacc cgtgatattg ctgaagagct tggcggcgaa
     3961 tgggctgacc gcttcctcgt gctttacggt atcgccgctc ccgattcgca gcgcatcgcc
     4021 ttctatcgcc ttcttgacga gttcttctga gcgggactct ggggttcgaa atgaccgacc
     4081 aagcgacgcc caacctgcca tcacgagatt tcgattccac cgccgccttc tatgaaaggt
     4141 tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc ggggatctca
     4201 tgctggagtt cttcgcccac cccaacttgt ttattgcagc ttataatggt tacaaataaa
     4261 gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt
     4321 tgtccaaact catcaatgta tcttatcatg tctgtatacc gtcgacctct agctagagct
     4381 tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac
     4441 acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac
     4501 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     4561 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg
     4621 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     4681 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     4741 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     4801 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     4861 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     4921 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     4981 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     5041 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     5101 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     5161 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     5221 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     5281 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     5341 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     5401 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     5461 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     5521 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     5581 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     5641 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     5701 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     5761 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     5821 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     5881 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     5941 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     6001 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     6061 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     6121 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     6181 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     6241 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     6301 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     6361 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     6421 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     6481 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     6541 cacctgacgt cgacggatcg ggagatctcc cgatccccta tggtgcactc tcagtacaat
     6601 ctgctctgat gccgcatagt taagccagta tctgctccct gcttgtgtgt tggaggtc
//
