LOCUS       sequence-348187-        6658 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Transient mammalian expression of codon optimized SARS-CoV-2 
            Nucleocapsid (R203M). 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-R203M
FEATURES             Location/Qualifiers
     source          1..6658
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        119..498
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        499..702
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     653..673
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     747..766
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        747..765
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2133..2150)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2139..2363
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2409..2837
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2496..2515)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2706..2727
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2846..2866)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2851..3180
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3031..3166
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3093..3112
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3247..4041
                     /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(3301..3320)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3911..3930
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4215..4336
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4252..4271)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4306..4325
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4385..4401)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4385..4401)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4398..4420)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4409..4425
                     /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(4433..4439,4440..4457,4458..4463))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4478..4499
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4616..4633)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4787..5375)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4867..4886)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5546..6337,6338..6406))
                     /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     6169..6188
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6407..6511)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6586..6605)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 tgcgcgagca aaatttaagc tacaacaagg caaggcttga ccgacaattg catgaagaat
       61 ctgcttaggg ttaggcgttt tgcgctgctt cgcgatgtac gggccagata tacgcgttga
      121 cattgattat tgactagtta ttaatagtaa tcaattacgg ggtcattagt tcatagccca
      181 tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac
      241 gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact
      301 ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa
      361 gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg
      421 cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta
      481 gtcatcgcta ttaccatggt gatgcggttt tggcagtaca tcaatgggcg tggatagcgg
      541 tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg
      601 caccaaaatc aacgggactt tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg
      661 ggcggtaggc gtgtacggtg ggaggtctat ataagcagag ctctctggct aactagagaa
      721 cccactgctt actggcttat cgaaattaat acgactcact atagggagac ccaagctggc
      781 tagccttcca tttcaggtgt cgtgaggatc tatttccggt gaattcgccg ccaccatgag
      841 cgacaacggc ccccaaaacc aacggaatgc ccctcggata acatttggtg gccctagtga
      901 ttccacagga agtaaccaaa acggagaacg gtctggagcc cgctctaaac agaggcgacc
      961 acagggattg ccaaataaca cagcgtcatg gtttacagcc ctgactcaac atggaaagga
     1021 ggacttgaaa tttccacgag gacagggtgt accaataaac acaaacagta gtccagacga
     1081 tcagatcggg tattatagaa gggccacgag acgcatcaga ggtggagatg gtaaaatgaa
     1141 agatctcagt ccgcgctggt acttttatta tttgggaacc ggcccagaag cgggcctccc
     1201 atatggagcc aataaggacg ggattatatg ggtggctaca gagggagccc tcaatacgcc
     1261 aaaagatcac atcggaacaa gaaaccctgc taataatgcc gcaatcgtgc tgcagttgcc
     1321 tcaggggacg acgcttccta aaggctttta cgcagaagga tcacgcggcg gcagccaagc
     1381 atcctcaagg tctagttcca gaagtcgaaa cagctccaga aactccacac cagggtccag
     1441 tatgggcaca agtccggcgc ggatggcggg caacggcgga gacgccgcac tcgctctgtt
     1501 gctcctggac cggctcaacc aacttgaatc caagatgagt ggtaagggac agcagcaaca
     1561 aggtcaaacc gtaaccaaga aaagcgctgc agaagcttct aaaaaacctc gacaaaaacg
     1621 gaccgctacg aaggcatata acgttactca agccttcggg agacgagggc cggagcaaac
     1681 ccaggggaat tttggagatc aggagctcat ccggcaaggg acagattaca aacattggcc
     1741 tcaaatcgct cagttcgcgc cgtccgcaag tgcttttttc ggaatgtctc gcatcggtat
     1801 ggaagtaaca ccatccggga catggctcac ttataccggc gccatcaagc tggacgacaa
     1861 agatccaaat ttcaaagatc aggtgattct cttgaataaa cacatagacg cgtataagac
     1921 gtttccaccc accgaaccca agaaggacaa aaagaaaaaa gctgatgaaa cacaagcgtt
     1981 gccccagcga cagaagaagc agcaaaccgt caccctcctg ccagccgctg atctcgatga
     2041 cttctccaag caacttcaac agtccatgag ctcagccgat tctactcagg cgtaactcga
     2101 gtctagaggg cccgtttaaa cccgctgatc agcctcgact gtgccttcta gttgccagcc
     2161 atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt
     2221 cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct
     2281 ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc
     2341 tggggatgcg gtgggctcta tggcttctga ggcggaaaga accagctggg gctctagggg
     2401 gtatccccac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
     2461 cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt
     2521 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt
     2581 ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg
     2641 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt
     2701 taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt
     2761 tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca
     2821 aaaatttaac gcgaattaat tctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca
     2881 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaggtgt
     2941 ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca
     3001 gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc
     3061 cattctccgc cccatggctg actaattttt tttatttatg cagaggccga ggccgcctct
     3121 gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa
     3181 aagctcccgg gagcttgtat atccattttc ggatctgatc aagagacagg atgaggatcg
     3241 tttcgcatga ttgaacaaga tggattgcac gcaggttctc cggccgcttg ggtggagagg
     3301 ctattcggct atgactgggc acaacagaca atcggctgct ctgatgccgc cgtgttccgg
     3361 ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg acctgtccgg tgccctgaat
     3421 gaactgcagg acgaggcagc gcggctatcg tggctggcca cgacgggcgt tccttgcgca
     3481 gctgtgctcg acgttgtcac tgaagcggga agggactggc tgctattggg cgaagtgccg
     3541 gggcaggatc tcctgtcatc tcaccttgct cctgccgaga aagtatccat catggctgat
     3601 gcaatgcggc ggctgcatac gcttgatccg gctacctgcc cattcgacca ccaagcgaaa
     3661 catcgcatcg agcgagcacg tactcggatg gaagccggtc ttgtcgatca ggatgatctg
     3721 gacgaagagc atcaggggct cgcgccagcc gaactgttcg ccaggctcaa ggcgcgcatg
     3781 cccgacggcg aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg
     3841 gaaaatggcc gcttttctgg attcatcgac tgtggccggc tgggtgtggc ggaccgctat
     3901 caggacatag cgttggctac ccgtgatatt gctgaagagc ttggcggcga atgggctgac
     3961 cgcttcctcg tgctttacgg tatcgccgct cccgattcgc agcgcatcgc cttctatcgc
     4021 cttcttgacg agttcttctg agcgggactc tggggttcga aatgaccgac caagcgacgc
     4081 ccaacctgcc atcacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg
     4141 gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt
     4201 tcttcgccca ccccaacttg tttattgcag cttataatgg ttacaaataa agcaatagca
     4261 tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac
     4321 tcatcaatgt atcttatcat gtctgtatac cgtcgacctc tagctagagc ttggcgtaat
     4381 catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac
     4441 gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa
     4501 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat
     4561 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc
     4621 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
     4681 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     4741 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     4801 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     4861 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     4921 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     4981 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     5041 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     5101 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     5161 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     5221 ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     5281 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     5341 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     5401 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     5461 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     5521 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
     5581 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
     5641 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
     5701 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
     5761 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
     5821 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
     5881 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
     5941 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
     6001 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
     6061 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
     6121 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
     6181 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
     6241 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
     6301 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
     6361 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
     6421 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     6481 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg
     6541 tcgacggatc gggagatctc ccgatcccct atggtgcact ctcagtacaa tctgctctga
     6601 tgccgcatag ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg ctgagtag
//
