LOCUS       sequence-348184-        6658 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Transient mammalian expression of codon optimized SARS-CoV-2 
            Nucleocapsid (D377Y). 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-D377Y
FEATURES             Location/Qualifiers
     source          1..6658
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        49..428
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        429..632
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     583..603
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     677..696
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        677..695
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2063..2080)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2069..2293
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2339..2767
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2426..2445)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2636..2657
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2776..2796)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2781..3110
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2961..3096
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3023..3042
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3177..3971
                     /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(3231..3250)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3841..3860
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4145..4266
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4182..4201)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4236..4255
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4315..4331)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4315..4331)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4328..4350)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4339..4355
                     /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(4363..4369,4370..4387,4388..4393))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4408..4429
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4546..4563)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4717..5305)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4797..4816)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5476..6267,6268..6336))
                     /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     6099..6118
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6337..6441)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6516..6535)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 ttaggcgttt tgcgctgctt cgcgatgtac gggccagata tacgcgttga cattgattat
       61 tgactagtta ttaatagtaa tcaattacgg ggtcattagt tcatagccca tatatggagt
      121 tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc
      181 cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac
      241 gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata
      301 tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattatgccc
      361 agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta
      421 ttaccatggt gatgcggttt tggcagtaca tcaatgggcg tggatagcgg tttgactcac
      481 ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg caccaaaatc
      541 aacgggactt tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc
      601 gtgtacggtg ggaggtctat ataagcagag ctctctggct aactagagaa cccactgctt
      661 actggcttat cgaaattaat acgactcact atagggagac ccaagctggc tagccttcca
      721 tttcaggtgt cgtgaggatc tatttccggt gaattcgccg ccaccatgag cgacaacggc
      781 ccccaaaacc aacggaatgc ccctcggata acatttggtg gccctagtga ttccacagga
      841 agtaaccaaa acggagaacg gtctggagcc cgctctaaac agaggcgacc acagggattg
      901 ccaaataaca cagcgtcatg gtttacagcc ctgactcaac atggaaagga ggacttgaaa
      961 tttccacgag gacagggtgt accaataaac acaaacagta gtccagacga tcagatcggg
     1021 tattatagaa gggccacgag acgcatcaga ggtggagatg gtaaaatgaa agatctcagt
     1081 ccgcgctggt acttttatta tttgggaacc ggcccagaag cgggcctccc atatggagcc
     1141 aataaggacg ggattatatg ggtggctaca gagggagccc tcaatacgcc aaaagatcac
     1201 atcggaacaa gaaaccctgc taataatgcc gcaatcgtgc tgcagttgcc tcaggggacg
     1261 acgcttccta aaggctttta cgcagaagga tcacgcggcg gcagccaagc atcctcaagg
     1321 tctagttcca gaagtcgaaa cagctccaga aactccacac cagggtccag taggggcaca
     1381 agtccggcgc ggatggcggg caacggcgga gacgccgcac tcgctctgtt gctcctggac
     1441 cggctcaacc aacttgaatc caagatgagt ggtaagggac agcagcaaca aggtcaaacc
     1501 gtaaccaaga aaagcgctgc agaagcttct aaaaaacctc gacaaaaacg gaccgctacg
     1561 aaggcatata acgttactca agccttcggg agacgagggc cggagcaaac ccaggggaat
     1621 tttggagatc aggagctcat ccggcaaggg acagattaca aacattggcc tcaaatcgct
     1681 cagttcgcgc cgtccgcaag tgcttttttc ggaatgtctc gcatcggtat ggaagtaaca
     1741 ccatccggga catggctcac ttataccggc gccatcaagc tggacgacaa agatccaaat
     1801 ttcaaagatc aggtgattct cttgaataaa cacatagacg cgtataagac gtttccaccc
     1861 accgaaccca agaaggacaa aaagaaaaaa gcttacgaaa cacaagcgtt gccccagcga
     1921 cagaagaagc agcaaaccgt caccctcctg ccagccgctg atctcgatga cttctccaag
     1981 caacttcaac agtccatgag ctcagccgat tctactcagg cgtaactcga gtctagaggg
     2041 cccgtttaaa cccgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt
     2101 tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa
     2161 taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg
     2221 gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg
     2281 gtgggctcta tggcttctga ggcggaaaga accagctggg gctctagggg gtatccccac
     2341 gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct
     2401 acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg
     2461 ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt
     2521 gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca
     2581 tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga
     2641 ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa
     2701 gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac
     2761 gcgaattaat tctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag
     2821 caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc
     2881 caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag
     2941 tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc
     3001 cccatggctg actaattttt tttatttatg cagaggccga ggccgcctct gcctctgagc
     3061 tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa aagctcccgg
     3121 gagcttgtat atccattttc ggatctgatc aagagacagg atgaggatcg tttcgcatga
     3181 ttgaacaaga tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct
     3241 atgactgggc acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc
     3301 aggggcgccc ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcagg
     3361 acgaggcagc gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg
     3421 acgttgtcac tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc
     3481 tcctgtcatc tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc
     3541 ggctgcatac gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg
     3601 agcgagcacg tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc
     3661 atcaggggct cgcgccagcc gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg
     3721 aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc
     3781 gcttttctgg attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag
     3841 cgttggctac ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg
     3901 tgctttacgg tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg
     3961 agttcttctg agcgggactc tggggttcga aatgaccgac caagcgacgc ccaacctgcc
     4021 atcacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt
     4081 ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca
     4141 ccccaacttg tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt
     4201 cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt
     4261 atcttatcat gtctgtatac cgtcgacctc tagctagagc ttggcgtaat catggtcata
     4321 gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag
     4381 cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg
     4441 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
     4501 acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc
     4561 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
     4621 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
     4681 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     4741 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     4801 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     4861 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
     4921 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     4981 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     5041 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     5101 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac
     5161 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     5221 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     5281 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     5341 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
     5401 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
     5461 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     5521 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
     5581 cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga
     5641 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     5701 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     5761 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
     5821 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     5881 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     5941 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     6001 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     6061 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
     6121 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     6181 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
     6241 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
     6301 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
     6361 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     6421 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tcgacggatc
     6481 gggagatctc ccgatcccct atggtgcact ctcagtacaa tctgctctga tgccgcatag
     6541 ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg cgcgagcaaa
     6601 atttaagcta caacaaggca aggcttgacc gacaattgca tgaagaatct gcttaggg
//
