LOCUS       sequence-376958-        6658 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Transient mammalian expression of codon optimized SARS-CoV-2 
            Nucleocapsid (D3L, R203K, G204R, 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-B.1.1.7
FEATURES             Location/Qualifiers
     source          1..6658
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     629..648
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        629..647
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2015..2032)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2021..2245
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2291..2719
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2378..2397)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2588..2609
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2728..2748)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2733..3062
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2913..3048
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2975..2994
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3129..3923
                     /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(3183..3202)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3793..3812
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4097..4218
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4134..4153)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4188..4207
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4267..4283)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4267..4283)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4280..4302)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4291..4307
                     /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(4315..4321,4322..4339,4340..4345))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4360..4381
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4498..4515)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4669..5257)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4749..4768)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5428..6219,6220..6288))
                     /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     6051..6070
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6289..6393)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6468..6487)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg ctaactagag
      601 aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag acccaagctg
      661 gctagccttc catttcaggt gtcgtgagga tctatttccg gtgaattcgc cgccaccatg
      721 agcctgaacg gcccccaaaa ccaacggaat gcccctcgga taacatttgg tggccctagt
      781 gattccacag gaagtaacca aaacggagaa cggtctggag cccgctctaa acagaggcga
      841 ccacagggat tgccaaataa cacagcgtca tggtttacag ccctgactca acatggaaag
      901 gaggacttga aatttccacg aggacagggt gtaccaataa acacaaacag tagtccagac
      961 gatcagatcg ggtattatag aagggccacg agacgcatca gaggtggaga tggtaaaatg
     1021 aaagatctca gtccgcgctg gtacttttat tatttgggaa ccggcccaga agcgggcctc
     1081 ccatatggag ccaataagga cgggattata tgggtggcta cagagggagc cctcaatacg
     1141 ccaaaagatc acatcggaac aagaaaccct gctaataatg ccgcaatcgt gctgcagttg
     1201 cctcagggga cgacgcttcc taaaggcttt tacgcagaag gatcacgcgg cggcagccaa
     1261 gcatcctcaa ggtctagttc cagaagtcga aacagctcca gaaactccac accagggtcc
     1321 agtaagcgaa caagtccggc gcggatggcg ggcaacggcg gagacgccgc actcgctctg
     1381 ttgctcctgg accggctcaa ccaacttgaa tccaagatgt tcggtaaggg acagcagcaa
     1441 caaggtcaaa ccgtaaccaa gaaaagcgct gcagaagctt ctaaaaaacc tcgacaaaaa
     1501 cggaccgcta cgaaggcata taacgttact caagccttcg ggagacgagg gccggagcaa
     1561 acccagggga attttggaga tcaggagctc atccggcaag ggacagatta caaacattgg
     1621 cctcaaatcg ctcagttcgc gccgtccgca agtgcttttt tcggaatgtc tcgcatcggt
     1681 atggaagtaa caccatccgg gacatggctc acttataccg gcgccatcaa gctggacgac
     1741 aaagatccaa atttcaaaga tcaggtgatt ctcttgaata aacacataga cgcgtataag
     1801 acgtttccac ccaccgaacc caagaaggac aaaaagaaaa aagctgatga aacacaagcg
     1861 ttgccccagc gacagaagaa gcagcaaacc gtcaccctcc tgccagccgc tgatctcgat
     1921 gacttctcca agcaacttca acagtccatg agctcagccg attctactca ggcgtaactc
     1981 gagtctagag ggcccgttta aacccgctga tcagcctcga ctgtgccttc tagttgccag
     2041 ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact
     2101 gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt
     2161 ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat
     2221 gctggggatg cggtgggctc tatggcttct gaggcggaaa gaaccagctg gggctctagg
     2281 gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc
     2341 agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc
     2401 tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg
     2461 ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca
     2521 cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc
     2581 tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct
     2641 tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa
     2701 caaaaattta acgcgaatta attctgtgga atgtgtgtca gttagggtgt ggaaagtccc
     2761 caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt
     2821 gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt
     2881 cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg
     2941 cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct
     3001 ctgcctctga gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca
     3061 aaaagctccc gggagcttgt atatccattt tcggatctga tcaagagaca ggatgaggat
     3121 cgtttcgcat gattgaacaa gatggattgc acgcaggttc tccggccgct tgggtggaga
     3181 ggctattcgg ctatgactgg gcacaacaga caatcggctg ctctgatgcc gccgtgttcc
     3241 ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga
     3301 atgaactgca ggacgaggca gcgcggctat cgtggctggc cacgacgggc gttccttgcg
     3361 cagctgtgct cgacgttgtc actgaagcgg gaagggactg gctgctattg ggcgaagtgc
     3421 cggggcagga tctcctgtca tctcaccttg ctcctgccga gaaagtatcc atcatggctg
     3481 atgcaatgcg gcggctgcat acgcttgatc cggctacctg cccattcgac caccaagcga
     3541 aacatcgcat cgagcgagca cgtactcgga tggaagccgg tcttgtcgat caggatgatc
     3601 tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc aaggcgcgca
     3661 tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg
     3721 tggaaaatgg ccgcttttct ggattcatcg actgtggccg gctgggtgtg gcggaccgct
     3781 atcaggacat agcgttggct acccgtgata ttgctgaaga gcttggcggc gaatgggctg
     3841 accgcttcct cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc gccttctatc
     3901 gccttcttga cgagttcttc tgagcgggac tctggggttc gaaatgaccg accaagcgac
     3961 gcccaacctg ccatcacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt
     4021 cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga
     4081 gttcttcgcc caccccaact tgtttattgc agcttataat ggttacaaat aaagcaatag
     4141 catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa
     4201 actcatcaat gtatcttatc atgtctgtat accgtcgacc tctagctaga gcttggcgta
     4261 atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat
     4321 acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt
     4381 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
     4441 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc
     4501 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     4561 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     4621 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     4681 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     4741 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     4801 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     4861 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     4921 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     4981 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     5041 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     5101 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     5161 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     5221 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     5281 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     5341 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     5401 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     5461 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     5521 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
     5581 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     5641 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     5701 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
     5761 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     5821 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     5881 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     5941 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     6001 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
     6061 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     6121 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     6181 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     6241 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     6301 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     6361 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga
     6421 cgtcgacgga tcgggagatc tcccgatccc ctatggtgca ctctcagtac aatctgctct
     6481 gatgccgcat agttaagcca gtatctgctc cctgcttgtg tgttggaggt cgctgagtag
     6541 tgcgcgagca aaatttaagc tacaacaagg caaggcttga ccgacaattg catgaagaat
     6601 ctgcttaggg ttaggcgttt tgcgctgctt cgcgatgtac gggccagata tacgcgtt
//
