LOCUS       sequence-348181-        6658 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Transient mammalian expression of codon optimized SARS-CoV-2 
            Nucleocapsid (T205I). 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-T205I
FEATURES             Location/Qualifiers
     source          1..6658
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        111..490
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        491..694
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     645..665
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     739..758
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        739..757
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2125..2142)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2131..2355
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2401..2829
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2488..2507)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2698..2719
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2838..2858)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2843..3172
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3023..3158
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3085..3104
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3239..4033
                     /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(3293..3312)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3903..3922
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4207..4328
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4244..4263)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4298..4317
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4377..4393)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4377..4393)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4390..4412)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4401..4417
                     /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(4425..4431,4432..4449,4450..4455))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4470..4491
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4608..4625)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4779..5367)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4859..4878)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5538..6329,6330..6398))
                     /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     6161..6180
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6399..6503)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6578..6597)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 caaaatttaa gctacaacaa ggcaaggctt gaccgacaat tgcatgaaga atctgcttag
       61 ggttaggcgt tttgcgctgc ttcgcgatgt acgggccaga tatacgcgtt gacattgatt
      121 attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga
      181 gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg
      241 cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg
      301 acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca
      361 tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc
      421 ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc
      481 tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc
      541 acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa
      601 tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag
      661 gcgtgtacgg tgggaggtct atataagcag agctctctgg ctaactagag aacccactgc
      721 ttactggctt atcgaaatta atacgactca ctatagggag acccaagctg gctagccttc
      781 catttcaggt gtcgtgagga tctatttccg gtgaattcgc cgccaccatg agcgacaacg
      841 gcccccaaaa ccaacggaat gcccctcgga taacatttgg tggccctagt gattccacag
      901 gaagtaacca aaacggagaa cggtctggag cccgctctaa acagaggcga ccacagggat
      961 tgccaaataa cacagcgtca tggtttacag ccctgactca acatggaaag gaggacttga
     1021 aatttccacg aggacagggt gtaccaataa acacaaacag tagtccagac gatcagatcg
     1081 ggtattatag aagggccacg agacgcatca gaggtggaga tggtaaaatg aaagatctca
     1141 gtccgcgctg gtacttttat tatttgggaa ccggcccaga agcgggcctc ccatatggag
     1201 ccaataagga cgggattata tgggtggcta cagagggagc cctcaatacg ccaaaagatc
     1261 acatcggaac aagaaaccct gctaataatg ccgcaatcgt gctgcagttg cctcagggga
     1321 cgacgcttcc taaaggcttt tacgcagaag gatcacgcgg cggcagccaa gcatcctcaa
     1381 ggtctagttc cagaagtcga aacagctcca gaaactccac accagggtcc agtaggggca
     1441 tcagtccggc gcggatggcg ggcaacggcg gagacgccgc actcgctctg ttgctcctgg
     1501 accggctcaa ccaacttgaa tccaagatga gtggtaaggg acagcagcaa caaggtcaaa
     1561 ccgtaaccaa gaaaagcgct gcagaagctt ctaaaaaacc tcgacaaaaa cggaccgcta
     1621 cgaaggcata taacgttact caagccttcg ggagacgagg gccggagcaa acccagggga
     1681 attttggaga tcaggagctc atccggcaag ggacagatta caaacattgg cctcaaatcg
     1741 ctcagttcgc gccgtccgca agtgcttttt tcggaatgtc tcgcatcggt atggaagtaa
     1801 caccatccgg gacatggctc acttataccg gcgccatcaa gctggacgac aaagatccaa
     1861 atttcaaaga tcaggtgatt ctcttgaata aacacataga cgcgtataag acgtttccac
     1921 ccaccgaacc caagaaggac aaaaagaaaa aagctgatga aacacaagcg ttgccccagc
     1981 gacagaagaa gcagcaaacc gtcaccctcc tgccagccgc tgatctcgat gacttctcca
     2041 agcaacttca acagtccatg agctcagccg attctactca ggcgtaactc gagtctagag
     2101 ggcccgttta aacccgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg
     2161 tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct
     2221 aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg
     2281 gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggatg
     2341 cggtgggctc tatggcttct gaggcggaaa gaaccagctg gggctctagg gggtatcccc
     2401 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
     2461 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
     2521 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
     2581 gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc
     2641 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
     2701 gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat
     2761 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
     2821 acgcgaatta attctgtgga atgtgtgtca gttagggtgt ggaaagtccc caggctcccc
     2881 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt gtggaaagtc
     2941 cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat
     3001 agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc
     3061 gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct ctgcctctga
     3121 gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca aaaagctccc
     3181 gggagcttgt atatccattt tcggatctga tcaagagaca ggatgaggat cgtttcgcat
     3241 gattgaacaa gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg
     3301 ctatgactgg gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc
     3361 gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca
     3421 ggacgaggca gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct
     3481 cgacgttgtc actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga
     3541 tctcctgtca tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg
     3601 gcggctgcat acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat
     3661 cgagcgagca cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga
     3721 gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc aaggcgcgca tgcccgacgg
     3781 cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg
     3841 ccgcttttct ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat
     3901 agcgttggct acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct
     3961 cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga
     4021 cgagttcttc tgagcgggac tctggggttc gaaatgaccg accaagcgac gcccaacctg
     4081 ccatcacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt
     4141 ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc
     4201 caccccaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat
     4261 ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat
     4321 gtatcttatc atgtctgtat accgtcgacc tctagctaga gcttggcgta atcatggtca
     4381 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga
     4441 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg
     4501 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     4561 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
     4621 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     4681 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     4741 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     4801 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     4861 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     4921 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     4981 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     5041 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     5101 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     5161 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga
     5221 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     5281 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     5341 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     5401 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     5461 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     5521 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     5581 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     5641 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     5701 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     5761 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     5821 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     5881 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     5941 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     6001 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     6061 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     6121 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     6181 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     6241 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     6301 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     6361 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     6421 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     6481 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtcgacgga
     6541 tcgggagatc tcccgatccc ctatggtgca ctctcagtac aatctgctct gatgccgcat
     6601 agttaagcca gtatctgctc cctgcttgtg tgttggaggt cgctgagtag tgcgcgag
//
