LOCUS       sequence-406192-        7344 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  E. coli expression of full-length SARS-CoV-2 nucleocapsid with an 
            N-terminal mCherry tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7344)
  AUTHORS   Dumelie JG, Chen Q, Miller D, Attarwala N, Gross SS, Jaffrey SR
  TITLE     Biomolecular condensates create phospholipid-enriched 
            microenvironments.
  JOURNAL   Nat Chem Biol. 2023 Nov 16. doi: 10.1038/s41589-023-01474-4.
  PUBMED    37973889
REFERENCE   2  (bases 1 to 7344)
  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: "Nat Chem 
            Biol. 2023 Nov 16. doi: 10.1038/s41589-023-01474-4."
COMMENT     Sequence Label: pET28C-mCherry-SARS-CoV-2-Nucleocapsid
FEATURES             Location/Qualifiers
     source          1..7344
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      45..500
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(132..151)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     342..363
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(592..1407)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(592..1407)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     1315..1334
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1399..1418
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1529..2117
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2018..2037
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2271..2288
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2303..2442
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2368..2387)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2528..2550
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2544..2735)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3544..4626)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     4588..4607
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4627..4704)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4910..4929
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5013..5032
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5013..5031
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5032..5056
                     /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)."
     RBS             5071..5093
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5112..5129
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             5139..5156
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             5160..5192
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     CDS             5199..5906
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(5344..5362)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     5612..5631
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             7221..7238
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(7291..7309)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      join(7305..7344,1..8)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 gttttttgct gaaaggagga actatatccg gattggcgaa tgggacgcgc cctgtagcgg
       61 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
      121 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
      181 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
      241 cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac
      301 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
      361 tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat
      421 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa
      481 aatattaacg cttacaattt aggtggcact tttcggggaa atgtgcgcgg aacccctatt
      541 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgaattaatt cttagaaaaa
      601 ctcatcgagc atcaaatgaa actgcaattt attcatatca ggattatcaa taccatattt
      661 ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg aggcagttcc ataggatggc
      721 aagatcctgg tatcggtctg cgattccgac tcgtccaaca tcaatacaac ctattaattt
      781 cccctcgtca aaaataaggt tatcaagtga gaaatcacca tgagtgacga ctgaatccgg
      841 tgagaatggc aaaagtttat gcatttcttt ccagacttgt tcaacaggcc agccattacg
      901 ctcgtcatca aaatcactcg catcaaccaa accgttattc attcgtgatt gcgcctgagc
      961 gagacgaaat acgcgatcgc tgttaaaagg acaattacaa acaggaatcg aatgcaaccg
     1021 gcgcaggaac actgccagcg catcaacaat attttcacct gaatcaggat attcttctaa
     1081 tacctggaat gctgttttcc cggggatcgc agtggtgagt aaccatgcat catcaggagt
     1141 acggataaaa tgcttgatgg tcggaagagg cataaattcc gtcagccagt ttagtctgac
     1201 catctcatct gtaacatcat tggcaacgct acctttgcca tgtttcagaa acaactctgg
     1261 cgcatcgggc ttcccataca atcgatagat tgtcgcacct gattgcccga cattatcgcg
     1321 agcccattta tacccatata aatcagcatc catgttggaa tttaatcgcg gcctagagca
     1381 agacgtttcc cgttgaatat ggctcataac accccttgta ttactgttta tgtaagcaga
     1441 cagttttatt gttcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     1501 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     1561 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     1621 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc
     1681 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
     1741 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     1801 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     1861 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     1921 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     1981 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     2041 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     2101 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     2161 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta
     2221 ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag
     2281 tgagcgagga agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta
     2341 tttcacaccg caatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca
     2401 gtatacactc cgctatcgct acgtgactgg gtcatggctg cgccccgaca cccgccaaca
     2461 cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg
     2521 accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg
     2581 cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca
     2641 tccgcgtcca gctcgttgag tttctccaga agcgttaatg tctggcttct gataaagcgg
     2701 gccatgttaa gggcggtttt ttcctgtttg gtcactgatg cctccgtgta agggggattt
     2761 ctgttcatgg gggtaatgat accgatgaaa cgagagagga tgctcacgat acgggttact
     2821 gatgatgaac atgcccggtt actggaacgt tgtgagggta aacaactggc ggtatggatg
     2881 cggcgggacc agagaaaaat cactcagggt caatgccagc gcttcgttaa tacagatgta
     2941 ggtgttccac agggtagcca gcagcatcct gcgatgcaga tccggaacat aatggtgcag
     3001 ggcgctgact tccgcgtttc cagactttac gaaacacgga aaccgaagac cattcatgtt
     3061 gttgctcagg tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt
     3121 gattcattct gctaaccagt aaggcaaccc cgccagccta gccgggtcct caacgacagg
     3181 agcacgatca tgcgcacccg tggggccgcc atgccggcga taatggcctg cttctcgccg
     3241 aaacgtttgg tggcgggacc agtgacgaag gcttgagcga gggcgtgcaa gattccgaat
     3301 accgcaagcg acaggccgat catcgtcgcg ctccagcgaa agcggtcctc gccgaaaatg
     3361 acccagagcg ctgccggcac ctgtcctacg agttgcatga taaagaagac agtcataagt
     3421 gcggcgacga tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt gaaggctctc
     3481 aagggcatcg gtcgagatcc cggtgcctaa tgagtgagct aacttacatt aattgcgttg
     3541 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     3601 caacgcgcgg ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc ttttcaccag
     3661 tgagacgggc aacagctgat tgcccttcac cgcctggccc tgagagagtt gcagcaagcg
     3721 gtccacgctg gtttgcccca gcaggcgaaa atcctgtttg atggtggtta acggcgggat
     3781 ataacatgag ctgtcttcgg tatcgtcgta tcccactacc gagatatccg caccaacgcg
     3841 cagcccggac tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag
     3901 catcgcagtg ggaacgatgc cctcattcag catttgcatg gtttgttgaa aaccggacat
     3961 ggcactccag tcgccttccc gttccgctat cggctgaatt tgattgcgag tgagatattt
     4021 atgccagcca gccagacgca gacgcgccga gacagaactt aatgggcccg ctaacagcgc
     4081 gatttgctgg tgacccaatg cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg
     4141 ggagaaaata atactgttga tgggtgtctg gtcagagaca tcaagaaata acgccggaac
     4201 attagtgcag gcagcttcca cagcaatggc atcctggtca tccagcggat agttaatgat
     4261 cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc
     4321 gcttcgttct accatcgaca ccaccacgct ggcacccagt tgatcggcgc gagatttaat
     4381 cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag
     4441 caacgactgt ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc
     4501 catcgccgct tccacttttt cccgcgtttt cgcagaaacg tggctggcct ggttcaccac
     4561 gcgggaaacg gtctgataag agacaccggc atactctgcg acatcgtata acgttactgg
     4621 tttcacattc accaccctga attgactctc ttccgggcgc tatcatgcca taccgcgaaa
     4681 ggttttgcgc cattcgatgg tgtccgggat ctcgacgctc tcccttatgc gactcctgca
     4741 ttaggaagca gcccagtagt aggttgaggc cgttgagcac cgccgccgca aggaatggtg
     4801 catgcaagga gatggcgccc aacagtcccc cggccacggg gcctgccacc atacccacgc
     4861 cgaaacaagc gctcatgagc ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg
     4921 cgatataggc gccagcaacc gcacctgtgg cgccggtgat gccggccacg atgcgtccgg
     4981 cgtagaggat cgagatctcg atcccgcgaa attaatacga ctcactatag gggaattgtg
     5041 agcggataac aattcccctc tagaaataat tttgtttaac tttaagaagg agatatacca
     5101 tgggcagcag ccatcatcat catcatcaca gcagcggcct ggtgccgcgc ggcagccata
     5161 tggctagcat gactggtgga cagcaaatgg gtcggatcat ggtgagcaag ggcgaggagg
     5221 ataacatggc catcatcaag gagttcatgc gcttcaaggt gcacatggag ggctccgtga
     5281 acggccacga gttcgagatc gagggcgagg gcgagggccg cccctacgag ggcacccaga
     5341 ccgccaagct gaaggtgacc aagggtggcc ccctgccctt cgcctgggac atcctgtccc
     5401 ctcagttcat gtacggctcc aaggcctacg tgaagcaccc cgccgacatc cccgactact
     5461 tgaagctgtc cttccccgag ggcttcaagt gggagcgcgt gatgaacttc gaggacggcg
     5521 gcgtggtgac cgtgacccag gactcctccc tgcaggacgg cgagttcatc tacaaggtga
     5581 agctgcgcgg caccaacttc ccctccgacg gccccgtaat gcagaagaag accatgggct
     5641 gggaggcctc ctccgagcgg atgtaccccg aggacggcgc cctgaagggc gagatcaagc
     5701 agaggctgaa gctgaaggac ggcggccact acgacgctga ggtcaagacc acctacaagg
     5761 ccaagaagcc cgtgcagctg cccggcgcct acaacgtcaa catcaagttg gacatcacct
     5821 cccacaacga ggactacacc atcgtggaac agtacgaacg cgccgagggc cgccactcca
     5881 ccggcggcat ggacgagctg tacaagggtg cgcctggttc tgcaggctcc gcagctggcg
     5941 gatcggggat catgagcgat aacggccccc aaaaccaacg gaatgcccct cggataacat
     6001 ttggtggccc tagtgattcc acaggaagta accaaaacgg agaacggtct ggagcccgct
     6061 ctaaacagag gcgaccacag ggattgccaa ataacacagc gtcatggttt acagccctga
     6121 ctcaacatgg aaaggaggac ttgaaatttc cacgaggaca gggtgtacca ataaacacaa
     6181 acagtagtcc agacgatcag atcgggtatt atagaagggc cacgagacgc atcagaggtg
     6241 gagatggtaa aatgaaagat ctcagtccgc gctggtactt ttattatttg ggaaccggcc
     6301 cagaagcggg cctcccatat ggagccaata aggacgggat tatatgggtg gctacagagg
     6361 gagccctcaa tacgccaaaa gatcacatcg gaacaagaaa ccctgctaat aatgccgcaa
     6421 tcgtgctgca gttgcctcag gggacgacgc ttcctaaagg cttttacgca gaaggatcac
     6481 gcggcggcag ccaagcatcc tcaaggtcta gttccagaag tcgaaacagc tccagaaact
     6541 ccacaccagg gtccagtagg ggcacaagtc cggcgcggat ggcgggcaac ggcggagacg
     6601 ccgcactcgc tctgttgctc ctggaccggc tcaaccaact tgaatccaag atgagtggta
     6661 agggacagca gcaacaaggt caaaccgtaa ccaagaaaag cgctgcagaa gcttctaaaa
     6721 aacctcgaca aaaacggacc gctacgaagg catataacgt tactcaagcc ttcgggagac
     6781 gagggccgga gcaaacccag gggaattttg gagatcagga gctcatccgg caagggacag
     6841 attacaaaca ttggcctcaa atcgctcagt tcgcgccgtc cgcaagtgct tttttcggaa
     6901 tgtctcgcat cggtatggaa gtaacaccat ccgggacatg gctcacttat accggcgcca
     6961 tcaagctgga cgacaaagat ccaaatttca aagatcaggt gattctcttg aataaacaca
     7021 tagacgcgta taagacgttt ccacccaccg aacccaagaa ggacaaaaag aaaaaagctg
     7081 atgaaacaca agcgttgccc cagcgacaga agaagcagca aaccgtcacc ctcctgccag
     7141 ccgctgatct cgatgacttc tccaagcaac ttcaacagtc catgagctca gccgattcta
     7201 ctcaggcgct ctaagtcgag caccaccacc accaccactg agatccggct gctaacaaag
     7261 cccgaaagga agctgagttg gctgctgcca ccgctgagca ataactagca taaccccttg
     7321 gggcctctaa acgggtcttg aggg
//
