LOCUS       sequence-307842-        7710 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  express MBP tagged SARS-CoV-2 Nucleocapsid protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7710)
  AUTHORS   Perdikari TM, Murthy AC, Ryan VH, Watters S, Naik MT, Fawzi NL
  TITLE     SARS-CoV-2 nucleocapsid protein phase-separates with RNA and with 
            human hnRNPs.
  JOURNAL   EMBO J. 2020 Nov 17:e106478. doi: 10.15252/embj.2020106478.
  PUBMED    33200826
REFERENCE   2  (bases 1 to 7710)
  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: "EMBO J. 
            2020 Nov 17:e106478. doi: 10.15252/embj.2020106478."
COMMENT     Sequence Label: SARS-CoV-2_N_FL
FEATURES             Location/Qualifiers
     source          1..7710
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /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             59..81
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             106..123
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             136..1233
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="KIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEE
                     KFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLI
                     AYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAA
                     DGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGET
                     AMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLE
                     NYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFW
                     YAVRTAVINAASGRQTVDEALKDAQT"
     regulatory      1090..1099
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     1207..1230
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     protein_bind    1252..1276
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1285..1305
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             complement(1895..1912)
                     /codon_start=1
                     /product="tetracysteine peptide that binds biarsenical 
                     labeling reagents"
                     /label=tetracysteine tag
                     /translation="CCPGCC"
     CDS             2575..2592
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2645..2663)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2659..2706
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2743..3198
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2830..2849)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3040..3061
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3290..4105)
                     /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(3290..4105)
                     /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     4013..4032
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4097..4116
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4227..4815
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4716..4735
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4969..4986
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5001..5140
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5066..5085)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5226..5248
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5242..5433)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(6242..7324)
                     /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     7286..7305
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7325..7402)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7608..7627
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atataccatg ggatctgata aaattcatca tcatcatcat
      121 cacagcagcg gtaccaaaat cgaagaaggt aaactggtaa tctggattaa cggcgataaa
      181 ggctataacg gtctcgctga agtcggtaag aaattcgaga aagataccgg aattaaagtc
      241 accgttgagc atccggataa actggaagag aaattcccac aggttgcggc aactggcgat
      301 ggccctgaca ttatcttctg ggcacacgac cgctttggtg gctacgctca atctggcctg
      361 ttggctgaaa tcaccccgga caaagcgttc caggacaagc tgtatccgtt tacctgggat
      421 gccgtacgtt acaacggcaa gctgattgct tacccgatcg ctgttgaagc gttatcgctg
      481 atttataaca aagatctgct gccgaacccg ccaaaaacct gggaagagat cccggcgctg
      541 gataaagaac tgaaagcgaa aggtaagagc gcgctgatgt tcaacctgca agaaccgtac
      601 ttcacctggc cgctgattgc tgctgacggg ggttatgcgt tcaagtatga aaacggcaag
      661 tacgacatta aagacgtggg cgtggataac gctggcgcga aagcgggtct gaccttcctg
      721 gttgacctga ttaaaaacaa acacatgaat gcagacaccg attactccat cgcagaagct
      781 gcctttaata aaggcgaaac agcgatgacc atcaacggcc cgtgggcatg gtccaacatc
      841 gacaccagca aagtgaatta tggtgtaacg gtactgccga ccttcaaggg tcaaccatcc
      901 aaaccgttcg ttggcgtgct gagcgcaggt attaacgccg ccagtccgaa caaagagctg
      961 gcaaaagagt tcctcgaaaa ctatctgctg actgatgaag gtctggaagc ggttaataaa
     1021 gacaaaccgc tgggtgccgt agcgctgaag tcttacgagg aagagttggc gaaagatcca
     1081 cgtattgccg ccaccatgga aaacgcccag aaaggtgaaa tcatgccgaa catcccgcag
     1141 atgtccgctt tctggtatgc cgtgcgtact gcggtgatca acgccgccag cggtcgtcag
     1201 actgtcgatg aagccctgaa agacgcgcag actaattcgg gatctgatat cacaagtttg
     1261 tacaaaaaag ctgaaggcgg taccgagaac ctgtacttcc agggtcatat gagcgacaat
     1321 ggtccgcaga atcaacgtaa tgcgccgcgt atcacctttg gtggtccgag cgacagcacc
     1381 ggcagcaatc agaatggcga acgtagcggt gcgcgtagca aacagcgtcg tccgcagggt
     1441 ctgccgaaca acaccgcgag ctggtttacc gcgctgaccc aacacggcaa agaagacctg
     1501 aaattcccgc gtggtcaagg cgttccgatc aacaccaaca gcagcccgga tgaccagatt
     1561 ggctattacc gtcgtgcgac ccgtcgtatt cgtggcggtg atggcaagat gaaagacctg
     1621 agcccgcgtt ggtatttcta ctatctgggt accggcccgg aggcgggtct gccgtatggt
     1681 gcgaacaagg acggtatcat ttgggtggcg accgaaggcg cgctgaacac cccgaaggac
     1741 cacatcggta cccgtaaccc ggcgaacaac gcggcgatcg ttctgcagct gccgcaaggt
     1801 accaccctgc cgaaaggttt ctatgcggag ggtagccgtg gcggtagcca agcgagcagc
     1861 cgtagcagca gccgtagccg taacagcagc cgtaacagca ccccgggcag cagccgtggt
     1921 accagcccgg cgcgtatggc gggcaacggt ggtgacgcgg cgctggcgct gctgctgctg
     1981 gatcgtctga accaactgga gagcaagatg agcggcaagg gtcaacagca acaaggtcaa
     2041 accgttacca agaaaagcgc ggcggaggcg agcaaaaagc cgcgtcagaa acgtaccgcg
     2101 accaaagcgt acaacgtgac ccaagcgttt ggtcgtcgtg gtccggaaca aacccagggt
     2161 aacttcggtg atcaggaact gattcgtcaa ggtaccgact acaaacactg gccgcaaatc
     2221 gcgcagttcg cgccgagcgc gagcgcgttc tttggtatga gccgtatcgg tatggaagtt
     2281 accccgagcg gtacctggct gacctatacc ggtgcgatca agctggacga taaggatccg
     2341 aacttcaagg accaggttat cctgctgaac aagcacatcg acgcgtacaa aaccttcccg
     2401 ccgaccgagc cgaagaaaga caagaagaag aaagcggacg agacccaagc gctgccgcaa
     2461 cgtcagaaaa agcaacagac cgttaccctg ctgccggcgg cggatctgga cgactttagc
     2521 aaacaactgc aacagagcat gagcagcgcg gatagcaccc aggcgtaact cgagcaccac
     2581 caccaccacc actgagatcc ggctgctaac aaagcccgaa aggaagctga gttggctgct
     2641 gccaccgctg agcaataact agcataaccc cttggggcct ctaaacgggt cttgaggggt
     2701 tttttgctga aaggaggaac tatatccgga ttggcgaatg ggacgcgccc tgtagcggcg
     2761 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc
     2821 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc
     2881 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg
     2941 accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg
     3001 tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg
     3061 gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt
     3121 cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa
     3181 tattaacgct tacaatttag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
     3241 tttatttttc taaatacatt caaatatgta tccgctcatg aattaattct tagaaaaact
     3301 catcgagcat caaatgaaac tgcaatttat tcatatcagg attatcaata ccatattttt
     3361 gaaaaagccg tttctgtaat gaaggagaaa actcaccgag gcagttccat aggatggcaa
     3421 gatcctggta tcggtctgcg attccgactc gtccaacatc aatacaacct attaatttcc
     3481 cctcgtcaaa aataaggtta tcaagtgaga aatcaccatg agtgacgact gaatccggtg
     3541 agaatggcaa aagtttatgc atttctttcc agacttgttc aacaggccag ccattacgct
     3601 cgtcatcaaa atcactcgca tcaaccaaac cgttattcat tcgtgattgc gcctgagcga
     3661 gacgaaatac gcgatcgctg ttaaaaggac aattacaaac aggaatcgaa tgcaaccggc
     3721 gcaggaacac tgccagcgca tcaacaatat tttcacctga atcaggatat tcttctaata
     3781 cctggaatgc tgttttcccg gggatcgcag tggtgagtaa ccatgcatca tcaggagtac
     3841 ggataaaatg cttgatggtc ggaagaggca taaattccgt cagccagttt agtctgacca
     3901 tctcatctgt aacatcattg gcaacgctac ctttgccatg tttcagaaac aactctggcg
     3961 catcgggctt cccatacaat cgatagattg tcgcacctga ttgcccgaca ttatcgcgag
     4021 cccatttata cccatataaa tcagcatcca tgttggaatt taatcgcggc ctagagcaag
     4081 acgtttcccg ttgaatatgg ctcataacac cccttgtatt actgtttatg taagcagaca
     4141 gttttattgt tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     4201 cccgtagaaa agatcaaagg atcttcttga tatccttttt ttctgcgcgt aatctgctgc
     4261 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     4321 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta
     4381 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     4441 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     4501 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     4561 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     4621 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     4681 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     4741 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     4801 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     4861 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc
     4921 gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg
     4981 agcgaggaag cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt
     5041 tcacaccgca atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt
     5101 atacactccg ctatcgctac gtgactgggt catggctgcg ccccgacacc cgccaacacc
     5161 cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac
     5221 cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca
     5281 gctgcggtaa agctcatcag cgtggtcgtg aagcgattca cagatgtctg cctgttcatc
     5341 cgcgtccagc tcgttgagtt tctccagaag cgttaatgtc tggcttctga taaagcgggc
     5401 catgttaagg gcggtttttt cctgtttggt cactgatgcc tccgtgtaag ggggatttct
     5461 gttcatgggg gtaatgatac cgatgaaacg agagaggatg ctcacgatac gggttactga
     5521 tgatgaacat gcccggttac tggaacgttg tgagggtaaa caactggcgg tatggatgcg
     5581 gcgggaccag agaaaaatca ctcagggtca atgccagcgc ttcgttaata cagatgtagg
     5641 tgttccacag ggtagccagc agcatcctgc gatgcagatc cggaacataa tggtgcaggg
     5701 cgctgacttc cgcgtttcca gactttacga aacacggaaa ccgaagacca ttcatgttgt
     5761 tgctcaggtc gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga
     5821 ttcattctgc taaccagtaa ggcaaccccg ccagcctagc cgggtcctca acgacaggag
     5881 cacgatcatg cgcacccgtg gggccgccat gccggcgata atggcctgct tctcgccgaa
     5941 acgtttggtg gcgggaccag tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac
     6001 cgcaagcgac aggccgatca tcgtcgcgct ccagcgaaag cggtcctcgc cgaaaatgac
     6061 ccagagcgct gccggcacct gtcctacgag ttgcatgata aagaagacag tcataagtgc
     6121 ggcgacgata gtcatgcccc gcgcccaccg gaaggagctg actgggttga aggctctcaa
     6181 gggcatcggt cgagatcccg gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg
     6241 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
     6301 acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg
     6361 agacgggcaa cagctgattg cccttcaccg cctggccctg agagagttgc agcaagcggt
     6421 ccacgctggt ttgccccagc aggcgaaaat cctgtttgat ggtggttaac ggcgggatat
     6481 aacatgagct gtcttcggta tcgtcgtatc ccactaccga gatatccgca ccaacgcgca
     6541 gcccggactc ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca
     6601 tcgcagtggg aacgatgccc tcattcagca tttgcatggt ttgttgaaaa ccggacatgg
     6661 cactccagtc gccttcccgt tccgctatcg gctgaatttg attgcgagtg agatatttat
     6721 gccagccagc cagacgcaga cgcgccgaga cagaacttaa tgggcccgct aacagcgcga
     6781 tttgctggtg acccaatgcg accagatgct ccacgcccag tcgcgtaccg tcttcatggg
     6841 agaaaataat actgttgatg ggtgtctggt cagagacatc aagaaataac gccggaacat
     6901 tagtgcaggc agcttccaca gcaatggcat cctggtcatc cagcggatag ttaatgatca
     6961 gcccactgac gcgttgcgcg agaagattgt gcaccgccgc tttacaggct tcgacgccgc
     7021 ttcgttctac catcgacacc accacgctgg cacccagttg atcggcgcga gatttaatcg
     7081 ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca
     7141 acgactgttt gcccgccagt tgttgtgcca cgcggttggg aatgtaattc agctccgcca
     7201 tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc
     7261 gggaaacggt ctgataagag acaccggcat actctgcgac atcgtataac gttactggtt
     7321 tcacattcac caccctgaat tgactctctt ccgggcgcta tcatgccata ccgcgaaagg
     7381 ttttgcgcca ttcgatggtg tccgggatct cgacgctctc ccttatgcga ctcctgcatt
     7441 aggaagcagc ccagtagtag gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca
     7501 tgcaaggaga tggcgcccaa cagtcccccg gccacggggc ctgccaccat acccacgccg
     7561 aaacaagcgc tcatgagccc gaagtggcga gcccgatctt ccccatcggt gatgtcggcg
     7621 atataggcgc cagcaaccgc acctgtggcg ccggtgatgc cggccacgat gcgtccggcg
     7681 tagaggatcg agatctcgat cccgcgaaat
//
