LOCUS       sequence-485114-        6385 bp    DNA     circular SYN 02-SEP-2025
DEFINITION  GFP and the Ubl1 domain of the SARS-CoV-2 Nsp3 protein (aa 1-111).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6385)
  AUTHORS   Favetta B, Wang H, Cubuk J, Singh A, Barai M, Ramirez C, Gormley AJ,
            Murthy S, Dignon G, Soranno A, Shi Z, Schuster BS
  TITLE     Phosphorylation Toggles the SARS-CoV-2 Nucleocapsid Protein Between 
            Two Membrane-Associated Condensate States.
  JOURNAL   bioRxiv [Preprint]. 2025 Jun 10:2024.10.17.618867. doi: 
            10.1101/2024.10.17.618867.
  PUBMED    39464032
REFERENCE   2  (bases 1 to 6385)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Sep 2, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "bioRxiv 
            [Preprint]. 2025 Jun 10:2024.10.17.618867. doi: 
            10.1101/2024.10.17.618867."
COMMENT     Sequence Label: GFP-Nsp3(1-111)
FEATURES             Location/Qualifiers
     source          1..6385
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      115..703
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     604..623
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     857..874
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    889..1028
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(954..973)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1114..1136
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(1130..1321)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(2130..3212)
                     /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     3174..3193
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3213..3290)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     3496..3515
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     3599..3618
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3599..3617
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3618..3642
                     /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             3657..3679
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3698..3715
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             join(3725..3727,3728..4438)
                     /codon_start=1
                     /product="enhanced GFP (EGFP) derivative that exhibits 
                     robust folding and enhanced photostability (Zhong et al., 
                     2019)"
                     /label=SiriusGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(3767..3788)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     4375..4396
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             4448..4468
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             4847..4864
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4917..4935)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4931..4978
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      5015..5470
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5102..5121)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5312..5333
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(5563..6378)
                     /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(5563..6378)
                     /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     6286..6305
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     join(6370..6385,1..4)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
ORIGIN
        1 cttgtattac tgtttatgta agcagacagt tttattgttc atgaccaaaa tcccttaacg
       61 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
      121 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
      181 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
      241 agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa
      301 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
      361 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
      421 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
      481 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
      541 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
      601 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
      661 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
      721 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc
      781 ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag
      841 ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta
      901 ttttctcctt acgcatctgt gcggtatttc acaccgcaat ggtgcactct cagtacaatc
      961 tgctctgatg ccgcatagtt aagccagtat acactccgct atcgctacgt gactgggtca
     1021 tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc
     1081 cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt
     1141 caccgtcatc accgaaacgc gcgaggcagc tgcggtaaag ctcatcagcg tggtcgtgaa
     1201 gcgattcaca gatgtctgcc tgttcatccg cgtccagctc gttgagtttc tccagaagcg
     1261 ttaatgtctg gcttctgata aagcgggcca tgttaagggc ggttttttcc tgtttggtca
     1321 ctgatgcctc cgtgtaaggg ggatttctgt tcatgggggt aatgataccg atgaaacgag
     1381 agaggatgct cacgatacgg gttactgatg atgaacatgc ccggttactg gaacgttgtg
     1441 agggtaaaca actggcggta tggatgcggc gggaccagag aaaaatcact cagggtcaat
     1501 gccagcgctt cgttaataca gatgtaggtg ttccacaggg tagccagcag catcctgcga
     1561 tgcagatccg gaacataatg gtgcagggcg ctgacttccg cgtttccaga ctttacgaaa
     1621 cacggaaacc gaagaccatt catgttgttg ctcaggtcgc agacgttttg cagcagcagt
     1681 cgcttcacgt tcgctcgcgt atcggtgatt cattctgcta accagtaagg caaccccgcc
     1741 agcctagccg ggtcctcaac gacaggagca cgatcatgcg cacccgtggg gccgccatgc
     1801 cggcgataat ggcctgcttc tcgccgaaac gtttggtggc gggaccagtg acgaaggctt
     1861 gagcgagggc gtgcaagatt ccgaataccg caagcgacag gccgatcatc gtcgcgctcc
     1921 agcgaaagcg gtcctcgccg aaaatgaccc agagcgctgc cggcacctgt cctacgagtt
     1981 gcatgataaa gaagacagtc ataagtgcgg cgacgatagt catgccccgc gcccaccgga
     2041 aggagctgac tgggttgaag gctctcaagg gcatcggtcg agatcccggt gcctaatgag
     2101 tgagctaact tacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
     2161 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
     2221 gccagggtgg tttttctttt caccagtgag acgggcaaca gctgattgcc cttcaccgcc
     2281 tggccctgag agagttgcag caagcggtcc acgctggttt gccccagcag gcgaaaatcc
     2341 tgtttgatgg tggttaacgg cgggatataa catgagctgt cttcggtatc gtcgtatccc
     2401 actaccgaga tatccgcacc aacgcgcagc ccggactcgg taatggcgcg cattgcgccc
     2461 agcgccatct gatcgttggc aaccagcatc gcagtgggaa cgataccctc attcagcatt
     2521 tgcatggttt gttgaaaacc ggacatggca ctccagtcgc cttcccgttc cgctatcggc
     2581 tgaatttgat tgcgagtgag atatttatgc cagccagcca gacgcagacg cgccgagaca
     2641 gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc
     2701 acgcccagtc gcgtaccgtc ttcatgggag aaaataatac tgttgatggg tgtctggtca
     2761 gagacatcaa gaaataacgc cggaacatta gtgcaggcag cttccacagc aatggcatcc
     2821 tggtcatcca gcggatagtt aatgatcagc ccactgacgc gttgcgcgag aagattgtgc
     2881 accgccgctt tacaggcttc gacgccgctt cgttctacca tcgacaccac cacgctggca
     2941 cccagttgat cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc
     3001 agactggagg tggcaacgcc aatcagcaac gactgtttgc ccgccagttg ttgtgccacg
     3061 cggttgggaa tgtaattcag ctccgccatc gccgcttcca ctttttcccg cgttttcgca
     3121 gaaacgtggc tggcctggtt caccacgcgg gaaacggtct gataagagac accggcatac
     3181 tctgcgacat cgtataacgt tactggtttc acattcacca ccctgaattg actctcttcc
     3241 gggcgctatc atgccatacc gcgaaaggtt ttgcgccatt cgatggtgtc cgggatctcg
     3301 acgctctccc ttatgcgact cctgcattag gaagcagccc agtagtaggt tgaggccgtt
     3361 gagcaccgcc gccgcaagga atggtgcatg caaggagatg gcgcccaaca gtcccccggc
     3421 cacggggcct gccaccatac ccacgccgaa acaagcgctc atgagcccga agtggcgagc
     3481 ccgatcttcc ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc
     3541 ggtgatgccg gccacgatgc gtccggcgta gaggatcgag atctcgatcc cgcgaaatta
     3601 atacgactca ctatagggga attgtgagcg gataacaatt cccctctaga aataattttg
     3661 tttaacttta agaaggagat ataccatggg ttcttctcac catcaccatc accatggttc
     3721 ttctgtgagc aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga
     3781 cggcgacgta aacggccaca agttcagcgt gcgcggcgag ggcgagggcg atgccaccaa
     3841 cggcaagctg accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac
     3901 cctcgtgacc accctgacct acggcgtgca gtgcttcagc cgctaccccg accacatgaa
     3961 gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctc
     4021 cttcaaggac gacggcacct acaagacccg cgccgaggtg aagttcgagg gcgacaccct
     4081 ggtgaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca
     4141 caagctggag tacaacttca acagccacaa cgtctatatc acggccgaca agcagaagaa
     4201 cggcatcaag gcgaacttca agatccgcca caacgtcgag gacggcagcg tgcagctcgc
     4261 cgaccactac cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca
     4321 ctacctgagc acccagtcca agctgagcaa agaccccaac gagaagcgcg atcacatggt
     4381 cctgctggag ttcgtgaccg ccgccgggat cactctcggc atggacgagc tgtacaaggg
     4441 gatcgaggaa aacctgtact tccaatccaa tgcatcttct aatggcgcac cgacaaaagt
     4501 tacatttgga gacgataccg tgatcgaagt tcagggctac aaaagcgtga acatcacctt
     4561 cgagctggat gaacgtatcg ataaagtgct gaacgagaaa tgcagcgcat ataccgtgga
     4621 actgggtacc gaagtgaacg aatttgcctg tgttgttgca gatgcagtga tcaaaacctt
     4681 acagccggtt agcgaactgc tgacaccttt aggcattgat ctggatgaat ggagcatggc
     4741 aacctattat ctgttcgacg aaagcggcga gttcaaactg gcatcacaca tgtattgcag
     4801 cttctatccg cctgatgaat aataaaagct tgcggccgca ctcgagcacc accaccacca
     4861 ccactgagat ccggctgcta acaaagcccg aaaggaagct gagttggctg ctgccaccgc
     4921 tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct
     4981 gaaaggagga actatatccg gattggcgaa tgggacgcgc cctgtagcgg cgcattaagc
     5041 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc
     5101 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
     5161 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
     5221 aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc
     5281 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca
     5341 ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat
     5401 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa actagtaacg
     5461 tttacaattt caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt
     5521 ttctaaatac attcaaatat gtatccgctc atgaattaat tcttagaaaa actcatcgag
     5581 catcaaatga aactgcaatt tattcatatc aggattatca ataccatatt tttgaaaaag
     5641 ccgtttctgt aatgaaggag aaaactcacc gaggcagttc cataggatgg caagatcctg
     5701 gtatcggtct gcgattccga ctcgtccaac atcaatacaa cctattaatt tcccctcgtc
     5761 aaaaataagg ttatcaagtg agaaatcacc atgagtgacg actgaatccg gtgagaatgg
     5821 caaaagttta tgcatttctt tccagacttg ttcaacaggc cagccattac gctcgtcatc
     5881 aaaatcactc gcatcaacca aaccgttatt cattcgtgat tgcgcctgag cgagacgaaa
     5941 tacgcgatcg ctgttaaaag gacaattaca aacaggaatc gaatgcaacc ggcgcaggaa
     6001 cactgccagc gcatcaacaa tattttcacc tgaatcagga tattcttcta atacctggaa
     6061 tgctgttttc ccggggatcg cagtggtgag taaccatgca tcatcaggag tacggataaa
     6121 atgcttgatg gtcggaagag gcataaattc cgtcagccag tttagtctga ccatctcatc
     6181 tgtaacatca ttggcaacgc tacctttgcc atgtttcaga aacaactctg gcgcatcggg
     6241 cttcccatac aatcgataga ttgtcgcacc tgattgcccg acattatcgc gagcccattt
     6301 atacccatat aaatcagcat ccatgttgga atttaatcgc ggcctagagc aagacgtttc
     6361 ccgttgaata tggctcataa caccc
//
