LOCUS       sequence-418177-        6320 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses the C terminal of SARS-CoV-2 NSP3 fused with EGFP tag in 
            mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6320)
  AUTHORS   Li J, Gui Q, Liang FX, Sall J, Zhang Q, Duan Y, Dhabaria A, Askenazi
            M, Ueberheide B, Stapleford KA, Pagano M
  TITLE     The REEP5/TRAM1 complex binds SARS-CoV-2 NSP3 and promotes virus 
            replication.
  JOURNAL   J Virol. 2023 Sep 28:e0050723. doi: 10.1128/jvi.00507-23.
  PUBMED    37768083
REFERENCE   2  (bases 1 to 6320)
  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: "J Virol. 
            2023 Sep 28:e0050723. doi: 10.1128/jvi.00507-23."
COMMENT     Sequence Label: NSP3C-EGFP
FEATURES             Location/Qualifiers
     source          1..6320
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        62..365
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        366..569
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     520..540
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    623..647
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      2261..2270
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2267..2269,2270..2272,2273..2986)
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2312..2333)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2573..2592)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2920..2941
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    3109..3230
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3146..3165)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3200..3219
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3237..3692)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3374..3395)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3586..3605
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3719..3823
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3825..4182
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3825..4016
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3848..3868)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4033..4168
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4217..5011
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             4217..5011
                     /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
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4271..4290)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4881..4900
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5199..5218)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5243..5290
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5619..6207
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6108..6127
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ttagttatta atagtaatca attacggggt cattagttca tagcccatat atggagttcc
       61 gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat
      121 tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc
      181 aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc
      241 caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt
      301 acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta
      361 ccatggtgat gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg
      421 gatttccaag tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac
      481 gggactttcc aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg
      541 tacggtggga ggtctatata agcagagctg gtttagtgaa ccgtcagatc cgctagcgct
      601 accggactca gatctcgaga tgccaacttt gtacaaaaaa gttggcatgt ggttcctgct
      661 gctctccgtg tgcctgggtt ccctgatcta ctctaccgcc gctctgggag tgctgatgtc
      721 taacctgggt atgcctagct actgcactgg ttacagagag ggatacctga acagcaccaa
      781 cgtgaccatc gccacctact gcaccggcag catcccatgc tccgtgtgcc tgtctggact
      841 ggacagcctg gacacctacc cctccctgga aaccatccag atcaccatct ctagcttcaa
      901 gtgggacctg accgccttcg gtctggtggc tgagtggttc ctggcctaca tcctgttcac
      961 caggttcttc tacgtgctgg gcctggccgc tatcatgcag ctgttcttct cctacttcgc
     1021 tgtgcacttc atcagcaact cctggctgat gtggctgatc atcaacctgg tgcagatggc
     1081 tcccatctct gccatggtgc gcatgtacat cttcttcgcc agcttctact acgtgtggaa
     1141 gtcctacgtg cacgtggtgg acggttgcaa ctcctctacc tgcatgatgt gctacaagcg
     1201 caacagggct accagggtgg agtgcaccac catcgtgaac ggcgtgcgca ggtccttcta
     1261 cgtgtacgcc aacggcggca agggattctg caagctgcac aactggaact gcgtgaactg
     1321 cgacaccttc tgcgctggct ccaccttcat ctctgacgag gtggccagag acctgagcct
     1381 gcagttcaag cgtccaatca accccaccga ccagagctcc tacatcgtgg actctgtgac
     1441 cgtgaagaac ggtagcatcc acctgtactt cgacaaggct ggccagaaga cctacgagcg
     1501 ccactctctg agccacttcg tgaacctgga caacctgagg gccaacaaca ccaagggcag
     1561 cctgcctatc aacgtgatcg tgttcgacgg aaagtccaag tgcgaggaat ctagcgccaa
     1621 gtccgccagc gtgtactaca gccagctgat gtgccagcca atcctgctgc tggaccaggc
     1681 tctggtgtct gacgtgggag acagcgccga agtggccgtg aagatgttcg acgcctacgt
     1741 gaacaccttc tcctctacct tcaacgtgcc tatggagaag ctgaagaccc tggtggctac
     1801 tgctgaggct gaactggcta agaacgtgtc cctggacaac gtgctgagca ccttcatctc
     1861 cgccgctagg cagggtttcg tggactctga cgtggaaacc aaagacgtgg tggagtgcct
     1921 gaagctgtct caccagagcg acatcgaagt gaccggcgac tcctgcaaca actacatgct
     1981 gacctacaac aaggtggaga acatgacccc aagagacctg ggagcttgca tcgactgctc
     2041 cgcccgtcac atcaacgctc aggtggccaa gtctcacaac atcgccctga tctggaacgt
     2101 gaaggacttc atgagcctgt ccgagcagct gcgcaagcag atcaggagcg ccgctaagaa
     2161 gaacaacctg cccttcaagc tgacctgcgc taccaccaga caggtggtga acgtggtgac
     2221 caccaagatc gccctgaagg gcggagcgga tccaccggtc gccaccatgg tgagcaaggg
     2281 cgaggagctg ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg
     2341 ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc acctacggca agctgaccct
     2401 gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct
     2461 gacctacggc gtgcagtgct tcagccgcta ccccgaccac atgaagcagc acgacttctt
     2521 caagtccgcc atgcccgaag gctacgtcca ggagcgcacc atcttcttca aggacgacgg
     2581 caactacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga
     2641 gctgaagggc atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa
     2701 ctacaacagc cacaacgtct atatcatggc cgacaagcag aagaacggca tcaaggtgaa
     2761 cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc actaccagca
     2821 gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagcaccca
     2881 gtccaagctg agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt
     2941 gaccgccgcc gggatcactc tcggcatgga cgagctgtac aagtaaagcg gccgcgactc
     3001 tagatcataa tcagccatac cacatttgta gaggttttac ttgctttaaa aaacctccca
     3061 cacctccccc tgaacctgaa acataaaatg aatgcaattg ttgttgttaa cttgtttatt
     3121 gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt
     3181 ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta aggcgtaaat
     3241 tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt
     3301 taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg
     3361 gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt
     3421 caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat caccctaatc
     3481 aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg
     3541 atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa
     3601 aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc
     3661 cgccgcgctt aatgcgccgc tacagggcgc gtcaggtggc acttttcggg gaaatgtgcg
     3721 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca
     3781 ataaccctga taaatgcttc aataatattg aaaaaggaag agtcctgagg cggaaagaac
     3841 cagctgtgga atgtgtgtca gttagggtgt ggaaagtccc caggctcccc agcaggcaga
     3901 agtatgcaaa gcatgcatct caattagtca gcaaccaggt gtggaaagtc cccaggctcc
     3961 ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat agtcccgccc
     4021 ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc
     4081 tgactaattt tttttattta tgcagaggcc gaggccgcct cggcctctga gctattccag
     4141 aagtagtgag gaggcttttt tggaggccta ggcttttgca aagatcgatc aagagacagg
     4201 atgaggatcg tttcgcatga ttgaacaaga tggattgcac gcaggttctc cggccgcttg
     4261 ggtggagagg ctattcggct atgactgggc acaacagaca atcggctgct ctgatgccgc
     4321 cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg acctgtccgg
     4381 tgccctgaat gaactgcaag acgaggcagc gcggctatcg tggctggcca cgacgggcgt
     4441 tccttgcgca gctgtgctcg acgttgtcac tgaagcggga agggactggc tgctattggg
     4501 cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct cctgccgaga aagtatccat
     4561 catggctgat gcaatgcggc ggctgcatac gcttgatccg gctacctgcc cattcgacca
     4621 ccaagcgaaa catcgcatcg agcgagcacg tactcggatg gaagccggtc ttgtcgatca
     4681 ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg ccaggctcaa
     4741 ggcgagcatg cccgacggcg aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa
     4801 tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc tgggtgtggc
     4861 ggaccgctat caggacatag cgttggctac ccgtgatatt gctgaagagc ttggcggcga
     4921 atgggctgac cgcttcctcg tgctttacgg tatcgccgct cccgattcgc agcgcatcgc
     4981 cttctatcgc cttcttgacg agttcttctg agcgggactc tggggttcga aatgaccgac
     5041 caagcgacgc ccaacctgcc atcacgagat ttcgattcca ccgccgcctt ctatgaaagg
     5101 ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc
     5161 atgctggagt tcttcgccca ccctaggggg aggctaactg aaacacggaa ggagacaata
     5221 ccggaaggaa cccgcgctat gacggcaata aaaagacaga ataaaacgca cggtgttggg
     5281 tcgtttgttc ataaacgcgg ggttcggtcc cagggctggc actctgtcga taccccaccg
     5341 agaccccatt ggggccaata cgcccgcgtt tcttcctttt ccccacccca ccccccaagt
     5401 tcgggtgaag gcccagggct cgcagccaac gtcggggcgg caggccctgc catagcctca
     5461 ggttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag
     5521 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac
     5581 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     5641 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     5701 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     5761 actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     5821 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     5881 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     5941 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     6001 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     6061 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     6121 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     6181 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     6241 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
     6301 aaccgtatta ccgccatgca
//
