LOCUS       sequence-418192-        6224 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses SARS-CoV-2 NSP4 fused with EGFP in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6224)
  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 6224)
  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: NSP4-EGFP
FEATURES             Location/Qualifiers
     source          1..6224
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        61..364
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        365..568
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     519..539
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    622..646
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      2164..2173
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2170..2172,2173..2175,2176..2889)
                     /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(2215..2236)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2476..2495)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2823..2844
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    3012..3133
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3049..3068)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3103..3122
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3140..3595)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3277..3298)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3489..3508
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3622..3726
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3728..4085
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3728..3919
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3751..3771)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3936..4071
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4120..4914
                     /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             4120..4914
                     /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(4174..4193)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4784..4803
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5102..5121)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5146..5193
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5522..6110
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6011..6030
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
       61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      181 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      241 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      301 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      361 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      421 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      481 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      541 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta
      601 ccggactcag atctcgagat gccaactttg tacaaaaaag ttggcatgaa gatcgtgaac
      661 aactggctga agcagctgat caaggtgacc ctggtgttcc tgttcgtggc cgctatcttc
      721 tacctgatca cccctgtgca cgtgatgagc aagcacaccg acttcagctc cgagatcatc
      781 ggctacaagg ccatcgacgg cggagtgacc agagacatcg cttccaccga cacctgcttc
      841 gccaacaagc acgctgactt cgacacctgg ttctcccaga gaggtggctc ttacaccaac
      901 gacaaggcct gcccactgat cgccgctgtg atcacccgtg aagtgggatt cgtggtgcct
      961 ggcctgccag gaaccatcct gagaaccacc aacggtgact tcctgcactt cctgccacgc
     1021 gtgttcagcg ccgtgggcaa catctgctac accccttcta agctgatcga gtacaccgac
     1081 ttcgccacct ctgtttgcgt gctggccgct gaatgcacca tcttcaagga cgccagcgga
     1141 aagccagtgc cctactgcta cgacaccaac gtgctggagg gtagcgtggc ttacgaatcc
     1201 ctgcgcccag acaccagata cgtgctgatg gacggctcca tcatccagtt ccccaacacc
     1261 tacctggagg gaagcgtgcg cgtggtgacc accttcgact ccgaatactg cagacacgga
     1321 acctgcgagc gttccgaagc cggagtgtgc gtgtctacca gcggtcgctg ggtgctgaac
     1381 aacgactact acaggtctct gcctggcgtg ttctgcggag tggatgctgt gaacctgctg
     1441 accaacatgt tcaccccact gatccagccc atcggtgctc tggacatctc cgcctctatc
     1501 gtggctggag gtatcgtggc catcgtggtg acctgcctgg cttactactt catgaggttc
     1561 cgcagggcct tcggtgaata cagccacgtg gtggctttca acaccctgct gttcctgatg
     1621 tccttcaccg tgctgtgcct gacccccgtg tactctttcc tgccaggcgt gtacagcgtg
     1681 atctacctgt acctgacctt ctacctgacc aacgacgtgt ctttcctggc ccacatccag
     1741 tggatggtca tgttcacccc tctggtgcct ttctggatca ccatcgctta catcatctgc
     1801 atctctacca agcacttcta ctggttcttc agcaactacc tgaagagacg tgtggtgttc
     1861 aacggcgtga gcttctccac cttcgaggaa gccgctctgt gcaccttcct gctgaacaag
     1921 gagatgtacc tgaagctgcg cagcgacgtg ctgctgcctc tgacccagta caacaggtac
     1981 ctggccctgt acaacaagta caagtacttc tccggcgcta tggacaccac ctcttaccgt
     2041 gaagccgctt gctgccacct ggccaaggct ctgaacgact tctctaacag cggatccgac
     2101 gtgctgtacc agccccctca gacctctatc accagcgccg tgctgcaggc ggatccaccg
     2161 gtcgccacca tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc
     2221 gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat
     2281 gccacctacg gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc
     2341 tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg ctaccccgac
     2401 cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc
     2461 accatcttct tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc
     2521 gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc
     2581 ctggggcaca agctggagta caactacaac agccacaacg tctatatcat ggccgacaag
     2641 cagaagaacg gcatcaaggt gaacttcaag atccgccaca acatcgagga cggcagcgtg
     2701 cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc
     2761 gacaaccact acctgagcac ccagtccaag ctgagcaaag accccaacga gaagcgcgat
     2821 cacatggtcc tgctggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg
     2881 tacaagtaaa gcggccgcga ctctagatca taatcagcca taccacattt gtagaggttt
     2941 tacttgcttt aaaaaacctc ccacacctcc ccctgaacct gaaacataaa atgaatgcaa
     3001 ttgttgttgt taacttgttt attgcagctt ataatggtta caaataaagc aatagcatca
     3061 caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca
     3121 tcaatgtatc ttaaggcgta aattgtaagc gttaatattt tgttaaaatt cgcgttaaat
     3181 ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa
     3241 tcaaaagaat agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta
     3301 ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca
     3361 ctacgtgaac catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat
     3421 cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg
     3481 agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc
     3541 acgctgcgcg taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtcaggt
     3601 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca
     3661 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg
     3721 aagagtcctg aggcggaaag aaccagctgt ggaatgtgtg tcagttaggg tgtggaaagt
     3781 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca
     3841 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
     3901 agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt
     3961 ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg
     4021 cctcggcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt
     4081 gcaaagatcg atcaagagac aggatgagga tcgtttcgca tgattgaaca agatggattg
     4141 cacgcaggtt ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag
     4201 acaatcggct gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt
     4261 tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc aagacgaggc agcgcggcta
     4321 tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg
     4381 ggaagggact ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt
     4441 gctcctgccg agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat
     4501 ccggctacct gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg
     4561 atggaagccg gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca
     4621 gccgaactgt tcgccaggct caaggcgagc atgcccgacg gcgaggatct cgtcgtgacc
     4681 catggcgatg cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc
     4741 gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat
     4801 attgctgaag agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc
     4861 gctcccgatt cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgagcggga
     4921 ctctggggtt cgaaatgacc gaccaagcga cgcccaacct gccatcacga gatttcgatt
     4981 ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga
     5041 tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccctagg gggaggctaa
     5101 ctgaaacacg gaaggagaca ataccggaag gaacccgcgc tatgacggca ataaaaagac
     5161 agaataaaac gcacggtgtt gggtcgtttg ttcataaacg cggggttcgg tcccagggct
     5221 ggcactctgt cgatacccca ccgagacccc attggggcca atacgcccgc gtttcttcct
     5281 tttccccacc ccacccccca agttcgggtg aaggcccagg gctcgcagcc aacgtcgggg
     5341 cggcaggccc tgccatagcc tcaggttact catatatact ttagattgat ttaaaacttc
     5401 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc
     5461 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     5521 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     5581 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     5641 tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact
     5701 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     5761 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     5821 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     5881 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     5941 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     6001 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     6061 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     6121 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg
     6181 cgttatcccc tgattctgtg gataaccgta ttaccgccat gcat
//
