LOCUS       sequence-418190-        6214 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses SARS-CoV-2 NSP4 fused with mCherry in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6214)
  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 6214)
  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-mCherry
FEATURES             Location/Qualifiers
     source          1..6214
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        63..366
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        367..570
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     521..541
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    624..648
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      2166..2175
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2172..2882
                     /codon_start=1
                     /product="derivative of the monomeric red fluorescent 
                     protein mCherry"
                     /label=mCherry2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFNWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQCRTMGWEASTERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVDIKLDILSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(2317..2335)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     polyA_signal    3004..3125
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3041..3060)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3095..3114
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3132..3587)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3269..3290)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3481..3500
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3614..3718
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3720..4077
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3720..3911
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3743..3763)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3928..4063
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4112..4906
                     /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             4112..4906
                     /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(4166..4185)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4776..4795
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5094..5113)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5138..5185
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5514..6102
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6003..6022
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 attagttatt aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc
       61 cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca
      121 ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt
      181 caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg
      241 ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag
      301 tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt
      361 accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt tgactcacgg
      421 ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa
      481 cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg cggtaggcgt
      541 gtacggtggg aggtctatat aagcagagct ggtttagtga accgtcagat ccgctagcgc
      601 taccggactc agatctcgag atgccaactt tgtacaaaaa agttggcatg aagatcgtga
      661 acaactggct gaagcagctg atcaaggtga ccctggtgtt cctgttcgtg gccgctatct
      721 tctacctgat cacccctgtg cacgtgatga gcaagcacac cgacttcagc tccgagatca
      781 tcggctacaa ggccatcgac ggcggagtga ccagagacat cgcttccacc gacacctgct
      841 tcgccaacaa gcacgctgac ttcgacacct ggttctccca gagaggtggc tcttacacca
      901 acgacaaggc ctgcccactg atcgccgctg tgatcacccg tgaagtggga ttcgtggtgc
      961 ctggcctgcc aggaaccatc ctgagaacca ccaacggtga cttcctgcac ttcctgccac
     1021 gcgtgttcag cgccgtgggc aacatctgct acaccccttc taagctgatc gagtacaccg
     1081 acttcgccac ctctgcttgc gtgctggccg ctgaatgcac catcttcaag gacgccagcg
     1141 gaaagccagt gccctactgc tacgacacca acgtgctgga gggtagcgtg gcttacgaat
     1201 ccctgcgccc agacaccaga tacgtgctga tggacggctc catcatccag ttccccaaca
     1261 cctacctgga gggaagcgtg cgcgtggtga ccaccttcga ctccgaatac tgcagacacg
     1321 gaacctgcga gcgttccgaa gccggagtgt gcgtgtctac cagcggtcgc tgggtgctga
     1381 acaacgacta ctacaggtct ctgcctggcg tgttctgcgg agtggatgct gtgaacctgc
     1441 tgaccaacat gttcacccca ctgatccagc ccatcggtgc tctggacatc tccgcctcta
     1501 tcgtggctgg aggtatcgtg gccatcgtgg tgacctgcct ggcttactac ttcatgaggt
     1561 tccgcagggc cttcggtgaa tacagccacg tggtggcttt caacaccctg ctgttcctga
     1621 tgtccttcac cgtgctgtgc ctgacccccg tgtactcttt cctgccaggc gtgtacagcg
     1681 tgatctacct gtacctgacc ttctacctga ccaacgacgt gtctttcctg gcccacatcc
     1741 agtggatggt catgttcacc cctctggtgc ctttctggat caccatcgct tacatcatct
     1801 gcatctctac caagcacttc tactggttct tcagcaacta cctgaagaga cgtgtggtgt
     1861 tcaacggcgt gagcttctcc accttcgagg aagccgctct gtgcaccttc ctgctgaaca
     1921 aggagatgta cctgaagctg cgcagcgacg tgctgctgcc tctgacccag tacaacaggt
     1981 acctggccct gtacaacaag tacaagtact tctccggcgc tatggacacc acctcttacc
     2041 gtgaagccgc ttgctgccac ctggccaagg ctctgaacga cttctctaac agcggatccg
     2101 acgtgctgta ccagccccct cagacctcta tcaccagcgc cgtgctgcag gcggatccac
     2161 cggtcgccac catggtgagc aagggcgagg aggataacat ggccatcatc aaggagttca
     2221 tgcgcttcaa ggtgcacatg gagggctccg tgaacggcca cgagttcgag atcgagggcg
     2281 agggcgaggg ccgcccctac gagggcaccc agaccgccaa gctgaaggtg accaagggtg
     2341 gccccctgcc cttcgcctgg gacatcctgt cccctcagtt catgtacggc tccaaggcct
     2401 acgtgaagca ccccgccgac atccccgact acttgaagct gtccttcccc gagggcttca
     2461 attgggagcg cgtgatgaac ttcgaggacg gcggcgtggt gaccgtgacc caggactcct
     2521 ccctgcagga cggcgagttc atctacaagg tgaagctgcg cggcaccaac ttcccctccg
     2581 acggccccgt aatgcagtgt cgtaccatgg gctgggaggc ctccactgag cggatgtacc
     2641 ccgaggacgg cgccctgaag ggcgagatca agcagaggct gaagctgaag gacggcggcc
     2701 actacgacgc tgaggtcaag accacctaca aggccaagaa gcccgtgcag ctgcccggcg
     2761 cctacaacgt cgacatcaag ttggacatcc tttcccacaa cgaggactac accatcgtgg
     2821 aacagtacga acgcgccgag ggccgccact ccaccggcgg catggacgag ctgtacaagt
     2881 aagcggccgc gactctagat cataatcagc cataccacat ttgtagaggt tttacttgct
     2941 ttaaaaaacc tcccacacct ccccctgaac ctgaaacata aaatgaatgc aattgttgtt
     3001 gttaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc
     3061 acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta
     3121 tcttaaggcg taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta
     3181 aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga
     3241 atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa
     3301 cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga
     3361 accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc
     3421 taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga
     3481 agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg
     3541 cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtcag gtggcacttt
     3601 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
     3661 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtcc
     3721 tgaggcggaa agaaccagct gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc
     3781 tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac caggtgtgga
     3841 aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca
     3901 accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat
     3961 tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc cgcctcggcc
     4021 tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaagat
     4081 cgatcaagag acaggatgag gatcgtttcg catgattgaa caagatggat tgcacgcagg
     4141 ttctccggcc gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg
     4201 ctgctctgat gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa
     4261 gaccgacctg tccggtgccc tgaatgaact gcaagacgag gcagcgcggc tatcgtggct
     4321 ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga
     4381 ctggctgcta ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc
     4441 cgagaaagta tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac
     4501 ctgcccattc gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc
     4561 cggtcttgtc gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact
     4621 gttcgccagg ctcaaggcga gcatgcccga cggcgaggat ctcgtcgtga cccatggcga
     4681 tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg
     4741 ccggctgggt gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga
     4801 agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga
     4861 ttcgcagcgc atcgccttct atcgccttct tgacgagttc ttctgagcgg gactctgggg
     4921 ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac gagatttcga ttccaccgcc
     4981 gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc
     5041 cagcgcgggg atctcatgct ggagttcttc gcccacccta gggggaggct aactgaaaca
     5101 cggaaggaga caataccgga aggaacccgc gctatgacgg caataaaaag acagaataaa
     5161 acgcacggtg ttgggtcgtt tgttcataaa cgcggggttc ggtcccaggg ctggcactct
     5221 gtcgataccc caccgagacc ccattggggc caatacgccc gcgtttcttc cttttcccca
     5281 ccccaccccc caagttcggg tgaaggccca gggctcgcag ccaacgtcgg ggcggcaggc
     5341 cctgccatag cctcaggtta ctcatatata ctttagattg atttaaaact tcatttttaa
     5401 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
     5461 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     5521 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     5581 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     5641 gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac
     5701 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     5761 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     5821 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     5881 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     5941 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     6001 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     6061 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     6121 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
     6181 cctgattctg tggataaccg tattaccgcc atgc
//
