LOCUS       sequence-418188-        6962 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses SARS-CoV-2 NSP4 fused with Flag in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6962)
  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 6962)
  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-WT-Flag
FEATURES             Location/Qualifiers
     source          1..6962
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    916..940
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      2452..2461
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2461..2484
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     promoter        complement(2515..2533)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2516..2533)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2553..2570)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2559..2783
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2829..3257
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2916..2935)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3126..3147
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3266..3286)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3271..3600
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3451..3586
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3513..3532
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3667..4461
                     /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
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3721..3740)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4331..4350
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4635..4756
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4672..4691)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4726..4745
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4805..4821)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4805..4821)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4818..4840)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4829..4845
                     /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)."
     promoter        complement(join(4853..4859,4860..4877,4878..4883))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4898..4919
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5036..5053)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5207..5795)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5287..5306)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5966..6757,6758..6826))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     6589..6608
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6827..6931)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttggtacc
      901 gagctcggat ccatgccaac tttgtacaaa aaagttggca tgaagatcgt gaacaactgg
      961 ctgaagcagc tgatcaaggt gaccctggtg ttcctgttcg tggccgctat cttctacctg
     1021 atcacccctg tgcacgtgat gagcaagcac accgacttca gctccgagat catcggctac
     1081 aaggccatcg acggcggagt gaccagagac atcgcttcca ccgacacctg cttcgccaac
     1141 aagcacgctg acttcgacac ctggttctcc cagagaggtg gctcttacac caacgacaag
     1201 gcctgcccac tgatcgccgc tgtgatcacc cgtgaagtgg gattcgtggt gcctggcctg
     1261 ccaggaacca tcctgagaac caccaacggt gacttcctgc acttcctgcc acgcgtgttc
     1321 agcgccgtgg gcaacatctg ctacacccct tctaagctga tcgagtacac cgacttcgcc
     1381 acctctgctt gcgtgctggc cgctgaatgc accatcttca aggacgccag cggaaagcca
     1441 gtgccctact gctacgacac caacgtgctg gagggtagcg tggcttacga atccctgcgc
     1501 ccagacacca gatacgtgct gatggacggc tccatcatcc agttccccaa cacctacctg
     1561 gagggaagcg tgcgcgtggt gaccaccttc gactccgaat actgcagaca cggaacctgc
     1621 gagcgttccg aagccggagt gtgcgtgtct accagcggtc gctgggtgct gaacaacgac
     1681 tactacaggt ctctgcctgg cgtgttctgc ggagtggatg ctgtgaacct gctgaccaac
     1741 atgttcaccc cactgatcca gcccatcggt gctctggaca tctccgcctc tatcgtggct
     1801 ggaggtatcg tggccatcgt ggtgacctgc ctggcttact acttcatgag gttccgcagg
     1861 gccttcggtg aatacagcca cgtggtggct ttcaacaccc tgctgttcct gatgtccttc
     1921 accgtgctgt gcctgacccc cgtgtactct ttcctgccag gcgtgtacag cgtgatctac
     1981 ctgtacctga ccttctacct gaccaacgac gtgtctttcc tggcccacat ccagtggatg
     2041 gtcatgttca cccctctggt gcctttctgg atcaccatcg cttacatcat ctgcatctct
     2101 accaagcact tctactggtt cttcagcaac tacctgaaga gacgtgtggt gttcaacggc
     2161 gtgagcttct ccaccttcga ggaagccgct ctgtgcacct tcctgctgaa caaggagatg
     2221 tacctgaagc tgcgcagcga cgtgctgctg cctctgaccc agtacaacag gtacctggcc
     2281 ctgtacaaca agtacaagta cttctccggc gctatggaca ccacctctta ccgtgaagcc
     2341 gcttgctgcc acctggccaa ggctctgaac gacttctcta acagcggatc cgacgtgctg
     2401 taccagcccc ctcagacctc tatcaccagc gccgtgctgc aggaattcgc cgccaccatg
     2461 gattataaag atgatgatga taaatgactc gagcatgcat ctagagggcc ctattctata
     2521 gtgtcaccta aatgctagag ctcgctgatc agcctcgact gtgccttcta gttgccagcc
     2581 atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt
     2641 cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct
     2701 ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc
     2761 tggggatgcg gtgggctcta tggcttctga ggcggaaaga accagctggg gctctagggg
     2821 gtatccccac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
     2881 cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt
     2941 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt
     3001 ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg
     3061 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt
     3121 taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt
     3181 tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca
     3241 aaaatttaac gcgaattaat tctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca
     3301 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaggtgt
     3361 ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca
     3421 gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc
     3481 cattctccgc cccatggctg actaattttt tttatttatg cagaggccga ggccgcctct
     3541 gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa
     3601 aagctcccgg gagcttgtat atccattttc ggatctgatc aagagacagg atgaggatcg
     3661 tttcgcatga ttgaacaaga tggattgcac gcaggttctc cggccgcttg ggtggagagg
     3721 ctattcggct atgactgggc acaacagaca atcggctgct ctgatgccgc cgtgttccgg
     3781 ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg acctgtccgg tgccctgaat
     3841 gaactgcagg acgaggcagc gcggctatcg tggctggcca cgacgggcgt tccttgcgca
     3901 gctgtgctcg acgttgtcac tgaagcggga agggactggc tgctattggg cgaagtgccg
     3961 gggcaggatc tcctgtcatc tcaccttgct cctgccgaga aagtatccat catggctgat
     4021 gcaatgcggc ggctgcatac gcttgatccg gctacctgcc cattcgacca ccaagcgaaa
     4081 catcgcatcg agcgagcacg tactcggatg gaagccggtc ttgtcgatca ggatgatctg
     4141 gacgaagagc atcaggggct cgcgccagcc gaactgttcg ccaggctcaa ggcgcgcatg
     4201 cccgacggcg aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg
     4261 gaaaatggcc gcttttctgg attcatcgac tgtggccggc tgggtgtggc ggaccgctat
     4321 caggacatag cgttggctac ccgtgatatt gctgaagagc ttggcggcga atgggctgac
     4381 cgcttcctcg tgctttacgg tatcgccgct cccgattcgc agcgcatcgc cttctatcgc
     4441 cttcttgacg agttcttctg agcgggactc tggggttcga aatgaccgac caagcgacgc
     4501 ccaacctgcc atcacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg
     4561 gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt
     4621 tcttcgccca ccccaacttg tttattgcag cttataatgg ttacaaataa agcaatagca
     4681 tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac
     4741 tcatcaatgt atcttatcat gtctgtatac cgtcgacctc tagctagagc ttggcgtaat
     4801 catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac
     4861 gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa
     4921 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat
     4981 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc
     5041 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
     5101 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     5161 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     5221 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     5281 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     5341 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     5401 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     5461 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     5521 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     5581 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     5641 ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     5701 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     5761 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     5821 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     5881 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     5941 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
     6001 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
     6061 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
     6121 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
     6181 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
     6241 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
     6301 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
     6361 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
     6421 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
     6481 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
     6541 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
     6601 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
     6661 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
     6721 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
     6781 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
     6841 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     6901 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg
     6961 tc
//
