LOCUS       sequence-418199-        6917 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses SARS-CoV-2 NSP4-Delta-220-234 fused with Flag in mammalian
            cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6917)
  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 6917)
  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-Delta-220-234-Flag
FEATURES             Location/Qualifiers
     source          1..6917
                     /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      2407..2416
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2416..2439
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     promoter        complement(2470..2488)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2471..2488)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2508..2525)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2514..2738
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2784..3212
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2871..2890)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3081..3102
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3221..3241)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3226..3555
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3406..3541
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3468..3487
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3622..4416
                     /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(3676..3695)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4286..4305
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4590..4711
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4627..4646)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4681..4700
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4760..4776)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4760..4776)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4773..4795)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4784..4800
                     /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(4808..4814,4815..4832,4833..4838))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4853..4874
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4991..5008)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5162..5750)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5242..5261)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5921..6712,6713..6781))
                     /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     6544..6563
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6782..6886)
                     /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 gactccgaag tgtctaccag cggtcgctgg
     1621 gtgctgaaca acgactacta caggtctctg cctggcgtgt tctgcggagt ggatgctgtg
     1681 aacctgctga ccaacatgtt caccccactg atccagccca tcggtgctct ggacatctcc
     1741 gcctctatcg tggctggagg tatcgtggcc atcgtggtga cctgcctggc ttactacttc
     1801 atgaggttcc gcagggcctt cggtgaatac agccacgtgg tggctttcaa caccctgctg
     1861 ttcctgatgt ccttcaccgt gctgtgcctg acccccgtgt actctttcct gccaggcgtg
     1921 tacagcgtga tctacctgta cctgaccttc tacctgacca acgacgtgtc tttcctggcc
     1981 cacatccagt ggatggtcat gttcacccct ctggtgcctt tctggatcac catcgcttac
     2041 atcatctgca tctctaccaa gcacttctac tggttcttca gcaactacct gaagagacgt
     2101 gtggtgttca acggcgtgag cttctccacc ttcgaggaag ccgctctgtg caccttcctg
     2161 ctgaacaagg agatgtacct gaagctgcgc agcgacgtgc tgctgcctct gacccagtac
     2221 aacaggtacc tggccctgta caacaagtac aagtacttct ccggcgctat ggacaccacc
     2281 tcttaccgtg aagccgcttg ctgccacctg gccaaggctc tgaacgactt ctctaacagc
     2341 ggatccgacg tgctgtacca gccccctcag acctctatca ccagcgccgt gctgcaggaa
     2401 ttcgccgcca ccatggatta taaagatgat gatgataaat gactcgagca tgcatctaga
     2461 gggccctatt ctatagtgtc acctaaatgc tagagctcgc tgatcagcct cgactgtgcc
     2521 ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg
     2581 tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag
     2641 gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga
     2701 caatagcagg catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag
     2761 ctggggctct agggggtatc cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     2821 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
     2881 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     2941 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta
     3001 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     3061 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat
     3121 ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa
     3181 tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg
     3241 tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag
     3301 tcagcaacca ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg
     3361 catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact
     3421 ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag
     3481 gccgaggccg cctctgcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc
     3541 ctaggctttt gcaaaaagct cccgggagct tgtatatcca ttttcggatc tgatcaagag
     3601 acaggatgag gatcgtttcg catgattgaa caagatggat tgcacgcagg ttctccggcc
     3661 gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat
     3721 gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg
     3781 tccggtgccc tgaatgaact gcaggacgag gcagcgcggc tatcgtggct ggccacgacg
     3841 ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta
     3901 ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta
     3961 tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc
     4021 gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc
     4081 gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg
     4141 ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg
     4201 ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt
     4261 gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc
     4321 ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc
     4381 atcgccttct atcgccttct tgacgagttc ttctgagcgg gactctgggg ttcgaaatga
     4441 ccgaccaagc gacgcccaac ctgccatcac gagatttcga ttccaccgcc gccttctatg
     4501 aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg
     4561 atctcatgct ggagttcttc gcccacccca acttgtttat tgcagcttat aatggttaca
     4621 aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt
     4681 gtggtttgtc caaactcatc aatgtatctt atcatgtctg tataccgtcg acctctagct
     4741 agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa
     4801 ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga
     4861 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     4921 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct
     4981 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     5041 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     5101 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     5161 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     5221 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     5281 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     5341 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     5401 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     5461 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     5521 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     5581 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
     5641 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     5701 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     5761 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     5821 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     5881 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     5941 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     6001 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     6061 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     6121 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     6181 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     6241 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     6301 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     6361 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     6421 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     6481 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     6541 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     6601 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     6661 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     6721 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     6781 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     6841 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     6901 aagtgccacc tgacgtc
//
