LOCUS       sequence-418173-        7058 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses the C terminal of SARS-CoV-2 NSP3 fused with Flag in 
            mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7058)
  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 7058)
  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-Flag
FEATURES             Location/Qualifiers
     source          1..7058
                     /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      2548..2557
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2557..2580
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     promoter        complement(2611..2629)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2612..2629)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2649..2666)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2655..2879
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2925..3353
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3012..3031)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3222..3243
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3362..3382)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3367..3696
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3547..3682
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3609..3628
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3763..4557
                     /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(3817..3836)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4427..4446
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4731..4852
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4768..4787)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4822..4841
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4901..4917)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4901..4917)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4914..4936)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4925..4941
                     /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(4949..4955,4956..4973,4974..4979))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4994..5015
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5132..5149)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5303..5891)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5383..5402)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6062..6853,6854..6922))
                     /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     6685..6704
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6923..7027)
                     /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 tgtggttcct gctgctctcc
      961 gtgtgcctgg gttccctgat ctactctacc gccgctctgg gagtgctgat gtctaacctg
     1021 ggtatgccta gctactgcac tggttacaga gagggatacc tgaacagcac caacgtgacc
     1081 atcgccacct actgcaccgg cagcatccca tgctccgtgt gcctgtctgg actggacagc
     1141 ctggacacct acccctccct ggaaaccatc cagatcacca tctctagctt caagtgggac
     1201 ctgaccgcct tcggtctggt ggctgagtgg ttcctggcct acatcctgtt caccaggttc
     1261 ttctacgtgc tgggcctggc cgctatcatg cagctgttct tctcctactt cgctgtgcac
     1321 ttcatcagca actcctggct gatgtggctg atcatcaacc tggtgcagat ggctcccatc
     1381 tctgccatgg tgcgcatgta catcttcttc gccagcttct actacgtgtg gaagtcctac
     1441 gtgcacgtgg tggacggttg caactcctct acctgcatga tgtgctacaa gcgcaacagg
     1501 gctaccaggg tggagtgcac caccatcgtg aacggcgtgc gcaggtcctt ctacgtgtac
     1561 gccaacggcg gcaagggatt ctgcaagctg cacaactgga actgcgtgaa ctgcgacacc
     1621 ttctgcgctg gctccacctt catctctgac gaggtggcca gagacctgag cctgcagttc
     1681 aagcgtccaa tcaaccccac cgaccagagc tcctacatcg tggactctgt gaccgtgaag
     1741 aacggtagca tccacctgta cttcgacaag gctggccaga agacctacga gcgccactct
     1801 ctgagccact tcgtgaacct ggacaacctg agggccaaca acaccaaggg cagcctgcct
     1861 atcaacgtga tcgtgttcga cggaaagtcc aagtgcgagg aatctagcgc caagtccgcc
     1921 agcgtgtact acagccagct gatgtgccag ccaatcctgc tgctggacca ggctctggtg
     1981 tctgacgtgg gagacagcgc cgaagtggcc gtgaagatgt tcgacgccta cgtgaacacc
     2041 ttctcctcta ccttcaacgt gcctatggag aagctgaaga ccctggtggc tactgctgag
     2101 gctgaactgg ctaagaacgt gtccctggac aacgtgctga gcaccttcat ctccgccgct
     2161 aggcagggtt tcgtggactc tgacgtggaa accaaagacg tggtggagtg cctgaagctg
     2221 tctcaccaga gcgacatcga agtgaccggc gactcctgca acaactacat gctgacctac
     2281 aacaaggtgg agaacatgac cccaagagac ctgggagctt gcatcgactg ctccgcccgt
     2341 cacatcaacg ctcaggtggc caagtctcac aacatcgccc tgatctggaa cgtgaaggac
     2401 ttcatgagcc tgtccgagca gctgcgcaag cagatcagga gcgccgctaa gaagaacaac
     2461 ctgcccttca agctgacctg cgctaccacc agacaggtgg tgaacgtggt gaccaccaag
     2521 atcgccctga agggcggaga attcgccgcc accatggatt ataaagatga tgatgataaa
     2581 tgactcgagc atgcatctag agggccctat tctatagtgt cacctaaatg ctagagctcg
     2641 ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt
     2701 gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat
     2761 tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag
     2821 caagggggag gattgggaag acaatagcag gcatgctggg gatgcggtgg gctctatggc
     2881 ttctgaggcg gaaagaacca gctggggctc tagggggtat ccccacgcgc cctgtagcgg
     2941 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     3001 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     3061 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     3121 cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac
     3181 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
     3241 tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat
     3301 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attaattctg
     3361 tggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccagcagg cagaagtatg
     3421 caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg ctccccagca
     3481 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc gcccctaact
     3541 ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta
     3601 atttttttta tttatgcaga ggccgaggcc gcctctgcct ctgagctatt ccagaagtag
     3661 tgaggaggct tttttggagg cctaggcttt tgcaaaaagc tcccgggagc ttgtatatcc
     3721 attttcggat ctgatcaaga gacaggatga ggatcgtttc gcatgattga acaagatgga
     3781 ttgcacgcag gttctccggc cgcttgggtg gagaggctat tcggctatga ctgggcacaa
     3841 cagacaatcg gctgctctga tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt
     3901 ctttttgtca agaccgacct gtccggtgcc ctgaatgaac tgcaggacga ggcagcgcgg
     3961 ctatcgtggc tggccacgac gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa
     4021 gcgggaaggg actggctgct attgggcgaa gtgccggggc aggatctcct gtcatctcac
     4081 cttgctcctg ccgagaaagt atccatcatg gctgatgcaa tgcggcggct gcatacgctt
     4141 gatccggcta cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact
     4201 cggatggaag ccggtcttgt cgatcaggat gatctggacg aagagcatca ggggctcgcg
     4261 ccagccgaac tgttcgccag gctcaaggcg cgcatgcccg acggcgagga tctcgtcgtg
     4321 acccatggcg atgcctgctt gccgaatatc atggtggaaa atggccgctt ttctggattc
     4381 atcgactgtg gccggctggg tgtggcggac cgctatcagg acatagcgtt ggctacccgt
     4441 gatattgctg aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc
     4501 gccgctcccg attcgcagcg catcgccttc tatcgccttc ttgacgagtt cttctgagcg
     4561 ggactctggg gttcgaaatg accgaccaag cgacgcccaa cctgccatca cgagatttcg
     4621 attccaccgc cgccttctat gaaaggttgg gcttcggaat cgttttccgg gacgccggct
     4681 ggatgatcct ccagcgcggg gatctcatgc tggagttctt cgcccacccc aacttgttta
     4741 ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat
     4801 ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct
     4861 gtataccgtc gacctctagc tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt
     4921 gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag
     4981 cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt
     5041 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag
     5101 gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
     5161 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
     5221 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
     5281 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     5341 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     5401 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     5461 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
     5521 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     5581 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     5641 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     5701 cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct
     5761 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     5821 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     5881 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     5941 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
     6001 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
     6061 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
     6121 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
     6181 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa
     6241 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
     6301 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
     6361 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
     6421 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
     6481 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
     6541 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
     6601 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
     6661 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc
     6721 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
     6781 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
     6841 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
     6901 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
     6961 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
     7021 ttccgcgcac atttccccga aaagtgccac ctgacgtc
//
