LOCUS       sequence-418203-        6332 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses SARS-CoV-2 NSP6 fused with Flag in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6332)
  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 6332)
  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: NSP6-Flag
FEATURES             Location/Qualifiers
     source          1..6332
                     /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      1822..1831
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1831..1854
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     promoter        complement(1885..1903)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1886..1903)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1923..1940)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1929..2153
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2199..2627
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2286..2305)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2496..2517
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2636..2656)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2641..2970
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2821..2956
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2883..2902
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3037..3831
                     /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(3091..3110)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3701..3720
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4005..4126
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4042..4061)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4096..4115
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4175..4191)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4175..4191)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4188..4210)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4199..4215
                     /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(4223..4229,4230..4247,4248..4253))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4268..4289
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4406..4423)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4577..5165)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4657..4676)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5336..6127,6128..6196))
                     /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     5959..5978
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6197..6301)
                     /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 tgagcgccgt gaagagaacc
      961 atcaagggaa cccaccactg gctgctgctg accatcctga cctccctgct ggtgctggtg
     1021 cagtctaccc agtggagcct gttcttcttc ctgtacgaga acgccttcct gcccttcgct
     1081 atgggtatca tcgccatgtc cgccttcgct atgatgttcg tgaagcacaa gcacgctttc
     1141 ctgtgcctgt tcctgctgcc atccctggcc accgtggctt acttcaacat ggtgtacatg
     1201 cccgcctctt gggtcatgcg catcatgacc tggctggaca tggtggacac cagcctgtcc
     1261 ggcttcaagc tgaaggactg cgtgatgtac gcctctgctg tggtgctgct gatcctgatg
     1321 accgctagaa ccgtgtacga tgatggtgct agaagggtgt ggaccctgat gaacgtgctg
     1381 accctggtgt acaaggtgta ctacggcaac gccctggacc aggctatcag catgtgggct
     1441 ctgatcatct ctgtgaccag caactactcc ggagtggtga ccaccgtgat gttcctggcc
     1501 agaggtatcg tgttcatgtg cgtggagtac tgccctatct tcttcatcac cggcaacacc
     1561 ctgcagtgca tcatgctggt gtactgcttc ctgggttact tctgcacctg ctatttcggc
     1621 ctgttctgcc tgctgaacag atacttccgt ctgaccctgg gcgtgtacga ctacctggtg
     1681 tccacccagg aattccgtta catgaactct cagggactgc tgccccctaa gaacagcatc
     1741 gacgctttca agctgaacat caagctgctg ggagtgggcg gaaagccatg catcaaggtg
     1801 gccaccgtgc aggaattcgc cgccaccatg gattataaag atgatgatga taaatgactc
     1861 gagcatgcat ctagagggcc ctattctata gtgtcaccta aatgctagag ctcgctgatc
     1921 agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc
     1981 cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc
     2041 gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg
     2101 ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga
     2161 ggcggaaaga accagctggg gctctagggg gtatccccac gcgccctgta gcggcgcatt
     2221 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
     2281 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     2341 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     2401 caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
     2461 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
     2521 aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc
     2581 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaattaat tctgtggaat
     2641 gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
     2701 atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga
     2761 agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc
     2821 atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt
     2881 tttatttatg cagaggccga ggccgcctct gcctctgagc tattccagaa gtagtgagga
     2941 ggcttttttg gaggcctagg cttttgcaaa aagctcccgg gagcttgtat atccattttc
     3001 ggatctgatc aagagacagg atgaggatcg tttcgcatga ttgaacaaga tggattgcac
     3061 gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca
     3121 atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt
     3181 gtcaagaccg acctgtccgg tgccctgaat gaactgcagg acgaggcagc gcggctatcg
     3241 tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga
     3301 agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct
     3361 cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg
     3421 gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg
     3481 gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc
     3541 gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat
     3601 ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac
     3661 tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt
     3721 gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct
     3781 cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg agcgggactc
     3841 tggggttcga aatgaccgac caagcgacgc ccaacctgcc atcacgagat ttcgattcca
     3901 ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga
     3961 tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaacttg tttattgcag
     4021 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt
     4081 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctgtatac
     4141 cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt
     4201 gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg
     4261 gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt
     4321 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     4381 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     4441 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     4501 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     4561 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     4621 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     4681 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     4741 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     4801 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     4861 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     4921 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     4981 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc
     5041 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     5101 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     5161 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     5221 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     5281 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     5341 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     5401 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     5461 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     5521 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     5581 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     5641 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     5701 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     5761 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     5821 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     5881 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     5941 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     6001 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     6061 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     6121 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     6181 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
     6241 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     6301 gcacatttcc ccgaaaagtg ccacctgacg tc
//
