LOCUS       sequence-372759-        5875 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses FLAG/HA-NSP1 in mammalian cells (SARS-CoV-2 NSP1).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5875)
  AUTHORS   Anastasakis DG, Benhalevy D, Cuburu N, Altan-Bonnet N, Hafner M
  TITLE     Epigenetic repression of antiviral genes by SARS-CoV-2 NSP1.
  JOURNAL   PLoS One. 2024 Jan 26;19(1):e0297262. doi: 
            10.1371/journal.pone.0297262. eCollection 2024.
  PUBMED    38277395
REFERENCE   2  (bases 1 to 5875)
  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: "PLoS One.";
            date: "2024-01-26"; volume: "19(1):e0297262. doi"; pages: " 
            10.1371/journal.pone.0297262. eCollection 2024"
COMMENT     Sequence Label: pEXP(FLAG/HA-NSP1-WT)
FEATURES             Location/Qualifiers
     source          1..5875
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        114..493
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        494..697
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     648..668
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    699..717
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    720..738
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     748..772
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             872..895
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             896..922
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     protein_bind    938..962
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(1508..1532)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1585..1626
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(1706..1723)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1712..1936
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2069..2088)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2220..2267
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             2275..3297
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             2275..3297
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     polyA_signal    3427..3548
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3464..3483)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3518..3537
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3597..3613)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3597..3613)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3610..3632)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3621..3637
                     /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(3645..3651,3652..3669,3670..3675))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3690..3711
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3828..3845)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3999..4587)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4079..4098)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4758..5549,5550..5618))
                     /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     5381..5400
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5619..5723)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5798..5817)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gagcaaaatt taagctacaa caaggcaagg cttgaccgac aattgcatga agaatctgct
       61 tagggttagg cgttttgcgc tgcttcgcga tgtacgggcc agatatacgc gttgacattg
      121 attattgact agttattaat agtaatcaat tacggggtca ttagttcata gcccatatat
      181 ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc
      241 ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca
      301 ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta
      361 tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta
      421 tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat
      481 cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat agcggtttga
      541 ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt tttggcacca
      601 aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg
      661 taggcgtgta cggtgggagg tctatataag cagagctctc cctatcagtg atagagatct
      721 ccctatcagt gatagagatc gtcgacgagc tcgtttagtg aaccgtcaga tcgcctggag
      781 acgccatcca cgctgttttg acctccatag aagacaccgg gaccgatcca gcctccggac
      841 tctagcgttt aaacttaagc ttggtaccat ggactacaag gacgacgatg acaagtaccc
      901 ttatgacgtg cccgattacg ctttgaacca gatatcaaca agtttgtaca aaaaagcagg
      961 cttcatggag agccttgtcc ctggtttcaa cgagaaaaca cacgtccaac tcagtttgcc
     1021 tgttttacag gttcgcgacg tgctcgtacg tggctttgga gactccgtgg aggaggtctt
     1081 atcagaggca cgtcaacatc ttaaagatgg cacttgtggc ttagtagaag ttgaaaaagg
     1141 cgttttgcct caacttgaac agccctatgt gttcatcaaa cgttcggatg ctcgaactgc
     1201 acctcatggt catgttatgg ttgagctggt agcagaactc gaaggcattc agtacggtcg
     1261 tagtggtgag acacttggtg tccttgtccc tcatgtgggc gaaataccag tggcttaccg
     1321 caaggttctt cttcgtaaga acggtaataa aggagctggt ggccatagtt acggcgccga
     1381 tctaaagtca tttgacttag gcgacgagct tggcactgat ccttatgaag attttcaaga
     1441 aaactggaac actaaacata gcagtggtgt tacccgtgaa ctcatgcgtg agcttaacgg
     1501 agggtgaacc cagctttctt gtacaaagtg gttgatatcc agcacagtgg cggccgctca
     1561 agtctagagg gcccgcggtt cgaaggtaag cctatcccta accctctcct cggtctcgat
     1621 tctacgcgta cctgatccgc ggccgcatag ataactgatc cagtgtgcct cgagtctaga
     1681 gggcccgttt aaacccgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt
     1741 gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc
     1801 taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt
     1861 ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat
     1921 gcggtgggct ctatggcttc tgaggcggaa agaaccagct ggggctctag ggggtatccc
     1981 cacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc
     2041 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
     2101 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt
     2161 agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtacctag
     2221 aagttcctat tccgaagttc ctattctcta gaaagtatag gaacttcctt ggccaaaaag
     2281 cctgaactca ccgcgacgtc tgtcgagaag tttctgatcg aaaagttcga cagcgtctcc
     2341 gacctgatgc agctctcgga gggcgaagaa tctcgtgctt tcagcttcga tgtaggaggg
     2401 cgtggatatg tcctgcgggt aaatagctgc gccgatggtt tctacaaaga tcgttatgtt
     2461 tatcggcact ttgcatcggc cgcgctcccg attccggaag tgcttgacat tggggaattc
     2521 agcgagagcc tgacctattg catctcccgc cgtgcacagg gtgtcacgtt gcaagacctg
     2581 cctgaaaccg aactgcccgc tgttctgcag ccggtcgcgg aggccatgga tgcgatcgct
     2641 gcggccgatc ttagccagac gagcgggttc ggcccattcg gaccgcaagg aatcggtcaa
     2701 tacactacat ggcgtgattt catatgcgcg attgctgatc cccatgtgta tcactggcaa
     2761 actgtgatgg acgacaccgt cagtgcgtcc gtcgcgcagg ctctcgatga gctgatgctt
     2821 tgggccgagg actgccccga agtccggcac ctcgtgcacg cggatttcgg ctccaacaat
     2881 gtcctgacgg acaatggccg cataacagcg gtcattgact ggagcgaggc gatgttcggg
     2941 gattcccaat acgaggtcgc caacatcttc ttctggaggc cgtggttggc ttgtatggag
     3001 cagcagacgc gctacttcga gcggaggcat ccggagcttg caggatcgcc gcggctccgg
     3061 gcgtatatgc tccgcattgg tcttgaccaa ctctatcaga gcttggttga cggcaatttc
     3121 gatgatgcag cttgggcgca gggtcgatgc gacgcaatcg tccgatccgg agccgggact
     3181 gtcgggcgta cacaaatcgc ccgcagaagc gcggccgtct ggaccgatgg ctgtgtagaa
     3241 gtactcgccg atagtggaaa ccgacgcccc agcactcgtc cgagggcaaa ggaatagcac
     3301 gtactacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt
     3361 ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc
     3421 caccccaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat
     3481 ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat
     3541 gtatcttatc atgtctgtat accgtcgacc tctagctaga gcttggcgta atcatggtca
     3601 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga
     3661 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg
     3721 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     3781 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
     3841 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     3901 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgttagcaaa aggccagcaa
     3961 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     4021 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     4081 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     4141 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     4201 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     4261 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     4321 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     4381 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga
     4441 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     4501 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     4561 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     4621 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     4681 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     4741 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     4801 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     4861 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     4921 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     4981 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     5041 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     5101 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     5161 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     5221 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     5281 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     5341 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     5401 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     5461 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     5521 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     5581 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     5641 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     5701 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtcgacgga
     5761 tcgggagatc tcccgatccc ctatggtgca ctctcagtac aatctgctct gatgccgcat
     5821 agttaagcca gtatctgctc cctgcttgtg tgttggaggt cgctgagtag tgcgc
//
