LOCUS       sequence-307611-        5705 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mammalian expression of C-terminally His8-Flag tagged SARS-CoV-2 
            Nsp1 under control of a tetracycline-inducible promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5705)
  AUTHORS   Bresson S, Robertson N, Sani E, Turowski TW, Shchepachev V, 
            Kompauerova M, Spanos C, Helwak A, Tollervey D
  TITLE     Integrative vectors for regulated expression of SARS-CoV-2 proteins 
            implicated in RNA metabolism.
  JOURNAL   Wellcome Open Res. 2020 Nov 3;5:261. doi: 
            10.12688/wellcomeopenres.16322.1. eCollection 2020.
  PUBMED    33313418
REFERENCE   2  (bases 1 to 5705)
  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: "Wellcome 
            Open Res."; date: "2020-11-3"; volume: "5:261. doi"; pages: " 
            10.12688/wellcomeopenres.16322.1. eCollection 2020"
COMMENT     Sequence Label: pcDNA5-FRT-TO-Nsp1-HF
FEATURES             Location/Qualifiers
     source          1..5705
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        100..479
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        480..683
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     634..654
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    685..703
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    706..724
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     734..758
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             1422..1445
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             1458..1481
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(1522..1539)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1528..1752
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(1885..1904)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2036..2083
                     /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             2091..3113
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             2091..3113
                     /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    3243..3364
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3280..3299)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3334..3353
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3413..3429)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3413..3429)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3426..3448)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3437..3453
                     /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(3461..3467,3468..3485,3486..3491))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3506..3527
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3644..3661)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3815..4403)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3895..3914)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4574..5365,5366..5434))
                     /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     5197..5216
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5435..5539)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5614..5633)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 ctacaacaag gcaaggcttg accgacaatt gcatgaagaa tctgcttagg gttaggcgtt
       61 ttgcgctgct tcgcgatgta cgggccagat atacgcgttg acattgatta ttgactagtt
      121 attaatagta atcaattacg gggtcattag ttcatagccc atatatggag ttccgcgtta
      181 cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt
      241 caataatgac gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg
      301 tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagta
      361 cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga
      421 ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgg
      481 tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca cggggatttc
      541 caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact
      601 ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg cgtgtacggt
      661 gggaggtcta tataagcaga gctctcccta tcagtgatag agatctccct atcagtgata
      721 gagatcgtcg acgagctcgt ttagtgaacc gtcagatcgc ctggagacgc catccacgct
      781 gttttgacct ccatagaaga caccgggacc gatccagcct ccggactcta gcgtttaaac
      841 ttaagcttgg taccgagctc ggatccggga tggaatcctt ggtgccgggt tttaatgaga
      901 aaacacacgt ccagttgtcc ctcccagttt tgcaggtgcg agatgtgctt gtccgggggt
      961 tcggggactc cgtggaggag gtactcagcg aagctagaca gcatctcaag gacggaactt
     1021 gcggactcgt tgaagttgaa aagggggtac tgccacagct tgagcagccc tacgtcttca
     1081 ttaagcgatc tgatgccaga acggctcctc atggacacgt aatggtggag ttggtcgctg
     1141 agcttgaggg aatacagtac ggcagatctg gagagacgct gggcgtactc gtaccgcatg
     1201 ttggtgagat ccctgttgca tacaggaaag tattgttgcg aaaaaacggt aataaaggag
     1261 cgggtggtca tagctacggg gcggatctta aatcttttga ccttggggat gaactgggca
     1321 ctgatccgta cgaagatttt caggagaatt ggaataccaa acattcaagc ggcgtgacga
     1381 gggaacttat gagagagctg aacggcgggg catcgggcgg acatcaccac catcatcacc
     1441 atcacgccgc agccgcagat tataaagatg acgatgacaa gtagctcgag tctagagggc
     1501 ccgtttaaac ccgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt
     1561 gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat
     1621 aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg
     1681 tggggcagga cagcaagggg gaggattggg aagacaatag caggcatgct ggggatgcgg
     1741 tgggctctat ggcttctgag gcggaaagaa ccagctgggg ctctaggggg tatccccacg
     1801 cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta
     1861 cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt
     1921 tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg
     1981 ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt acctagaagt
     2041 tcctattccg aagttcctat tctctagaaa gtataggaac ttccttggcc aaaaagcctg
     2101 aactcaccgc gacgtctgtc gagaagtttc tgatcgaaaa gttcgacagc gtctccgacc
     2161 tgatgcagct ctcggagggc gaagaatctc gtgctttcag cttcgatgta ggagggcgtg
     2221 gatatgtcct gcgggtaaat agctgcgccg atggtttcta caaagatcgt tatgtttatc
     2281 ggcactttgc atcggccgcg ctcccgattc cggaagtgct tgacattggg gaattcagcg
     2341 agagcctgac ctattgcatc tcccgccgtg cacagggtgt cacgttgcaa gacctgcctg
     2401 aaaccgaact gcccgctgtt ctgcagccgg tcgcggaggc catggatgcg atcgctgcgg
     2461 ccgatcttag ccagacgagc gggttcggcc cattcggacc gcaaggaatc ggtcaataca
     2521 ctacatggcg tgatttcata tgcgcgattg ctgatcccca tgtgtatcac tggcaaactg
     2581 tgatggacga caccgtcagt gcgtccgtcg cgcaggctct cgatgagctg atgctttggg
     2641 ccgaggactg ccccgaagtc cggcacctcg tgcacgcgga tttcggctcc aacaatgtcc
     2701 tgacggacaa tggccgcata acagcggtca ttgactggag cgaggcgatg ttcggggatt
     2761 cccaatacga ggtcgccaac atcttcttct ggaggccgtg gttggcttgt atggagcagc
     2821 agacgcgcta cttcgagcgg aggcatccgg agcttgcagg atcgccgcgg ctccgggcgt
     2881 atatgctccg cattggtctt gaccaactct atcagagctt ggttgacggc aatttcgatg
     2941 atgcagcttg ggcgcagggt cgatgcgacg caatcgtccg atccggagcc gggactgtcg
     3001 ggcgtacaca aatcgcccgc agaagcgcgg ccgtctggac cgatggctgt gtagaagtac
     3061 tcgccgatag tggaaaccga cgccccagca ctcgtccgag ggcaaaggaa tagcacgtac
     3121 tacgagattt cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc
     3181 gggacgccgg ctggatgatc ctccagcgcg gggatctcat gctggagttc ttcgcccacc
     3241 ccaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca
     3301 caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat
     3361 cttatcatgt ctgtataccg tcgacctcta gctagagctt ggcgtaatca tggtcatagc
     3421 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca
     3481 taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct
     3541 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
     3601 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc
     3661 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     3721 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     3781 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     3841 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     3901 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     3961 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     4021 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     4081 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     4141 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     4201 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag
     4261 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     4321 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     4381 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     4441 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     4501 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
     4561 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
     4621 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
     4681 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
     4741 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
     4801 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
     4861 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg
     4921 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
     4981 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
     5041 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
     5101 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
     5161 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa
     5221 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
     5281 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
     5341 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
     5401 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
     5461 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     5521 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc gacggatcgg
     5581 gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt
     5641 aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg cgagcaaaat
     5701 ttaag
//
