LOCUS       sequence-307609-        5715 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mammalian expression of N-terminally Flag-His8 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 5715)
  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 5715)
  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-FH-Nsp1
FEATURES             Location/Qualifiers
     source          1..5715
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        121..500
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        501..704
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     655..675
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    706..724
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    727..745
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     755..779
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             876..899
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             912..935
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     primer_bind     complement(1553..1570)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1559..1783
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(1916..1935)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2067..2114
                     /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             2122..3144
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             2122..3144
                     /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    3274..3395
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3311..3330)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3365..3384
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3444..3460)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3444..3460)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3457..3479)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3468..3484
                     /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(3492..3498,3499..3516,3517..3522))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3537..3558
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3675..3692)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3846..4434)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3926..3945)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4605..5396,5397..5465))
                     /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     5228..5247
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5466..5570)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5645..5664)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 agtgcgcgag caaaatttaa gctacaacaa ggcaaggctt gaccgacaat tgcatgaaga
       61 atctgcttag ggttaggcgt tttgcgctgc ttcgcgatgt acgggccaga tatacgcgtt
      121 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
      181 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      241 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      301 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      361 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      421 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      481 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      541 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      601 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      661 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctccct atcagtgata
      721 gagatctccc tatcagtgat agagatcgtc gacgagctcg tttagtgaac cgtcagatcg
      781 cctggagacg ccatccacgc tgttttgacc tccatagaag acaccgggac cgatccagcc
      841 tccggactct agcgtttaaa cttaagcttg agatggacta taaagatgac gatgacaagg
      901 ccgcagccgc acatcaccac catcatcacc atcacggatc cgggatggaa tccttggtgc
      961 cgggttttaa tgagaaaaca cacgtccagt tgtccctccc agttttgcag gtgcgagatg
     1021 tgcttgtccg ggggttcggg gactccgtgg aggaggtact cagcgaagct agacagcatc
     1081 tcaaggacgg aacttgcgga ctcgttgaag ttgaaaaggg ggtactgcca cagcttgagc
     1141 agccctacgt cttcattaag cgatctgatg ccagaacggc tcctcatgga cacgtaatgg
     1201 tggagttggt cgctgagctt gagggaatac agtacggcag atctggagag acgctgggcg
     1261 tactcgtacc gcatgttggt gagatccctg ttgcatacag gaaagtattg ttgcgaaaaa
     1321 acggtaataa aggagcgggt ggtcatagct acggggcgga tcttaaatct tttgaccttg
     1381 gggatgaact gggcactgat ccgtacgaag attttcagga gaattggaat accaaacatt
     1441 caagcggcgt gacgagggaa cttatgagag agctgaacgg cggggcctag gagatatcca
     1501 gcacagtggc ggccgctcga gtctagaggg cccgtttaaa cccgctgatc agcctcgact
     1561 gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg
     1621 gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg
     1681 agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg
     1741 gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga ggcggaaaga
     1801 accagctggg gctctagggg gtatccccac gcgccctgta gcggcgcatt aagcgcggcg
     1861 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct
     1921 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
     1981 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
     2041 gattagggtg atggttcacg tacctagaag ttcctattcc gaagttccta ttctctagaa
     2101 agtataggaa cttccttggc caaaaagcct gaactcaccg cgacgtctgt cgagaagttt
     2161 ctgatcgaaa agttcgacag cgtctccgac ctgatgcagc tctcggaggg cgaagaatct
     2221 cgtgctttca gcttcgatgt aggagggcgt ggatatgtcc tgcgggtaaa tagctgcgcc
     2281 gatggtttct acaaagatcg ttatgtttat cggcactttg catcggccgc gctcccgatt
     2341 ccggaagtgc ttgacattgg ggaattcagc gagagcctga cctattgcat ctcccgccgt
     2401 gcacagggtg tcacgttgca agacctgcct gaaaccgaac tgcccgctgt tctgcagccg
     2461 gtcgcggagg ccatggatgc gatcgctgcg gccgatctta gccagacgag cgggttcggc
     2521 ccattcggac cgcaaggaat cggtcaatac actacatggc gtgatttcat atgcgcgatt
     2581 gctgatcccc atgtgtatca ctggcaaact gtgatggacg acaccgtcag tgcgtccgtc
     2641 gcgcaggctc tcgatgagct gatgctttgg gccgaggact gccccgaagt ccggcacctc
     2701 gtgcacgcgg atttcggctc caacaatgtc ctgacggaca atggccgcat aacagcggtc
     2761 attgactgga gcgaggcgat gttcggggat tcccaatacg aggtcgccaa catcttcttc
     2821 tggaggccgt ggttggcttg tatggagcag cagacgcgct acttcgagcg gaggcatccg
     2881 gagcttgcag gatcgccgcg gctccgggcg tatatgctcc gcattggtct tgaccaactc
     2941 tatcagagct tggttgacgg caatttcgat gatgcagctt gggcgcaggg tcgatgcgac
     3001 gcaatcgtcc gatccggagc cgggactgtc gggcgtacac aaatcgcccg cagaagcgcg
     3061 gccgtctgga ccgatggctg tgtagaagta ctcgccgata gtggaaaccg acgccccagc
     3121 actcgtccga gggcaaagga atagcacgta ctacgagatt tcgattccac cgccgccttc
     3181 tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc
     3241 ggggatctca tgctggagtt cttcgcccac cccaacttgt ttattgcagc ttataatggt
     3301 tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct
     3361 agttgtggtt tgtccaaact catcaatgta tcttatcatg tctgtatacc gtcgacctct
     3421 agctagagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc
     3481 acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga
     3541 gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     3601 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     3661 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
     3721 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     3781 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     3841 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     3901 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     3961 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     4021 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     4081 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     4141 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     4201 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     4261 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca
     4321 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     4381 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     4441 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     4501 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     4561 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     4621 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     4681 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     4741 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     4801 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     4861 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     4921 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     4981 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     5041 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     5101 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     5161 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     5221 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     5281 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     5341 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     5401 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     5461 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     5521 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     5581 cgaaaagtgc cacctgacgt cgacggatcg ggagatctcc cgatccccta tggtgcactc
     5641 tcagtacaat ctgctctgat gccgcatagt taagccagta tctgctccct gcttgtgtgt
     5701 tggaggtcgc tgagt
//
