LOCUS       sequence-307608-        5661 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mammalian expression of untagged 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 5661)
  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 5661)
  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
FEATURES             Location/Qualifiers
     source          1..5661
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        86..465
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        466..669
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     620..640
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    671..689
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    692..710
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     720..744
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     primer_bind     complement(1464..1481)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1470..1694
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(1827..1846)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    1978..2025
                     /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             2033..3055
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             2033..3055
                     /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    3185..3306
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3222..3241)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3276..3295
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3355..3371)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3355..3371)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3368..3390)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3379..3395
                     /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(3403..3409,3410..3427,3428..3433))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3448..3469
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3586..3603)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3757..4345)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3837..3856)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4516..5307,5308..5376))
                     /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     5139..5158
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5377..5481)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5556..5575)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 ggcttgaccg acaattgcat gaagaatctg cttagggtta ggcgttttgc gctgcttcgc
       61 gatgtacggg ccagatatac gcgttgacat tgattattga ctagttatta atagtaatca
      121 attacggggt cattagttca tagcccatat atggagttcc gcgttacata acttacggta
      181 aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat
      241 gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg
      301 taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc ccctattgac
      361 gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt atgggacttt
      421 cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat gcggttttgg
      481 cagtacatca atgggcgtgg atagcggttt gactcacggg gatttccaag tctccacccc
      541 attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc aaaatgtcgt
      601 aacaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata
      661 agcagagctc tccctatcag tgatagagat ctccctatca gtgatagaga tcgtcgacga
      721 gctcgtttag tgaaccgtca gatcgcctgg agacgccatc cacgctgttt tgacctccat
      781 agaagacacc gggaccgatc cagcctccgg actctagcgt ttaaacttaa gcttggtacc
      841 gagctcggat ccgggatgga atccttggtg ccgggtttta atgagaaaac acacgtccag
      901 ttgtccctcc cagttttgca ggtgcgagat gtgcttgtcc gggggttcgg ggactccgtg
      961 gaggaggtac tcagcgaagc tagacagcat ctcaaggacg gaacttgcgg actcgttgaa
     1021 gttgaaaagg gggtactgcc acagcttgag cagccctacg tcttcattaa gcgatctgat
     1081 gccagaacgg ctcctcatgg acacgtaatg gtggagttgg tcgctgagct tgagggaata
     1141 cagtacggca gatctggaga gacgctgggc gtactcgtac cgcatgttgg tgagatccct
     1201 gttgcataca ggaaagtatt gttgcgaaaa aacggtaata aaggagcggg tggtcatagc
     1261 tacggggcgg atcttaaatc ttttgacctt ggggatgaac tgggcactga tccgtacgaa
     1321 gattttcagg agaattggaa taccaaacat tcaagcggcg tgacgaggga acttatgaga
     1381 gagctgaacg gcggggccta ggagatatcc agcacagtgg cggccgctcg agtctagagg
     1441 gcccgtttaa acccgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt
     1501 ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta
     1561 ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg
     1621 ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc
     1681 ggtgggctct atggcttctg aggcggaaag aaccagctgg ggctctaggg ggtatcccca
     1741 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     1801 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     1861 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
     1921 tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtacctagaa
     1981 gttcctattc cgaagttcct attctctaga aagtatagga acttccttgg ccaaaaagcc
     2041 tgaactcacc gcgacgtctg tcgagaagtt tctgatcgaa aagttcgaca gcgtctccga
     2101 cctgatgcag ctctcggagg gcgaagaatc tcgtgctttc agcttcgatg taggagggcg
     2161 tggatatgtc ctgcgggtaa atagctgcgc cgatggtttc tacaaagatc gttatgttta
     2221 tcggcacttt gcatcggccg cgctcccgat tccggaagtg cttgacattg gggaattcag
     2281 cgagagcctg acctattgca tctcccgccg tgcacagggt gtcacgttgc aagacctgcc
     2341 tgaaaccgaa ctgcccgctg ttctgcagcc ggtcgcggag gccatggatg cgatcgctgc
     2401 ggccgatctt agccagacga gcgggttcgg cccattcgga ccgcaaggaa tcggtcaata
     2461 cactacatgg cgtgatttca tatgcgcgat tgctgatccc catgtgtatc actggcaaac
     2521 tgtgatggac gacaccgtca gtgcgtccgt cgcgcaggct ctcgatgagc tgatgctttg
     2581 ggccgaggac tgccccgaag tccggcacct cgtgcacgcg gatttcggct ccaacaatgt
     2641 cctgacggac aatggccgca taacagcggt cattgactgg agcgaggcga tgttcgggga
     2701 ttcccaatac gaggtcgcca acatcttctt ctggaggccg tggttggctt gtatggagca
     2761 gcagacgcgc tacttcgagc ggaggcatcc ggagcttgca ggatcgccgc ggctccgggc
     2821 gtatatgctc cgcattggtc ttgaccaact ctatcagagc ttggttgacg gcaatttcga
     2881 tgatgcagct tgggcgcagg gtcgatgcga cgcaatcgtc cgatccggag ccgggactgt
     2941 cgggcgtaca caaatcgccc gcagaagcgc ggccgtctgg accgatggct gtgtagaagt
     3001 actcgccgat agtggaaacc gacgccccag cactcgtccg agggcaaagg aatagcacgt
     3061 actacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt
     3121 ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca
     3181 ccccaacttg tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt
     3241 cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt
     3301 atcttatcat gtctgtatac cgtcgacctc tagctagagc ttggcgtaat catggtcata
     3361 gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag
     3421 cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg
     3481 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
     3541 acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc
     3601 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
     3661 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
     3721 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     3781 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     3841 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     3901 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
     3961 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     4021 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     4081 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     4141 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac
     4201 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     4261 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     4321 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     4381 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
     4441 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
     4501 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     4561 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
     4621 cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga
     4681 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     4741 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     4801 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
     4861 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     4921 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     4981 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     5041 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     5101 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
     5161 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     5221 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
     5281 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
     5341 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
     5401 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     5461 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tcgacggatc
     5521 gggagatctc ccgatcccct atggtgcact ctcagtacaa tctgctctga tgccgcatag
     5581 ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg cgcgagcaaa
     5641 atttaagcta caacaaggca a
//
