LOCUS       sequence-307864-        6003 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mammalian expression of untagged SARS-CoV-2 Nsp8-Nsp7 fusion protein
            under control of a tetracycline-inducible promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6003)
  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 6003)
  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: pcDNA-FRT-TO-Nsp8-Nsp7_GC3opt
FEATURES             Location/Qualifiers
     source          1..6003
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        150..529
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        530..733
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     684..704
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    735..753
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    756..774
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     784..808
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     primer_bind     complement(1870..1887)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1876..2100
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2233..2252)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2384..2431
                     /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             2439..3461
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             2439..3461
                     /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    3591..3712
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3628..3647)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3682..3701
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3761..3777)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3761..3777)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3774..3796)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3785..3801
                     /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(3809..3815,3816..3833,3834..3839))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3854..3875
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3992..4009)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4163..4751)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4243..4262)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4922..5713,5714..5782))
                     /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     5545..5564
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5783..5887)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5962..5981)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 ccctgcttgt gtgttggagg tcgctgagta gtgcgcgagc aaaatttaag ctacaacaag
       61 gcaaggcttg accgacaatt gcatgaagaa tctgcttagg gttaggcgtt ttgcgctgct
      121 tcgcgatgta cgggccagat atacgcgttg acattgatta ttgactagtt attaatagta
      181 atcaattacg gggtcattag ttcatagccc atatatggag ttccgcgtta cataacttac
      241 ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caataatgac
      301 gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt
      361 acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagta cgccccctat
      421 tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttatggga
      481 ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgg tgatgcggtt
      541 ttggcagtac atcaatgggc gtggatagcg gtttgactca cggggatttc caagtctcca
      601 ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact ttccaaaatg
      661 tcgtaacaac tccgccccat tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta
      721 tataagcaga gctctcccta tcagtgatag agatctccct atcagtgata gagatcgtcg
      781 acgagctcgt ttagtgaacc gtcagatcgc ctggagacgc catccacgct gttttgacct
      841 ccatagaaga caccgggacc gatccagcct ccggactcta gcgtttaaac ttaagcttgg
      901 taccgagctc ggatccggga tggccattgc cagcgagttt agcagcctgc ctagctacgc
      961 cgcctttgcc acagctcaag aggcctatga acaggccgtg gccaatggcg attctgaggt
     1021 ggtgctgaag aagctgaaaa agtccctgaa cgtggccaag agcgagttcg acagagatgc
     1081 cgccatgcag cggaagctgg aaaagatggc cgaccaggcc atgacacaga tgtacaagca
     1141 ggccagaagc gaggacaagc gggccaaagt gacatctgcc atgcagacca tgctgttcac
     1201 catgctgaga aagctggaca acgacgccct gaacaacatc atcaacaacg ccagagatgg
     1261 ctgcgtgccc ctgaatatca tccctctgac aaccgccgcc aagctgatgg tggtcatccc
     1321 cgactacaac acctacaaga atacctgcga cggcaccacc ttcacctacg cctctgctct
     1381 gtgggagatc cagcaggtcg tggacgccga tagcaagatc gtgcagctga gcgagatcag
     1441 catggacaac agccccaatc tggcctggcc tctgattgtg acagccctga gagccaacag
     1501 cgccgtgaaa ctgcaaggct ctgcaggatc tgctgccggc tctggcgagt tcagcaagat
     1561 gagcgacgtg aagtgcacca gcgtggtgct gctgtctgtt ctgcagcagc tgagagtgga
     1621 aagcagcagc aaactgtggg cccagtgtgt ccagctgcac aatgacatcc tgctggccaa
     1681 ggataccacc gaggccttcg agaagatggt gtccctgctg agtgtgctgc tgagcatgca
     1741 aggcgccgtg gacatcaaca agctgtgcga ggaaatgctg gataaccggg ccacactgca
     1801 ggcctaggag atatccagca cagtggcggc cgctcgagtc tagagggccc gtttaaaccc
     1861 gctgatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg
     1921 tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa
     1981 ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca
     2041 gcaaggggga ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg
     2101 cttctgaggc ggaaagaacc agctggggct ctagggggta tccccacgcg ccctgtagcg
     2161 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg
     2221 ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc
     2281 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc
     2341 tcgaccccaa aaaacttgat tagggtgatg gttcacgtac ctagaagttc ctattccgaa
     2401 gttcctattc tctagaaagt ataggaactt ccttggccaa aaagcctgaa ctcaccgcga
     2461 cgtctgtcga gaagtttctg atcgaaaagt tcgacagcgt ctccgacctg atgcagctct
     2521 cggagggcga agaatctcgt gctttcagct tcgatgtagg agggcgtgga tatgtcctgc
     2581 gggtaaatag ctgcgccgat ggtttctaca aagatcgtta tgtttatcgg cactttgcat
     2641 cggccgcgct cccgattccg gaagtgcttg acattgggga attcagcgag agcctgacct
     2701 attgcatctc ccgccgtgca cagggtgtca cgttgcaaga cctgcctgaa accgaactgc
     2761 ccgctgttct gcagccggtc gcggaggcca tggatgcgat cgctgcggcc gatcttagcc
     2821 agacgagcgg gttcggccca ttcggaccgc aaggaatcgg tcaatacact acatggcgtg
     2881 atttcatatg cgcgattgct gatccccatg tgtatcactg gcaaactgtg atggacgaca
     2941 ccgtcagtgc gtccgtcgcg caggctctcg atgagctgat gctttgggcc gaggactgcc
     3001 ccgaagtccg gcacctcgtg cacgcggatt tcggctccaa caatgtcctg acggacaatg
     3061 gccgcataac agcggtcatt gactggagcg aggcgatgtt cggggattcc caatacgagg
     3121 tcgccaacat cttcttctgg aggccgtggt tggcttgtat ggagcagcag acgcgctact
     3181 tcgagcggag gcatccggag cttgcaggat cgccgcggct ccgggcgtat atgctccgca
     3241 ttggtcttga ccaactctat cagagcttgg ttgacggcaa tttcgatgat gcagcttggg
     3301 cgcagggtcg atgcgacgca atcgtccgat ccggagccgg gactgtcggg cgtacacaaa
     3361 tcgcccgcag aagcgcggcc gtctggaccg atggctgtgt agaagtactc gccgatagtg
     3421 gaaaccgacg ccccagcact cgtccgaggg caaaggaata gcacgtacta cgagatttcg
     3481 attccaccgc cgccttctat gaaaggttgg gcttcggaat cgttttccgg gacgccggct
     3541 ggatgatcct ccagcgcggg gatctcatgc tggagttctt cgcccacccc aacttgttta
     3601 ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat
     3661 ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct
     3721 gtataccgtc gacctctagc tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt
     3781 gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag
     3841 cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt
     3901 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag
     3961 gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
     4021 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
     4081 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
     4141 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     4201 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     4261 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     4321 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
     4381 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     4441 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     4501 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     4561 cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct
     4621 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     4681 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     4741 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     4801 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
     4861 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
     4921 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
     4981 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
     5041 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa
     5101 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
     5161 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
     5221 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
     5281 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
     5341 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
     5401 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
     5461 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
     5521 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc
     5581 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
     5641 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
     5701 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
     5761 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
     5821 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
     5881 ttccgcgcac atttccccga aaagtgccac ctgacgtcga cggatcggga gatctcccga
     5941 tcccctatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatct
     6001 gct
//
