LOCUS       sequence-307863-        5762 bp    DNA     circular SYN 15-APR-2025
DEFINITION  mammalian expression of C-terminally His8-Flag tagged SARS-CoV-2 
            Nsp8 under control of a tetracycline-inducible promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5762)
  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 5762)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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-Nsp8-HF_GC3opt
FEATURES             Location/Qualifiers
     source          1..5762
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        95..474
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        475..678
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     629..649
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    680..698
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    701..719
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     729..753
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             1474..1497
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             1510..1533
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(1574..1591)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1580..1804
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(1937..1956)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2088..2135
                     /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             2143..3165
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             2143..3165
                     /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    3295..3416
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3332..3351)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3386..3405
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3465..3481)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3465..3481)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3478..3500)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3489..3505
                     /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(3513..3519,3520..3537,3538..3543))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3558..3579
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3696..3713)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3867..4455)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3947..3966)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4626..5417,5418..5486))
                     /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     5249..5268
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5487..5591)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5666..5685)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 acaaggcaag gcttgaccga caattgcatg aagaatctgc ttagggttag gcgttttgcg
       61 ctgcttcgcg atgtacgggc cagatatacg cgttgacatt gattattgac tagttattaa
      121 tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg cgttacataa
      181 cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata
      241 atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag
      301 tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc
      361 cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta
      421 tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac catggtgatg
      481 cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg atttccaagt
      541 ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg ggactttcca
      601 aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt acggtgggag
      661 gtctatataa gcagagctct ccctatcagt gatagagatc tccctatcag tgatagagat
      721 cgtcgacgag ctcgtttagt gaaccgtcag atcgcctgga gacgccatcc acgctgtttt
      781 gacctccata gaagacaccg ggaccgatcc agcctccgga ctctagcgtt taaacttaag
      841 cttggtaccg agctcggatc cgggatggcc attgccagcg agtttagcag cctgcctagc
      901 tacgccgcct ttgccacagc tcaagaggcc tatgaacagg ccgtggccaa tggcgattct
      961 gaggtggtgc tgaagaagct gaaaaagtcc ctgaacgtgg ccaagagcga gttcgacaga
     1021 gatgccgcca tgcagcggaa gctggaaaag atggccgacc aggccatgac acagatgtac
     1081 aagcaggcca gaagcgagga caagcgggcc aaagtgacat ctgccatgca gaccatgctg
     1141 ttcaccatgc tgagaaagct ggacaacgac gccctgaaca acatcatcaa caacgccaga
     1201 gatggctgcg tgcccctgaa tatcatccct ctgacaaccg ccgccaagct gatggtggtc
     1261 atccccgact acaacaccta caagaatacc tgcgacggca ccaccttcac ctacgcctct
     1321 gctctgtggg agatccagca ggtcgtggac gccgatagca agatcgtgca gctgagcgag
     1381 atcagcatgg acaacagccc caatctggcc tggcctctga ttgtgacagc cctgagagcc
     1441 aacagcgccg tgaaactgca ggcatcgggc ggacatcacc accatcatca ccatcacgcc
     1501 gcagccgcag attataaaga tgacgatgac aagtagctcg agtctagagg gcccgtttaa
     1561 acccgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc
     1621 cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag
     1681 gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag
     1741 gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc ggtgggctct
     1801 atggcttctg aggcggaaag aaccagctgg ggctctaggg ggtatcccca cgcgccctgt
     1861 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc
     1921 agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc
     1981 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg
     2041 cacctcgacc ccaaaaaact tgattagggt gatggttcac gtacctagaa gttcctattc
     2101 cgaagttcct attctctaga aagtatagga acttccttgg ccaaaaagcc tgaactcacc
     2161 gcgacgtctg tcgagaagtt tctgatcgaa aagttcgaca gcgtctccga cctgatgcag
     2221 ctctcggagg gcgaagaatc tcgtgctttc agcttcgatg taggagggcg tggatatgtc
     2281 ctgcgggtaa atagctgcgc cgatggtttc tacaaagatc gttatgttta tcggcacttt
     2341 gcatcggccg cgctcccgat tccggaagtg cttgacattg gggaattcag cgagagcctg
     2401 acctattgca tctcccgccg tgcacagggt gtcacgttgc aagacctgcc tgaaaccgaa
     2461 ctgcccgctg ttctgcagcc ggtcgcggag gccatggatg cgatcgctgc ggccgatctt
     2521 agccagacga gcgggttcgg cccattcgga ccgcaaggaa tcggtcaata cactacatgg
     2581 cgtgatttca tatgcgcgat tgctgatccc catgtgtatc actggcaaac tgtgatggac
     2641 gacaccgtca gtgcgtccgt cgcgcaggct ctcgatgagc tgatgctttg ggccgaggac
     2701 tgccccgaag tccggcacct cgtgcacgcg gatttcggct ccaacaatgt cctgacggac
     2761 aatggccgca taacagcggt cattgactgg agcgaggcga tgttcgggga ttcccaatac
     2821 gaggtcgcca acatcttctt ctggaggccg tggttggctt gtatggagca gcagacgcgc
     2881 tacttcgagc ggaggcatcc ggagcttgca ggatcgccgc ggctccgggc gtatatgctc
     2941 cgcattggtc ttgaccaact ctatcagagc ttggttgacg gcaatttcga tgatgcagct
     3001 tgggcgcagg gtcgatgcga cgcaatcgtc cgatccggag ccgggactgt cgggcgtaca
     3061 caaatcgccc gcagaagcgc ggccgtctgg accgatggct gtgtagaagt actcgccgat
     3121 agtggaaacc gacgccccag cactcgtccg agggcaaagg aatagcacgt actacgagat
     3181 ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc
     3241 ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaacttg
     3301 tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa
     3361 gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat
     3421 gtctgtatac cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct
     3481 gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt
     3541 aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc
     3601 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     3661 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
     3721 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     3781 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     3841 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     3901 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     3961 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     4021 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     4081 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     4141 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     4201 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     4261 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
     4321 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     4381 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     4441 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     4501 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     4561 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     4621 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     4681 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
     4741 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
     4801 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     4861 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     4921 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     4981 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
     5041 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
     5101 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
     5161 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
     5221 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
     5281 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
     5341 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
     5401 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
     5461 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
     5521 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     5581 ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tcgacggatc gggagatctc
     5641 ccgatcccct atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt
     5701 atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg cgcgagcaaa atttaagcta
     5761 ca
//
