LOCUS       sequence-307835-        6047 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mammalian expression of C-terminally His8-Flag tagged Nsp7-Nsp8 
            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 6047)
  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 6047)
  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-Nsp7-Nsp8-HF_GC3opt
FEATURES             Location/Qualifiers
     source          1..6047
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        122..501
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        502..705
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     656..676
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    707..725
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    728..746
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     756..780
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             1786..1809
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             1822..1845
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(1886..1903)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1892..2116
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2249..2268)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2400..2447
                     /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             2455..3477
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             2455..3477
                     /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    3607..3728
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3644..3663)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3698..3717
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3777..3793)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3777..3793)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3790..3812)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3801..3817
                     /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(3825..3831,3832..3849,3850..3855))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3870..3891
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4008..4025)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4179..4767)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4259..4278)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4938..5729,5730..5798))
                     /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     5561..5580
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5799..5903)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5978..5997)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 tagtgcgcga gcaaaattta agctacaaca aggcaaggct tgaccgacaa ttgcatgaag
       61 aatctgctta gggttaggcg ttttgcgctg cttcgcgatg tacgggccag atatacgcgt
      121 tgacattgat tattgactag ttattaatag taatcaatta cggggtcatt agttcatagc
      181 ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc
      241 aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg
      301 actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat
      361 caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc
      421 tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta catctacgta
      481 ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag
      541 cggtttgact cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt
      601 tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa
      661 atgggcggta ggcgtgtacg gtgggaggtc tatataagca gagctctccc tatcagtgat
      721 agagatctcc ctatcagtga tagagatcgt cgacgagctc gtttagtgaa ccgtcagatc
      781 gcctggagac gccatccacg ctgttttgac ctccatagaa gacaccggga ccgatccagc
      841 ctccggactc tagcgtttaa acttaagctt ggtaccgagc tcggatccgg gatgagcaag
      901 atgagcgacg tgaagtgcac cagcgtggtg ctgctgtctg ttctgcagca gctgagagtg
      961 gaaagcagca gcaaactgtg ggcccagtgt gtccagctgc acaacgatat tctgctggcc
     1021 aaggacacca ccgaggcctt cgagaagatg gtgtctctgc tgagcgtgct gctgagcatg
     1081 caaggcgccg tggacatcaa caagctgtgc gaggaaatgc tggacaaccg ggccacactg
     1141 cagggctctg ctggatctgc tgctggctct ggcgaatttg ccattgccag cgagttcagc
     1201 agcctgccta gctatgccgc ctttgccaca gctcaagagg cctatgaaca ggccgtggcc
     1261 aatggcgatt ctgaggtggt gctgaagaag ctgaaaaagt ccctgaacgt ggccaagagc
     1321 gagttcgaca gagatgccgc catgcagcgg aagctggaaa agatggccga ccaggccatg
     1381 acacagatgt acaagcaggc cagaagcgag gacaagaggg ccaaagtgac ctctgccatg
     1441 cagaccatgc tgttcaccat gctgagaaag ctggataacg acgccctgaa caacatcatc
     1501 aacaacgcca gagatggctg cgtgcccctg aatatcatcc ctctgacaac cgccgccaag
     1561 ctgatggtgg tcatccccga ctacaacacc tacaagaata cctgcgacgg caccaccttc
     1621 acctacgcct ctgctctgtg ggagattcag caggtcgtgg acgccgacag caagatcgtg
     1681 cagctgagcg agatcagcat ggacaacagc cccaatctgg cctggcctct gattgtgaca
     1741 gccctgagag ccaacagcgc cgtgaaactg caggcatcgg gcggacatca ccaccatcat
     1801 caccatcacg ccgcagccgc agattataaa gatgacgatg acaagtagct cgagtctaga
     1861 gggcccgttt aaacccgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt
     1921 gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc
     1981 taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt
     2041 ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat
     2101 gcggtgggct ctatggcttc tgaggcggaa agaaccagct ggggctctag ggggtatccc
     2161 cacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc
     2221 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
     2281 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt
     2341 agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtacctag
     2401 aagttcctat tccgaagttc ctattctcta gaaagtatag gaacttcctt ggccaaaaag
     2461 cctgaactca ccgcgacgtc tgtcgagaag tttctgatcg aaaagttcga cagcgtctcc
     2521 gacctgatgc agctctcgga gggcgaagaa tctcgtgctt tcagcttcga tgtaggaggg
     2581 cgtggatatg tcctgcgggt aaatagctgc gccgatggtt tctacaaaga tcgttatgtt
     2641 tatcggcact ttgcatcggc cgcgctcccg attccggaag tgcttgacat tggggaattc
     2701 agcgagagcc tgacctattg catctcccgc cgtgcacagg gtgtcacgtt gcaagacctg
     2761 cctgaaaccg aactgcccgc tgttctgcag ccggtcgcgg aggccatgga tgcgatcgct
     2821 gcggccgatc ttagccagac gagcgggttc ggcccattcg gaccgcaagg aatcggtcaa
     2881 tacactacat ggcgtgattt catatgcgcg attgctgatc cccatgtgta tcactggcaa
     2941 actgtgatgg acgacaccgt cagtgcgtcc gtcgcgcagg ctctcgatga gctgatgctt
     3001 tgggccgagg actgccccga agtccggcac ctcgtgcacg cggatttcgg ctccaacaat
     3061 gtcctgacgg acaatggccg cataacagcg gtcattgact ggagcgaggc gatgttcggg
     3121 gattcccaat acgaggtcgc caacatcttc ttctggaggc cgtggttggc ttgtatggag
     3181 cagcagacgc gctacttcga gcggaggcat ccggagcttg caggatcgcc gcggctccgg
     3241 gcgtatatgc tccgcattgg tcttgaccaa ctctatcaga gcttggttga cggcaatttc
     3301 gatgatgcag cttgggcgca gggtcgatgc gacgcaatcg tccgatccgg agccgggact
     3361 gtcgggcgta cacaaatcgc ccgcagaagc gcggccgtct ggaccgatgg ctgtgtagaa
     3421 gtactcgccg atagtggaaa ccgacgcccc agcactcgtc cgagggcaaa ggaatagcac
     3481 gtactacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt
     3541 ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc
     3601 caccccaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat
     3661 ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat
     3721 gtatcttatc atgtctgtat accgtcgacc tctagctaga gcttggcgta atcatggtca
     3781 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga
     3841 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg
     3901 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     3961 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
     4021 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     4081 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     4141 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     4201 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     4261 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     4321 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     4381 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     4441 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     4501 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     4561 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga
     4621 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     4681 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     4741 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     4801 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     4861 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     4921 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     4981 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     5041 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     5101 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     5161 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     5221 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     5281 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     5341 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     5401 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     5461 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     5521 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     5581 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     5641 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     5701 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     5761 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     5821 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     5881 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtcgacgga
     5941 tcgggagatc tcccgatccc ctatggtgca ctctcagtac aatctgctct gatgccgcat
     6001 agttaagcca gtatctgctc cctgcttgtg tgttggaggt cgctgag
//
