LOCUS       sequence-307615-        7092 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mammalian expression of N-terminally Flag-His8 tagged SARS-CoV-2 
            Nsp2 under control of a tetracycline-inducible promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7092)
  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 7092)
  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-Nsp2
FEATURES             Location/Qualifiers
     source          1..7092
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        115..494
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        495..698
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     649..669
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    700..718
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    721..739
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     749..773
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             870..893
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             906..929
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     primer_bind     complement(2924..2941)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2930..3154
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3287..3306)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3438..3485
                     /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             3493..4515
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3493..4515
                     /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    4645..4766
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4682..4701)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4736..4755
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4815..4831)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4815..4831)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4828..4850)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4839..4855
                     /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(4863..4869,4870..4887,4888..4893))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4908..4929
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5046..5063)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5217..5805)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5297..5316)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5976..6767,6768..6836))
                     /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     6599..6618
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6837..6941)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7016..7035)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
       61 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      121 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      181 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      241 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      301 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      361 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      421 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      481 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      541 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      601 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      661 gtaggcgtgt acggtgggag gtctatataa gcagagctct ccctatcagt gatagagatc
      721 tccctatcag tgatagagat cgtcgacgag ctcgtttagt gaaccgtcag atcgcctgga
      781 gacgccatcc acgctgtttt gacctccata gaagacaccg ggaccgatcc agcctccgga
      841 ctctagcgtt taaacttaag cttgagatgg actataaaga tgacgatgac aaggccgcag
      901 ccgcacatca ccaccatcat caccatcacg gatccgggat ggcatacaca cgctatgttg
      961 ataataactt ttgtggccca gacggctatc cgctcgaatg cataaaggac cttcttgcac
     1021 gggccggaaa agcttcctgc actttgtctg agcagttgga cttcatcgat accaaaagag
     1081 gagtttattg ctgccgagag cacgagcacg aaatcgcgtg gtacactgag cgctccgaga
     1141 aaagctacga attgcagacg cctttcgaaa ttaaactcgc aaagaaattc gacaccttca
     1201 atggtgagtg cccaaatttt gtcttcccac ttaacagtat tatcaagacc atacagccga
     1261 gagtggagaa aaagaagctg gacggtttca tgggtcggat aaggagcgta taccctgttg
     1321 cgtcccccaa cgaatgcaac caaatgtgtt tgtcaacgct catgaaatgc gaccattgtg
     1381 gtgagactag ctggcaaact ggtgacttcg tcaaagctac ttgcgaattt tgtggcacgg
     1441 aaaaccttac caaagaaggc gctactactt gtgggtacct tccgcagaat gcggtggtaa
     1501 aaatctattg ccctgcgtgc cacaactctg aagtcggacc cgaacattct ctcgctgaat
     1561 atcacaacga atctggtctc aaaacgattc ttcgaaaagg cggacgcaca attgcgtttg
     1621 gcggctgcgt gttttcttac gtcggctgtc ataacaaatg cgcctactgg gtgccgcggg
     1681 cttcagccaa tattggttgt aaccataccg gagtggtcgg ggaagggagc gagggtctga
     1741 atgataacct gttggaaatt ttgcaaaagg aaaaggtaaa cattaatatc gttggagatt
     1801 ttaaattgaa cgaagaaatc gcgattattc tggcatcctt ttcagcatcc accagtgcct
     1861 tcgtagagac tgtgaaggga cttgactaca aggcttttaa gcagatagtc gagtcttgtg
     1921 ggaatttcaa ggtaacaaaa ggtaaagcga agaagggtgc ttggaatatt ggtgaacaga
     1981 aaagcatact cagcccattg tacgcatttg ccagtgaggc ggcacgagta gtgcgaagta
     2041 ttttcagcag gacgctcgaa acagcgcaga actccgtcag ggtgctccaa aaagccgcca
     2101 ttactatcct tgatggaatt agtcaataca gcttgagact gatagacgcg atgatgttta
     2161 cctccgactt ggcaacgaat aacttggtgg tgatggcgta tattacgggc ggcgttgttc
     2221 aacttacatc acagtggctc acaaatatat ttgggacggt ctacgagaag cttaagccgg
     2281 ttctcgactg gctggaagaa aagtttaaag agggggttga atttctccga gatggatggg
     2341 aaattgtcaa atttatttcc acatgtgcct gcgagatagt aggcgggcaa attgtcacat
     2401 gcgcgaaaga aattaaagaa agcgttcaaa catttttcaa gctcgtgaat aagttcttgg
     2461 ctctgtgtgc tgacagcata ataataggcg gggctaagct gaaagcgttg aatctcgggg
     2521 aaacatttgt tacgcatagt aaagggcttt atcggaagtg tgtgaaaagt agggaagaaa
     2581 ccggcctgct catgccgttg aaggctccaa aagagataat atttctggag ggcgaaactc
     2641 tgcccaccga ggttctgacc gaggaagtgg tactcaaaac tggggacctg cagcctcttg
     2701 agcaacccac gtccgaggct gttgaggccc ctcttgttgg cacacccgtg tgcataaacg
     2761 gtttgatgct cttggagatc aaggacacag aaaagtattg tgcactcgcg cccaatatga
     2821 tggttacgaa taataccttt acgctcaagg gaggtgccta ggagatatcc agcacagtgg
     2881 cggccgctcg agtctagagg gcccgtttaa acccgctgat cagcctcgac tgtgccttct
     2941 agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc
     3001 actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt
     3061 cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat
     3121 agcaggcatg ctggggatgc ggtgggctct atggcttctg aggcggaaag aaccagctgg
     3181 ggctctaggg ggtatcccca cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg
     3241 gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc
     3301 ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc
     3361 cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt
     3421 gatggttcac gtacctagaa gttcctattc cgaagttcct attctctaga aagtatagga
     3481 acttccttgg ccaaaaagcc tgaactcacc gcgacgtctg tcgagaagtt tctgatcgaa
     3541 aagttcgaca gcgtctccga cctgatgcag ctctcggagg gcgaagaatc tcgtgctttc
     3601 agcttcgatg taggagggcg tggatatgtc ctgcgggtaa atagctgcgc cgatggtttc
     3661 tacaaagatc gttatgttta tcggcacttt gcatcggccg cgctcccgat tccggaagtg
     3721 cttgacattg gggaattcag cgagagcctg acctattgca tctcccgccg tgcacagggt
     3781 gtcacgttgc aagacctgcc tgaaaccgaa ctgcccgctg ttctgcagcc ggtcgcggag
     3841 gccatggatg cgatcgctgc ggccgatctt agccagacga gcgggttcgg cccattcgga
     3901 ccgcaaggaa tcggtcaata cactacatgg cgtgatttca tatgcgcgat tgctgatccc
     3961 catgtgtatc actggcaaac tgtgatggac gacaccgtca gtgcgtccgt cgcgcaggct
     4021 ctcgatgagc tgatgctttg ggccgaggac tgccccgaag tccggcacct cgtgcacgcg
     4081 gatttcggct ccaacaatgt cctgacggac aatggccgca taacagcggt cattgactgg
     4141 agcgaggcga tgttcgggga ttcccaatac gaggtcgcca acatcttctt ctggaggccg
     4201 tggttggctt gtatggagca gcagacgcgc tacttcgagc ggaggcatcc ggagcttgca
     4261 ggatcgccgc ggctccgggc gtatatgctc cgcattggtc ttgaccaact ctatcagagc
     4321 ttggttgacg gcaatttcga tgatgcagct tgggcgcagg gtcgatgcga cgcaatcgtc
     4381 cgatccggag ccgggactgt cgggcgtaca caaatcgccc gcagaagcgc ggccgtctgg
     4441 accgatggct gtgtagaagt actcgccgat agtggaaacc gacgccccag cactcgtccg
     4501 agggcaaagg aatagcacgt actacgagat ttcgattcca ccgccgcctt ctatgaaagg
     4561 ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc
     4621 atgctggagt tcttcgccca ccccaacttg tttattgcag cttataatgg ttacaaataa
     4681 agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt
     4741 ttgtccaaac tcatcaatgt atcttatcat gtctgtatac cgtcgacctc tagctagagc
     4801 ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca
     4861 cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa
     4921 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     4981 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc
     5041 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     5101 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     5161 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     5221 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     5281 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     5341 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     5401 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     5461 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     5521 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     5581 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     5641 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     5701 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     5761 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     5821 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     5881 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     5941 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     6001 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     6061 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
     6121 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     6181 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     6241 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
     6301 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     6361 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     6421 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     6481 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     6541 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     6601 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     6661 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     6721 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     6781 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     6841 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     6901 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     6961 ccacctgacg tcgacggatc gggagatctc ccgatcccct atggtgcact ctcagtacaa
     7021 tctgctctga tgccgcatag ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg
     7081 ctgagtagtg cg
//
