LOCUS       sequence-307936-        5361 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mammalian expression of N-terminally Flag-His8 tagged SARS-CoV-2 
            Orf6 under control of a tetracycline-inducible promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5361)
  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 5361)
  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-Orf6
FEATURES             Location/Qualifiers
     source          1..5361
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        69..448
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        449..652
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     603..623
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    654..672
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    675..693
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     703..727
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             824..847
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             860..883
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     primer_bind     complement(1147..1164)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1153..1377
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(1510..1529)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    1661..1708
                     /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             1716..2738
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             1716..2738
                     /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    2868..2989
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2905..2924)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2959..2978
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3038..3054)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3038..3054)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3051..3073)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3062..3078
                     /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(3086..3092,3093..3110,3111..3116))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3131..3152
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3269..3286)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3440..4028)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3520..3539)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4199..4990,4991..5059))
                     /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     4822..4841
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5060..5164)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5239..5258)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 catgaagaat ctgcttaggg ttaggcgttt tgcgctgctt cgcgatgtac gggccagata
       61 tacgcgttga cattgattat tgactagtta ttaatagtaa tcaattacgg ggtcattagt
      121 tcatagccca tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg
      181 accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc
      241 aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc
      301 agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg
      361 gcccgcctgg cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat
      421 ctacgtatta gtcatcgcta ttaccatggt gatgcggttt tggcagtaca tcaatgggcg
      481 tggatagcgg tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag
      541 tttgttttgg caccaaaatc aacgggactt tccaaaatgt cgtaacaact ccgccccatt
      601 gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat ataagcagag ctctccctat
      661 cagtgataga gatctcccta tcagtgatag agatcgtcga cgagctcgtt tagtgaaccg
      721 tcagatcgcc tggagacgcc atccacgctg ttttgacctc catagaagac accgggaccg
      781 atccagcctc cggactctag cgtttaaact taagcttgag atggactata aagatgacga
      841 tgacaaggcc gcagccgcac atcaccacca tcatcaccat cacggatccg ggatgtttca
      901 tttggtggac tttcaggtta caatcgcgga gatcctgctc ataataatga ggacctttaa
      961 ggtgtcaata tggaaccttg attacattat caacctgatt attaagaacc tcagcaaaag
     1021 cctgacagaa aacaaataca gccagttgga tgaagaacaa cctatggaaa tcgactaagc
     1081 ctaggagata tccagcacag tggcggccgc tcgagtctag agggcccgtt taaacccgct
     1141 gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc
     1201 cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg
     1261 catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca
     1321 agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatggctt
     1381 ctgaggcgga aagaaccagc tggggctcta gggggtatcc ccacgcgccc tgtagcggcg
     1441 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc
     1501 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc
     1561 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg
     1621 accccaaaaa acttgattag ggtgatggtt cacgtaccta gaagttccta ttccgaagtt
     1681 cctattctct agaaagtata ggaacttcct tggccaaaaa gcctgaactc accgcgacgt
     1741 ctgtcgagaa gtttctgatc gaaaagttcg acagcgtctc cgacctgatg cagctctcgg
     1801 agggcgaaga atctcgtgct ttcagcttcg atgtaggagg gcgtggatat gtcctgcggg
     1861 taaatagctg cgccgatggt ttctacaaag atcgttatgt ttatcggcac tttgcatcgg
     1921 ccgcgctccc gattccggaa gtgcttgaca ttggggaatt cagcgagagc ctgacctatt
     1981 gcatctcccg ccgtgcacag ggtgtcacgt tgcaagacct gcctgaaacc gaactgcccg
     2041 ctgttctgca gccggtcgcg gaggccatgg atgcgatcgc tgcggccgat cttagccaga
     2101 cgagcgggtt cggcccattc ggaccgcaag gaatcggtca atacactaca tggcgtgatt
     2161 tcatatgcgc gattgctgat ccccatgtgt atcactggca aactgtgatg gacgacaccg
     2221 tcagtgcgtc cgtcgcgcag gctctcgatg agctgatgct ttgggccgag gactgccccg
     2281 aagtccggca cctcgtgcac gcggatttcg gctccaacaa tgtcctgacg gacaatggcc
     2341 gcataacagc ggtcattgac tggagcgagg cgatgttcgg ggattcccaa tacgaggtcg
     2401 ccaacatctt cttctggagg ccgtggttgg cttgtatgga gcagcagacg cgctacttcg
     2461 agcggaggca tccggagctt gcaggatcgc cgcggctccg ggcgtatatg ctccgcattg
     2521 gtcttgacca actctatcag agcttggttg acggcaattt cgatgatgca gcttgggcgc
     2581 agggtcgatg cgacgcaatc gtccgatccg gagccgggac tgtcgggcgt acacaaatcg
     2641 cccgcagaag cgcggccgtc tggaccgatg gctgtgtaga agtactcgcc gatagtggaa
     2701 accgacgccc cagcactcgt ccgagggcaa aggaatagca cgtactacga gatttcgatt
     2761 ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga
     2821 tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccccaac ttgtttattg
     2881 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt
     2941 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgta
     3001 taccgtcgac ctctagctag agcttggcgt aatcatggtc atagctgttt cctgtgtgaa
     3061 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
     3121 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
     3181 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     3241 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     3301 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     3361 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     3421 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     3481 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     3541 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     3601 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     3661 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     3721 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     3781 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     3841 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     3901 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     3961 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     4021 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     4081 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     4141 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     4201 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     4261 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     4321 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     4381 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     4441 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     4501 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     4561 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     4621 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     4681 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     4741 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     4801 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     4861 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     4921 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     4981 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     5041 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     5101 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     5161 cgcgcacatt tccccgaaaa gtgccacctg acgtcgacgg atcgggagat ctcccgatcc
     5221 cctatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agtatctgct
     5281 ccctgcttgt gtgttggagg tcgctgagta gtgcgcgagc aaaatttaag ctacaacaag
     5341 gcaaggcttg accgacaatt g
//
