LOCUS       sequence-308154-        5832 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mammalian expression of untagged eGFP under control of a 
            tetracycline-inducible promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5832)
  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 5832)
  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-eGFP
FEATURES             Location/Qualifiers
     source          1..5832
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     109..130
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(221..238)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    227..451
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(584..603)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    735..782
                     /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             790..1812
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             790..1812
                     /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    1942..2063
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1979..1998)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2033..2052
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2112..2128)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2112..2128)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2125..2147)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2136..2152
                     /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(2160..2166,2167..2184,2185..2190))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2205..2226
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2343..2360)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2514..3102)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2594..2613)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3273..4064,4065..4133))
                     /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     3896..3915
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4134..4238)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(4313..4332)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        4504..4883
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4884..5087
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     5038..5058
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    5089..5107
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5110..5128
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     5138..5162
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      5282..5291
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(5288..5290,5291..5293,5294..5832,1..175)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(5288..5290,5291..5293,5294..5832,1..172)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(5333..5354)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(5594..5613)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
ORIGIN
        1 ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct
       61 gagcacccag tccgccctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct
      121 ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtaca agtaaagcgg
      181 ccgctcgagt ctagagggcc cgtttaaacc cgctgatcag cctcgactgt gccttctagt
      241 tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact
      301 cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat
      361 tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc
      421 aggcatgctg gggatgcggt gggctctatg gcttctgagg cggaaagaac cagctggggc
      481 tctagggggt atccccacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt
      541 acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc
      601 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct
      661 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat
      721 ggttcacgta cctagaagtt cctattccga agttcctatt ctctagaaag tataggaact
      781 tccttggcca aaaagcctga actcaccgcg acgtctgtcg agaagtttct gatcgaaaag
      841 ttcgacagcg tctccgacct gatgcagctc tcggagggcg aagaatctcg tgctttcagc
      901 ttcgatgtag gagggcgtgg atatgtcctg cgggtaaata gctgcgccga tggtttctac
      961 aaagatcgtt atgtttatcg gcactttgca tcggccgcgc tcccgattcc ggaagtgctt
     1021 gacattgggg aattcagcga gagcctgacc tattgcatct cccgccgtgc acagggtgtc
     1081 acgttgcaag acctgcctga aaccgaactg cccgctgttc tgcagccggt cgcggaggcc
     1141 atggatgcga tcgctgcggc cgatcttagc cagacgagcg ggttcggccc attcggaccg
     1201 caaggaatcg gtcaatacac tacatggcgt gatttcatat gcgcgattgc tgatccccat
     1261 gtgtatcact ggcaaactgt gatggacgac accgtcagtg cgtccgtcgc gcaggctctc
     1321 gatgagctga tgctttgggc cgaggactgc cccgaagtcc ggcacctcgt gcacgcggat
     1381 ttcggctcca acaatgtcct gacggacaat ggccgcataa cagcggtcat tgactggagc
     1441 gaggcgatgt tcggggattc ccaatacgag gtcgccaaca tcttcttctg gaggccgtgg
     1501 ttggcttgta tggagcagca gacgcgctac ttcgagcgga ggcatccgga gcttgcagga
     1561 tcgccgcggc tccgggcgta tatgctccgc attggtcttg accaactcta tcagagcttg
     1621 gttgacggca atttcgatga tgcagcttgg gcgcagggtc gatgcgacgc aatcgtccga
     1681 tccggagccg ggactgtcgg gcgtacacaa atcgcccgca gaagcgcggc cgtctggacc
     1741 gatggctgtg tagaagtact cgccgatagt ggaaaccgac gccccagcac tcgtccgagg
     1801 gcaaaggaat agcacgtact acgagatttc gattccaccg ccgccttcta tgaaaggttg
     1861 ggcttcggaa tcgttttccg ggacgccggc tggatgatcc tccagcgcgg ggatctcatg
     1921 ctggagttct tcgcccaccc caacttgttt attgcagctt ataatggtta caaataaagc
     1981 aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg
     2041 tccaaactca tcaatgtatc ttatcatgtc tgtataccgt cgacctctag ctagagcttg
     2101 gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac
     2161 aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc
     2221 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     2281 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct
     2341 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
     2401 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     2461 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     2521 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     2581 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     2641 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     2701 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     2761 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     2821 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     2881 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     2941 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     3001 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     3061 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     3121 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     3181 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     3241 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     3301 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     3361 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
     3421 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     3481 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     3541 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
     3601 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     3661 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     3721 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     3781 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     3841 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
     3901 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     3961 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     4021 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     4081 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     4141 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     4201 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
     4261 cctgacgtcg acggatcggg agatctcccg atcccctatg gtgcactctc agtacaatct
     4321 gctctgatgc cgcatagtta agccagtatc tgctccctgc ttgtgtgttg gaggtcgctg
     4381 agtagtgcgc gagcaaaatt taagctacaa caaggcaagg cttgaccgac aattgcatga
     4441 agaatctgct tagggttagg cgttttgcgc tgcttcgcga tgtacgggcc agatatacgc
     4501 gttgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata
     4561 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
     4621 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
     4681 ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac
     4741 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
     4801 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
     4861 tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat
     4921 agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt
     4981 tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc
     5041 aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctctc cctatcagtg
     5101 atagagatct ccctatcagt gatagagatc gtcgacgagc tcgtttagtg aaccgtcaga
     5161 tcgcctggag acgccatcca cgctgttttg acctccatag aagacaccgg gaccgatcca
     5221 gcctccggac tctagcgttt aaacttaagc ttggtaccga gctcggatcc accggccggt
     5281 cgccaccatg gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga
     5341 gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc
     5401 cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg
     5461 gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct accccgacca
     5521 catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac
     5581 catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga
     5641 caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct
     5701 ggggcacaag ctggagtaca actacaacag ccacaacgtc tatatcatgg ccgacaagca
     5761 gaagaacggc atcaaggtga acttcaagat ccgccacaac atcgaggacg gcagcgtgca
     5821 gctcgccgac ca
//
