LOCUS       sequence-473274-        6426 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  transient overexpression of E2F6 in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6426)
  AUTHORS   Clijsters L, Hoencamp C, Calis JJA, Marzio A, Handgraaf SM, Cuitino 
            MC, Rosenberg BR, Leone G, Pagano M
  TITLE     Cyclin F Controls Cell-Cycle Transcriptional Outputs by Directing 
            the Degradation of the Three Activator E2Fs.
  JOURNAL   Mol Cell. 2019 Jun 20;74(6):1264-1277.e7. doi: 
            10.1016/j.molcel.2019.04.010. Epub 2019 May 23.
  PUBMED    31130363
REFERENCE   2  (bases 1 to 6426)
  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"; doi: 
            "10.1016/j.molcel.2019.04.010"; journalName: "Mol Cell"; date: 
            "2019-06-20- 20"; volume: "74"; issue: "6"; pages: "1264-1277.e7"
COMMENT     Sequence Label: pcDNA3 2xFLAG-2xSTREP E2F6
FEATURES             Location/Qualifiers
     source          1..6426
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      282..870
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     771..790
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1024..1041
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    1158..1179
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1194..1199,1200..1217,1218..1224)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1232..1248
                     /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)."
     primer_bind     1237..1259
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1256..1272
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1256..1272
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    1321..1442
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1332..1351)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1386..1405
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(1616..2410)
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(1727..1746)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2337..2356
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(2477..2806)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2491..2626
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2545..2564)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     2791..2811
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(2820..3248)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2930..2951)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3142..3161
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    3294..3518
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     3507..3524
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(4457..4480)
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             complement(4517..4540)
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             complement(4556..4579)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             complement(4595..4618)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(4659..4678)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4660..4678)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(4723..4926)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(4752..4772)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        4927..5306
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     5478..5497
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        5572..5676
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5677..5745,5746..6426,1..111)
                     /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     complement(5895..5914)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa
       61 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac
      121 caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc
      181 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
      241 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
      301 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
      361 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
      421 aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
      481 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
      541 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
      601 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
      661 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
      721 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
      781 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
      841 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
      901 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg
      961 gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag
     1021 cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc
     1081 gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc
     1141 agtgagcgca acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac
     1201 tttatgcttc cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga
     1261 aacagctatg accatgatta cgccaagctc tagctagagg tcgacggtat acagacatga
     1321 taagatacat tgatgagttt ggacaaacca caactagaat gcagtgaaaa aaatgcttta
     1381 tttgtgaaat ttgtgatgct attgctttat ttgtaaccat tataagctgc aataaacaag
     1441 ttggggtggg cgaagaactc cagcatgaga tccccgcgct ggaggatcat ccagccggcg
     1501 tcccggaaaa cgattccgaa gcccaacctt tcatagaagg cggcggtgga atcgaaatct
     1561 cgtgatggca ggttgggcgt cgcttggtcg gtcatttcga accccagagt cccgctcaga
     1621 agaactcgtc aagaaggcga tagaaggcga tgcgctgcga atcgggagcg gcgataccgt
     1681 aaagcacgag gaagcggtca gcccattcgc cgccaagctc ttcagcaata tcacgggtag
     1741 ccaacgctat gtcctgatag cggtccgcca cacccagccg gccacagtcg atgaatccag
     1801 aaaagcggcc attttccacc atgatattcg gcaagcaggc atcgccatgg gtcacgacga
     1861 gatcctcgcc gtcgggcatg cgcgccttga gcctggcgaa cagttcggct ggcgcgagcc
     1921 cctgatgctc ttcgtccaga tcatcctgat cgacaagacc ggcttccatc cgagtacgtg
     1981 ctcgctcgat gcgatgtttc gcttggtggt cgaatgggca ggtagccgga tcaagcgtat
     2041 gcagccgccg cattgcatca gccatgatgg atactttctc ggcaggagca aggtgagatg
     2101 acaggagatc ctgccccggc acttcgccca atagcagcca gtcccttccc gcttcagtga
     2161 caacgtcgag cacagctgcg caaggaacgc ccgtcgtggc cagccacgat agccgcgctg
     2221 cctcgtcctg cagttcattc agggcaccgg acaggtcggt cttgacaaaa agaaccgggc
     2281 gcccctgcgc tgacagccgg aacacggcgg catcagagca gccgattgtc tgttgtgccc
     2341 agtcatagcc gaatagcctc tccacccaag cggccggaga acctgcgtgc aatccatctt
     2401 gttcaatcat gcgaaacgat cctcatcctg tctcttgatc agatccgaaa atggatatac
     2461 aagctcccgg gagctttttg caaaagccta ggcctccaaa aaagcctcct cactacttct
     2521 ggaatagctc agaggcagag gcggcctcgg cctctgcata aataaaaaaa attagtcagc
     2581 catggggcgg agaatgggcg gaactgggcg gagttagggg cgggatgggc ggagttaggg
     2641 gcgggactat ggttgctgac taattgagat gcatgctttg catacttctg cctgctgggg
     2701 agcctgggga ctttccacac ctggttgctg actaattgag atgcatgctt tgcatacttc
     2761 tgcctgctgg ggagcctggg gactttccac accctaactg acacacattc cacagaatta
     2821 attcgcgtta aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa
     2881 aatcccttat aaatcaaaag aatagaccga gatagggttg agtgttgttc cagtttggaa
     2941 caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca
     3001 gggcgatggc ccactacgtg aaccatcacc ctaatcaagt tttttggggt cgaggtgccg
     3061 taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc
     3121 ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc
     3181 aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca
     3241 gggcgcgtgg ggataccccc tagagcccca gctggttctt tccgcctcag aagccataga
     3301 gcccaccgca tccccagcat gcctgctatt gtcttcccaa tcctccccct tgctgtcctg
     3361 ccccacccca ccccccagaa tagaatgaca cctactcaga caatgcgatg caatttcctc
     3421 attttattag gaaaggacag tgggagtggc accttccagg gtcaaggaag gcacggggga
     3481 ggggcaaaca acagatggct ggcaactaga aggcacagtc gaggctgatc agcgggttta
     3541 aacgggccct ctagactcag ttgcttactt caagcaattc ttcactttgc tgaggatttt
     3601 cttcttcctc agggccttct ggatgagtgc tctcagatga agaggtcccg acaccttcag
     3661 accttttgtt actggtctga ccctgctcca cttcacacaa atagacatcg ataggtccgt
     3721 tggtgctcct tatgtgcact gtgatagagt cttctctggg agctggaaca tccaatctgg
     3781 tttctgctgg agctttaact gcaatgacga tctgttcatg gaaggcctga atgctatgaa
     3841 tgtcttgata ggtcacatat gctagtcttt cattttcttt gtcatctgtt aactcaaaca
     3901 gctgctgagc acaatcctta attaactcat ccaaagcatc ttccattgct gataagtcag
     3961 aaagttcctc ctgtagcttc ttttgttggg gaactgctcc aaaattgcta agatcagatc
     4021 ctatccatct aatatggttc ttggatttct tttcaacgag gtcgattcca tctaagacat
     4081 tggtgatgtc atacactctc cgctttcgga ctcccagttt cgttgcaacc ttgtttaagt
     4141 caagaatacc cccgggagca gatctgacaa gatccataaa ttttcgagtt aaataaacca
     4201 gcgatacatc aaaacgaggt ctcttcactt ttagagcttt tctcatggac acatattgta
     4261 cattatcttc taaattaatc cttatttttg atggcagcag gccctccacg ttgatggggt
     4321 ctcggcaccg acggcgaacc gtctcctccg tcgggtccag gaggagactg ggtaacttcc
     4381 tcgccggccg ctgctgactg cgcggccgcc actgtgctgg atatctgcag aattccacca
     4441 cactggatcc agctcctttc tcgaactgcg ggtggctcca gcttcctcct ccgctgcctc
     4501 cgccgcttcc tcctcccttc tcgaactgag ggtggctcca gctggcggcc gaccctttat
     4561 catcatcatc tttataatcc atagatcctg gtaccttgtc atcgtcgtcc ttgtagtcca
     4621 tggtaagctt aagtttaaac gctagccagc ttgggtctcc ctatagtgag tcgtattaat
     4681 ttcgataagc cagtaagcag tgggttctct agttagccag agagctctgc ttatatagac
     4741 ctcccaccgt acacgcctac cgcccatttg cgtcaatggg gcggagttgt tacgacattt
     4801 tggaaagtcc cgttgatttt ggtgccaaaa caaactccca ttgacgtcaa tggggtggag
     4861 acttggaaat ccccgtgagt caaaccgcta tccacgccca ttgatgtact gccaaaaccg
     4921 catcaccatg gtaatagcga tgactaatac gtagatgtac tgccaagtag gaaagtccca
     4981 taaggtcatg tactgggcat aatgccaggc gggccattta ccgtcattga cgtcaatagg
     5041 gggcgtactt ggcatatgat acacttgatg tactgccaag tgggcagttt accgtaaata
     5101 ctccacccat tgacgtcaat ggaaagtccc tattggcgtt actatgggaa catacgtcat
     5161 tattgacgtc aatgggcggg ggtcgttggg cggtcagcca ggcgggccat ttaccgtaag
     5221 ttatgtaacg cggaactcca tatatgggct atgaactaat gaccccgtaa ttgattacta
     5281 ttaataacta gtcaataatc aatgtcaacg cgtatatctg gcccgtacat cgcgaagcag
     5341 cgcaaaacgc ctaaccctaa gcagattctt catgcaattg tcggtcaagc cttgccttgt
     5401 tgtagcttaa attttgctcg cgcactactc agcgacctcc aacacacaag cagggagcag
     5461 atactggctt aactatgcgg catcagagca gattgtactg agagtgcacc ataggggatc
     5521 gggagatctc ccgatccgtc gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc
     5581 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc
     5641 tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc
     5701 gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg
     5761 gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat
     5821 ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc
     5881 acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa
     5941 ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa
     6001 aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt
     6061 gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct
     6121 tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat
     6181 gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg
     6241 cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg
     6301 atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt
     6361 attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg
     6421 ccagat
//
