LOCUS       sequence-473265-        6621 bp    DNA     circular SYN 20-MAY-2025
DEFINITION  transient overexpression of E2F5 in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6621)
  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 6621)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 20, 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 E2F5
FEATURES             Location/Qualifiers
     source          1..6621
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(126..143)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(297..885)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(377..396)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1056..1847,1848..1916))
                     /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     1679..1698
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1917..2021)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(2096..2115)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        2287..2666
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2667..2870
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2821..2841
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2915..2934
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2915..2933
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2975..2998
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             3014..3037
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             3053..3076
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             3113..3136
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             3352..3369
                     /codon_start=1
                     /product="recognition and cleavage site for Genenase(TM) I,
                     a subtilisin BPN' variant engineered for increased 
                     substrate specificity (Carter and Wells, 1987)"
                     /label=Genenase(TM) I site
                     /translation="PGAAHY"
     primer_bind     complement(4264..4281)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4270..4494
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      4540..4968
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4627..4646)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4837..4858
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4977..4997)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4982..5311
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5162..5297
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     5224..5243
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             5378..6172
                     /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(5432..5451)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6042..6061
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    6346..6467
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6383..6402)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6437..6456
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6516..6532)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6516..6532)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6529..6551)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6540..6556
                     /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(6564..6570,6571..6588,6589..6594))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    join(6609..6621,1..9)
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
       61 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc
      121 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
      181 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
      241 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
      301 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
      361 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
      421 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
      481 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
      541 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
      601 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
      661 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
      721 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
      781 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
      841 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
      901 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
      961 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     1021 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     1081 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     1141 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
     1201 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     1261 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     1321 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
     1381 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     1441 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     1501 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     1561 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     1621 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     1681 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     1741 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     1801 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     1861 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     1921 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     1981 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     2041 ccacctgacg tcgacggatc gggagatctc ccgatcccct atggtgcact ctcagtacaa
     2101 tctgctctga tgccgcatag ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg
     2161 ctgagtagtg cgcgagcaaa atttaagcta caacaaggca aggcttgacc gacaattgca
     2221 tgaagaatct gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg gccagatata
     2281 cgcgttgaca ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc
     2341 atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac
     2401 cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa
     2461 tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag
     2521 tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc
     2581 ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct
     2641 acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg
     2701 gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt
     2761 tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga
     2821 cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagagct ctctggctaa
     2881 ctagagaacc cactgcttac tggcttatcg aaattaatac gactcactat agggagaccc
     2941 aagctggcta gcgtttaaac ttaagcttac catggactac aaggacgacg atgacaaggt
     3001 accaggatct atggattata aagatgatga tgataaaggg tcggccgcca gctggagcca
     3061 ccctcagttc gagaagggag gaggaagcgg cggaggcagc ggaggaggaa gctggagcca
     3121 cccgcagttc gagaaaggag ctggatccag tgtggtggaa ttctgcagat atccagcaca
     3181 gtggcggccg cgcgcggcgg cagagcccgc gagctcgggc cagcaggcgc cggcagggca
     3241 ggggcagggc cagcggccgc cgccgcagcc tccgcaggcg caagccccgc agccgccccc
     3301 gccgccgcag ctcgggggcg cggggggcgg cagcagcagg cacgagaaga gcctggggct
     3361 gctcactacc aagttcgtgt cgctgctgca ggaggccaag gacggcgttc tggatctcaa
     3421 agcggctgct gatactttgg ctgtgaggca aaaaaggaga atttatgata tcaccaatgt
     3481 cttagaggga attgacttga ttgaaaaaaa gtcaaaaaac agtatccagt ggaaaggtgt
     3541 aggtgctggc tgtaatacta aagaagtcat agatagatta agatatctta aagctgaaat
     3601 tgaagatcta gaactgaagg aaagagaact tgatcagcag aagttgtggc tacagcaaag
     3661 catcaaaaat gtgatggacg attccattaa taatagattt tcctatgtaa ctcatgaaga
     3721 catctgtaat tgctttaatg gtgatacact tttggccatt caggcacctt ctggtacaca
     3781 actggaggta cccattccag aaatgggtca gaatggacaa aagaaatacc agatcaatct
     3841 aaagagtcat tcaggaccta tccatgtgct gcttataaat aaagagtcga gttcatctaa
     3901 gcccgtggtt tttcctgttc ccccacctga tgacctcaca cagccttcct cccagtcctt
     3961 gactccagtg actccacaga aatccagcat ggcaactcaa aatctgcctg agcaacatgt
     4021 ctctgaaaga agccaggctc tgcagcagac atcagctaca gatatatctt cagcaggatc
     4081 tattagtgga gatatcattg atgagttaat gtcttctgac gtgtttcctc tcttaaggct
     4141 ttctcctacc ccggcagatg actacaactt taatttagat gataacgaag gagtttgtga
     4201 tctgtttgat gtccagatac taaattatta ggtctagagg gcccgtttaa acccgctgat
     4261 cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt
     4321 ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat
     4381 cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg
     4441 gggaggattg ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggcttctg
     4501 aggcggaaag aaccagctgg ggctctaggg ggtatcccca cgcgccctgt agcggcgcat
     4561 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag
     4621 cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc
     4681 aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc
     4741 ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt
     4801 ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa
     4861 caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg
     4921 cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaattaa ttctgtggaa
     4981 tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag
     5041 catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag
     5101 aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc
     5161 catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt
     5221 ttttatttat gcagaggccg aggccgcctc tgcctctgag ctattccaga agtagtgagg
     5281 aggctttttt ggaggcctag gcttttgcaa aaagctcccg ggagcttgta tatccatttt
     5341 cggatctgat caagagacag gatgaggatc gtttcgcatg attgaacaag atggattgca
     5401 cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac
     5461 aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt
     5521 tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag gacgaggcag cgcggctatc
     5581 gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg
     5641 aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc
     5701 tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc
     5761 ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat
     5821 ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc
     5881 cgaactgttc gccaggctca aggcgcgcat gcccgacggc gaggatctcg tcgtgaccca
     5941 tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga
     6001 ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat
     6061 tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc
     6121 tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct gagcgggact
     6181 ctggggttcg aaatgaccga ccaagcgacg cccaacctgc catcacgaga tttcgattcc
     6241 accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg
     6301 atcctccagc gcggggatct catgctggag ttcttcgccc accccaactt gtttattgca
     6361 gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt
     6421 tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctgtata
     6481 ccgtcgacct ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat
     6541 tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg
     6601 ggtgcctaat gagtgagcta a
//
