LOCUS       sequence-473587-        8527 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  insect expression of E2F3 for protein purification.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8527)
  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 8527)
  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: NLS-Cherry-PCNA-P2A-Venus-E2F3
FEATURES             Location/Qualifiers
     source          1..8527
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(96..684)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(176..195)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(855..1646,1647..1715))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     1478..1497
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1716..1820)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     1888..1906
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     LTR             2508..2683
                     /label=5' LTR (truncated)
                     /note="truncated long terminal repeat from Moloney murine 
                     sarcoma virus"
     misc_feature    2746..2945
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     CDS             3146..3562
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     3492..3514
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     3530..3546
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     CDS             3593..3613
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3650..4354
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
                     DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG
                     EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE
                     GRHSTGGMDELYK"
     primer_bind     complement(3792..3810)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     4060..4079
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             5192..5248
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             join(5300..5302,5303..6013)
                     /codon_start=1
                     /product="yellow fluorescent protein (YFP) with fast and 
                     efficient maturation (Nagai et al., 2002)"
                     /label=Venus
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     LICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIKA
                     NFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(5342..5363)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(5603..5622)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5950..5971
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     LTR             7493..8086
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     primer_bind     complement(8189..8211)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    8297..8437
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     8352..8371
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(8452..8469)
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag
       61 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
      121 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
      181 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
      241 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
      301 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
      361 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
      421 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
      481 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca
      541 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
      601 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
      661 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
      721 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
      781 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
      841 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
      901 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
      961 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat
     1021 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
     1081 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
     1141 aatagtttgc gcaacgttgt tgccattgct gcaggcatcg tggtgtcacg ctcgtcgttt
     1201 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
     1261 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
     1321 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
     1381 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
     1441 cggcgaccga gttgctcttg cccggcgtca acacgggata ataccgcgcc acatagcaga
     1501 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
     1561 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
     1621 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
     1681 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga
     1741 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
     1801 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc
     1861 attattatca tgacattaac ctataaaaat aggcgtatca cgaggccctt tcgtcttcaa
     1921 gaattgctag caattgctag caattgctag caattcatac cagatcaccg aaaactgtcc
     1981 tccaaatgtg tccccctcac actcccaaat tcgcgggctt ctgcctctta gaccactcta
     2041 ccctattccc cacactcacc ggagccaaag ccgcggccct tccgtttctt tgcttttgaa
     2101 agaccccacc cgtaggtggc aagctagctt aagtaacgcc actttgcaag gcatggaaaa
     2161 atacataact gagaatagaa aagttcagat caaggtcagg aacaaagaaa cagctgaata
     2221 ccaaacagga tatctgtggt aagcggttcc tgccccggct cagggccaag aacagatgag
     2281 acagctgagt gatgggccaa acaggatatc tgtggtaagc agttcctgcc ccggctcggg
     2341 gccaagaaca gatggtcccc agatgcggtc cagccctcag cagtttctag tgaatcatca
     2401 gatgtttcca gggtgcccca aggacctgaa aatgaccctg taccttattt gaactaacca
     2461 atcagttcgc ttctcgcttc tgttcgcgcg cttccgctct ccgagctcaa taaaagagcc
     2521 cacaacccct cactcggcgc gccagtcttc cgatagactg cgtcgcccgg gtacccgtat
     2581 tcccaataaa gcctcttgct gtttgcatcc gaatcgtggt ctcgctgttc cttgggaggg
     2641 tctcctctga gtgattgact acccacgacg ggggtctttc atttgggggc tcgtccggga
     2701 tttggagacc cctgcccagg gaccaccgac ccaccaccgg gaggtaagct ggccagcaac
     2761 ttatctgtgt ctgtccgatt gtctagtgtc tatgtttgat gttatgcgcc tgcgtctgta
     2821 ctagttagct aactagctct gtatctggcg gacccgtggt ggaactgacg agttctgaac
     2881 acccggccgc aaccctggga gacgtcccag ggactttggg ggccgttttt gtggcccgac
     2941 ctgaggaagg gagtcgatgt ggaatccgac cccgtcagga tatgtggttc tggtaggaga
     3001 cgagaaccta aaacagttcc cgcctccgtc tgaatttttg ctttcggttt ggaaccgaag
     3061 ccgcgcgtct tgtctgctgc agcgctgcag catcgttctg tgttgtctct gtctgactgt
     3121 gtttctgtat ttgtctgaaa attagggcca gactgttacc actcccttaa gtttgacctt
     3181 aggtcactgg aaagatgtcg agcggatcgc tcacaaccag tcggtagatg tcaagaagag
     3241 acgttgggtt accttctgct ctgcagaatg gccaaccttt aacgtcggat ggccgcgaga
     3301 cggcaccttt aaccgagacc tcatcaccca ggttaagatc aaggtctttt cacctggccc
     3361 gcatggacac ccagaccagg tcccctacat cgtgacctgg gaagccttgg cttttgaccc
     3421 ccctccctgg gtcaagccct ttgtacaccc taagcctccg cctcctcttc ctccatccgc
     3481 cccgtctctc ccccttgaac ctcctcgttc gaccccgcct cgatcctccc tttatccagc
     3541 cctcactcct tctctaggcg ccggaattgc cagatccacc atgggagcat caccaaaaaa
     3601 gaagagaaag gtacccgtgg ctggatctcg agaagcttct gtggcggccg tgagcaaggg
     3661 cgaggaggat aacatggcca tcatcaagga gttcatgcgc ttcaaggtgc acatggaggg
     3721 ctccgtgaac ggccacgagt tcgagatcga gggcgagggc gagggccgcc cctacgaggg
     3781 cacccagacc gccaagctga aggtgaccaa gggtggcccc ctgcccttcg cctgggacat
     3841 cctgtcccct cagttcatgt acggctccaa ggcctacgtg aagcaccccg ccgacatccc
     3901 cgactacttg aagctgtcct tccccgaggg cttcaagtgg gagcgcgtga tgaacttcga
     3961 ggacggcggc gtggtgaccg tgacccagga ctcctccctg caggacggcg agttcatcta
     4021 caaggtgaag ctgcgcggca ccaacttccc ctccgacggc cccgtaatgc agaagaagac
     4081 catgggctgg gaggcctcct ccgagcggat gtaccccgag gacggcgccc tgaagggcga
     4141 gatcaagcag aggctgaagc tgaaggacgg cggccactac gacgctgagg tcaagaccac
     4201 ctacaaggcc aagaagcccg tgcagctgcc cggcgcctac aacgtcaaca tcaagttgga
     4261 catcacctcc cacaacgagg actacaccat cgtggaacag tacgaacgcg ccgagggccg
     4321 ccactccacc ggcggcatgg acgagctgta caagtccgga ctcagatctc gagctcaaat
     4381 gttcgaggcg cgcctggtcc agggctccat cctcaagaag gtgttggagg cactcaagga
     4441 cctcatcaac gaggcctgct gggatattag ctccagcggt gtaaacctgc agagcatgga
     4501 ctcgtcccac gtctctttgg tgcagctcac cctgcggtct gagggcttcg acacctaccg
     4561 ctgcgaccgc aacctggcca tgggcgtgaa cctcaccagt atgtccaaaa tactaaaatg
     4621 cgccggcaat gaagatatca ttacactaag ggccgaagat aacgcggata ccttggcgct
     4681 agtatttgaa gcaccaaacc aggagaaagt ttcagactat gaaatgaagt tgatggattt
     4741 agatgttgaa caacttggaa ttccagaaca ggagtacagc tgtgtagtaa agatgccttc
     4801 tggtgaattt gcacgtatat gccgagatct cagccatatt ggagatgctg ttgtaatttc
     4861 ctgtgcaaaa gacggagtga aattttctgc aagtggagaa cttggaaatg gaaacattaa
     4921 attgtcacag acaagtaatg tcgataaaga ggaggaagct gttaccatag agatgaatga
     4981 accagttcaa ctaacttttg cactgaggta cctgaacttc tttacaaaag ccactccact
     5041 ctcttcaacg gtgacactca gtatgtctgc agatgtaccc cttgttgtag agtataaaat
     5101 tgcggatatg ggacacttaa aatactactt ggctcccaag atcgaggatg aagaaggatc
     5161 tgctcgagtc gagctcaagc tcggaagcgg agctactaac ttcagcctgc tgaagcaggc
     5221 tggagacgtg gaggagaacc ctggacctaa gcttcgaatt ctgcagtcga cggtaccgcg
     5281 ggcccgggat cgcaccctgg tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat
     5341 cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga
     5401 gggcgatgcc acctacggca agctgaccct gaagctgatc tgcaccaccg gcaagctgcc
     5461 cgtgccctgg cccaccctcg tgaccactct gggctacggc ctacagtgct tcgcccgcta
     5521 ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca
     5581 ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt
     5641 cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg
     5701 caacatcctg gggcacaagc tggagtacaa ctacaacagc cacaacgtct atatcaccgc
     5761 cgacaagcag aagaacggca tcaaggccaa cttcaagatc cgccacaaca tcgaggacgg
     5821 cggcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct
     5881 gctgcccgac aaccactacc tgagctacca gtccgccctg agcaaagacc ccaacgagaa
     5941 gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga
     6001 cgagctgtac aagtccggac tcagatctcg agcggatcgg agaaagggaa tccagcccgc
     6061 tctggagcag tacctggtga ccgccggggg tggggagggg gcggctgtcg tcgccgccgc
     6121 cgctgcagcc tccatggaca aaagggcact gctagccagc cccggcttcg ccgccgccgc
     6181 cccgggcgcg tacatccaga tcctcaccac gaacacttcc accacctcct gttcctcctc
     6241 cctccaaagc ggcgccgtag ccgccggccc cctcctcccc agtgcccccg gcgcggagca
     6301 gaccgccggc agcctcctct acaccacgcc gcacggaccc tccagcagag ccgggctgct
     6361 gcagcagcca ccagcgctgg gacgcggcgg cagcggcggc ggcggcggcc ctccggcaaa
     6421 gcgaaggctg gagctaggag aaagcggtca tcagtacctc tcagatggtt taaaaacccc
     6481 caagggcaaa ggaagagctg cactacgaag tccagatagt ccaaaaactc caaaatctcc
     6541 ctcagaaaaa acgcggtatg atacgtctct tggtctgctc accaagaagt tcattcagct
     6601 cctgagccag tcacccgatg gggtattgga tttgaacaag gcagcagaag tgctaaaagt
     6661 gcaaaagaga aggatttatg atatcaccaa cgttctggaa ggcatccacc tcattaagaa
     6721 gaagtctaaa aacaacgtcc aatggatggg ctgcagtctg tctgaggatg ggggcatgct
     6781 ggcccagtgt caaggcctgt caaaagaagt gaccgagctc agtcaggaag agaagaaatt
     6841 agatgaactg atccaaagct gcaccctgga cctcaaactg ttaaccgagg attcagagaa
     6901 tcaaaggtta gcttatgtta catatcaaga tattcgaaaa attagtggcc ttaaagacca
     6961 aactgttata gttgtgaaag cccctccaga aacaagactt gaagtgcctg actcaataga
     7021 gagcctacaa atacatttgg caagtaccca aggtcccatt gaggtttact tatgtccaga
     7081 agagactgaa acacacagtc caatgaaaac aaacaaccaa gaccacaatg ggaatatccc
     7141 taaacccgct tccaaagact tggcttcaac caactcagga catagcgatt gctcagtttc
     7201 tatgggaaac ctttctcctc tggcctcccc agccaacctc ttacagcaga ctgaggacca
     7261 aattccttcc aacctagaag gaccgtttgt gaacttactg cctcccctgc tgcaagagga
     7321 ctatctcctg agcctcgggg aggaggaagg catcagcgat ctcttcgatg cttacgattt
     7381 ggaaaagctc ccactggtgg aagacttcat gtgtagttga gatccccggt ggccgcctcg
     7441 gccaaacatc gataaaataa aagattttat ttagtctcca gaaaaagggg ggaatgaaag
     7501 accccacctg taggtttggc aagctagctt aagtaacgcc attttgcaag gcatggaaaa
     7561 atacataact gagaatagag aagttcagat caaggtcagg aacagatgga acagctgaat
     7621 atgggccaaa caggatatct gtggtaagca gttcctgccc cggctcaggg ccaagaacag
     7681 atggaacagc tgaatatggg ccaaacagga tatctgtggt aagcagttcc tgccccggct
     7741 cagggccaag aacagatggt ccccagatgc ggtccagccc tcagcagttt ctagagaacc
     7801 atcagatgtt tccagggtgc cccaaggacc tgaaatgacc ctgtgcctta tttgaactaa
     7861 ccaatcagtt cgcttctcgc ttctgttcgc gcgcttctgc tccccgagct caataaaaga
     7921 gcccacaacc cctcactcgg ggcgccagtc ctccgattga ctgagtcgcc cgggtacccg
     7981 tgtatccaat aaaccctctt gcagttgcat ccgacttgtg gtctcgctgt tccttgggag
     8041 ggtctcctct gagtgattga ctacccgtca gcgggggtct ttcatttggg ggctcgtccg
     8101 ggatcgggag acccctgccc agggaccacc gacccaccac cgggaggtaa gctggctgcc
     8161 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
     8221 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
     8281 ttggcgggtg tcggggcgca gccatgaccc agtcacgtag cgatagcgga gtgtatactg
     8341 gcttaactat gcggcatcag agcagattgt actgagagtg caccatatgc ggtgtgaaat
     8401 accgcacaga tgcgtaagga gaaaataccg catcaggcgc tcttccgctt cctcgctcac
     8461 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     8521 aatacgg
//
