LOCUS       sequence-473583-        8458 bp    DNA     circular SYN 21-MAY-2025
DEFINITION  insect expression of E2F1 for protein purification.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8458)
  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 8458)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 21, 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-E2F1
FEATURES             Location/Qualifiers
     source          1..8458
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(12..30)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        98..202
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(203..271,272..1063)
                     /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     complement(421..440)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1234..1822
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1723..1742
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1976..1993
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2008..2148
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2074..2093)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2234..2256
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     LTR             2359..2952
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     CDS             complement(join(4363..5073,5074..5076))
                     /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(4405..4426)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     4754..4773
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5013..5034
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             complement(5128..5184)
                     /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             complement(6022..6726)
                     /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(6297..6316)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     6566..6584
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     CDS             complement(6763..6783)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(6814..7230)
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     complement(6830..6846)
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     primer_bind     complement(6862..6884)
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     misc_feature    7431..7630
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     LTR             7693..7868
                     /label=5' LTR (truncated)
                     /note="truncated long terminal repeat from Moloney murine 
                     sarcoma virus"
ORIGIN
        1 aagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt
       61 ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt
      121 tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca
      181 ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt
      241 ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga
      301 tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa
      361 gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct
      421 gctatgtggc gcggtattat cccgtgttga cgccgggcaa gagcaactcg gtcgccgcat
      481 acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga
      541 tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc
      601 caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat
      661 gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa
      721 cgacgagcgt gacaccacga tgcctgcagc aatggcaaca acgttgcgca aactattaac
      781 tggcgaacta cttactctag cttcccggca acaattaata gactggatgg aggcggataa
      841 agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc
      901 tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc
      961 ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag
     1021 acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag accaagttta
     1081 ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa
     1141 gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
     1201 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     1261 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     1321 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     1381 ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     1441 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     1501 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     1561 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     1621 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     1681 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     1741 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
     1801 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
     1861 ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg
     1921 tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga
     1981 gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat tttctcctta cgcatctgtg
     2041 cggtatttca caccgcatat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt
     2101 aagccagtat acactccgct atcgctacgt gactgggtca tggctgcgcc ccgacacccg
     2161 ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa
     2221 gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc
     2281 gcgaggcagc cagcttacct cccggtggtg ggtcggtggt ccctgggcag gggtctcccg
     2341 atcccggacg agcccccaaa tgaaagaccc ccgctgacgg gtagtcaatc actcagagga
     2401 gaccctccca aggaacagcg agaccacaag tcggatgcaa ctgcaagagg gtttattgga
     2461 tacacgggta cccgggcgac tcagtcaatc ggaggactgg cgccccgagt gaggggttgt
     2521 gggctctttt attgagctcg gggagcagaa gcgcgcgaac agaagcgaga agcgaactga
     2581 ttggttagtt caaataaggc acagggtcat ttcaggtcct tggggcaccc tggaaacatc
     2641 tgatggttct ctagaaactg ctgagggctg gaccgcatct ggggaccatc tgttcttggc
     2701 cctgagccgg ggcaggaact gcttaccaca gatatcctgt ttggcccata ttcagctgtt
     2761 ccatctgttc ttggccctga gccggggcag gaactgctta ccacagatat cctgtttggc
     2821 ccatattcag ctgttccatc tgttcctgac cttgatctga acttctctat tctcagttat
     2881 gtatttttcc atgccttgca aaatggcgtt acttaagcta gcttgccaaa cctacaggtg
     2941 gggtctttca ttcccccctt tttctggaga ctaaataaaa tcttttattt tatcgatgtt
     3001 tggccgaggc ggccaccggg gatctcagaa atccaggggg gtgaggtccc caaagtcaca
     3061 gtcgaagagg tctctgatgc cctcgccctc ctcgaggccg aagtggtagt cgagggcctc
     3121 gtggggtggg gaaaggctga tgaactcctc agggaggagg ccggagaagt cctcccgcac
     3181 atgctccagg agcgagtcgg ccgccaccag cggggacagg cggtcctcgt ccacgggagc
     3241 ccgcaggctg cccatccggg acaacagcgg tttttgctcc aggctgagta gagactggct
     3301 gggatctgtg gtgagggatg aggggggaga tgatggtggt ggtgacacta tggtggcaga
     3361 gtcagtggcc ctgttctcct cctcagaagt gacctcctgg gatggggtct tcccagggct
     3421 gatcccacct acggtctcct cagggcacag gaaaacatcg atcgggcctt gtttgctctt
     3481 aagggagatc tgaaagttct ccgaagagtc cacggcttgg agctgggtct caggaggggc
     3541 tttgatcacc ataaccatct gctctgcagg gtctgcaatg ctacgaaggt cctgacacgt
     3601 cacgtaggcc aggcgctggc tgtcagtgtc ctcggagagc aggcgcagct gcgtagtaca
     3661 gatattcatc aggtggtcca gctgctgctc gctctcctgc agctgtcgga ggtcctgggt
     3721 caacccctca agccgtccgc cgacgcccac tgtggtgtgg ctgcccagcc actggatgtg
     3781 gttcttggac ttcttggcaa tgagctggat gccctcaagg acgttggtga tgtcatagat
     3841 gcgccgcttc tgcaccttca gcacctcggc agcccagttc aggtcgacga caccgtcagc
     3901 cgagtggctc agcagctcca ggaagcgctt ggtggtcaga ttcagtgagg tctcatagcg
     3961 tgacttctcc cccggggatt tcacaccttt tcctggatgg cggcctctgc cccgagctgg
     4021 cccactgctc tcggccaggt actgatggtc agtttccagg tccagcctcc gcttcaccgg
     4081 cgggcggccg agcgcgggcc gcggcgcact gggtgtgggc cggggcgcct gcggtgtggc
     4141 gaagagcagc aggtcagggt cgcaggggcc ggcggcgggc gccgcggggc cggtgggagc
     4201 cggcggggcg ctggcgtcct gcgcggcgga gatgatgacg atctgcgagg agtcgagcag
     4261 ccgcagcgcg ccggccccga gcagggcctc cagcgccggc gcgcatgggc cgcccgcagg
     4321 ggccccggcc aaggcgcgat ccgctcgaga tctgagtccg gacttgtaca gctcgtccat
     4381 gccgagagtg atcccggcgg cggtcacgaa ctccagcagg accatgtgat cgcgcttctc
     4441 gttggggtct ttgctcaggg cggactggta gctcaggtag tggttgtcgg gcagcagcac
     4501 ggggccgtcg ccgatggggg tgttctgctg gtagtggtcg gcgagctgca cgccgccgtc
     4561 ctcgatgttg tggcggatct tgaagttggc cttgatgccg ttcttctgct tgtcggcggt
     4621 gatatagacg ttgtggctgt tgtagttgta ctccagcttg tgccccagga tgttgccgtc
     4681 ctccttgaag tcgatgccct tcagctcgat gcggttcacc agggtgtcgc cctcgaactt
     4741 cacctcggcg cgggtcttgt agttgccgtc gtccttgaag aagatggtgc gctcctggac
     4801 gtagccttcg ggcatggcgg acttgaagaa gtcgtgctgc ttcatgtggt cggggtagcg
     4861 ggcgaagcac tgtaggccgt agcccagagt ggtcacgagg gtgggccagg gcacgggcag
     4921 cttgccggtg gtgcagatca gcttcagggt cagcttgccg taggtggcat cgccctcgcc
     4981 ctcgccggac acgctgaact tgtggccgtt tacgtcgccg tccagctcga ccaggatggg
     5041 caccaccccg gtgaacagct cctcgccctt gctcaccagg gtgcgatccc gggcccgcgg
     5101 taccgtcgac tgcagaattc gaagcttagg tccagggttc tcctccacgt ctccagcctg
     5161 cttcagcagg ctgaagttag tagctccgct tccgagcttg agctcgactc gagcagatcc
     5221 ttcttcatcc tcgatcttgg gagccaagta gtattttaag tgtcccatat ccgcaatttt
     5281 atactctaca acaaggggta catctgcaga catactgagt gtcaccgttg aagagagtgg
     5341 agtggctttt gtaaagaagt tcaggtacct cagtgcaaaa gttagttgaa ctggttcatt
     5401 catctctatg gtaacagctt cctcctcttt atcgacatta cttgtctgtg acaatttaat
     5461 gtttccattt ccaagttctc cacttgcaga aaatttcact ccgtcttttg cacaggaaat
     5521 tacaacagca tctccaatat ggctgagatc tcggcatata cgtgcaaatt caccagaagg
     5581 catctttact acacagctgt actcctgttc tggaattcca agttgttcaa catctaaatc
     5641 catcaacttc atttcatagt ctgaaacttt ctcctggttt ggtgcttcaa atactagcgc
     5701 caaggtatcc gcgttatctt cggcccttag tgtaatgata tcttcattgc cggcgcattt
     5761 tagtattttg gacatactgg tgaggttcac gcccatggcc aggttgcggt cgcagcggta
     5821 ggtgtcgaag ccctcagacc gcagggtgag ctgcaccaaa gagacgtggg acgagtccat
     5881 gctctgcagg tttacaccgc tggagctaat atcccagcag gcctcgttga tgaggtcctt
     5941 gagtgcctcc aacaccttct tgaggatgga gccctggacc aggcgcgcct cgaacatttg
     6001 agctcgagat ctgagtccgg acttgtacag ctcgtccatg ccgccggtgg agtggcggcc
     6061 ctcggcgcgt tcgtactgtt ccacgatggt gtagtcctcg ttgtgggagg tgatgtccaa
     6121 cttgatgttg acgttgtagg cgccgggcag ctgcacgggc ttcttggcct tgtaggtggt
     6181 cttgacctca gcgtcgtagt ggccgccgtc cttcagcttc agcctctgct tgatctcgcc
     6241 cttcagggcg ccgtcctcgg ggtacatccg ctcggaggag gcctcccagc ccatggtctt
     6301 cttctgcatt acggggccgt cggaggggaa gttggtgccg cgcagcttca ccttgtagat
     6361 gaactcgccg tcctgcaggg aggagtcctg ggtcacggtc accacgccgc cgtcctcgaa
     6421 gttcatcacg cgctcccact tgaagccctc ggggaaggac agcttcaagt agtcggggat
     6481 gtcggcgggg tgcttcacgt aggccttgga gccgtacatg aactgagggg acaggatgtc
     6541 ccaggcgaag ggcagggggc cacccttggt caccttcagc ttggcggtct gggtgccctc
     6601 gtaggggcgg ccctcgccct cgccctcgat ctcgaactcg tggccgttca cggagccctc
     6661 catgtgcacc ttgaagcgca tgaactcctt gatgatggcc atgttatcct cctcgccctt
     6721 gctcacggcc gccacagaag cttctcgaga tccagccacg ggtacctttc tcttcttttt
     6781 tggtgatgct cccatggtgg atctggcaat tccggcgcct agagaaggag tgagggctgg
     6841 ataaagggag gatcgaggcg gggtcgaacg aggaggttca agggggagag acggggcgga
     6901 tggaggaaga ggaggcggag gcttagggtg tacaaagggc ttgacccagg gaggggggtc
     6961 aaaagccaag gcttcccagg tcacgatgta ggggacctgg tctgggtgtc catgcgggcc
     7021 aggtgaaaag accttgatct taacctgggt gatgaggtct cggttaaagg tgccgtctcg
     7081 cggccatccg acgttaaagg ttggccattc tgcagagcag aaggtaaccc aacgtctctt
     7141 cttgacatct accgactggt tgtgagcgat ccgctcgaca tctttccagt gacctaaggt
     7201 caaacttaag ggagtggtaa cagtctggcc ctaattttca gacaaataca gaaacacagt
     7261 cagacagaga caacacagaa cgatgctgca gcgctgcagc agacaagacg cgcggcttcg
     7321 gttccaaacc gaaagcaaaa attcagacgg aggcgggaac tgttttaggt tctcgtctcc
     7381 taccagaacc acatatcctg acggggtcgg attccacatc gactcccttc ctcaggtcgg
     7441 gccacaaaaa cggcccccaa agtccctggg acgtctccca gggttgcggc cgggtgttca
     7501 gaactcgtca gttccaccac gggtccgcca gatacagagc tagttagcta actagtacag
     7561 acgcaggcgc ataacatcaa acatagacac tagacaatcg gacagacaca gataagttgc
     7621 tggccagctt acctcccggt ggtgggtcgg tggtccctgg gcaggggtct ccaaatcccg
     7681 gacgagcccc caaatgaaag acccccgtcg tgggtagtca atcactcaga ggagaccctc
     7741 ccaaggaaca gcgagaccac gattcggatg caaacagcaa gaggctttat tgggaatacg
     7801 ggtacccggg cgacgcagtc tatcggaaga ctggcgcgcc gagtgagggg ttgtgggctc
     7861 ttttattgag ctcggagagc ggaagcgcgc gaacagaagc gagaagcgaa ctgattggtt
     7921 agttcaaata aggtacaggg tcattttcag gtccttgggg caccctggaa acatctgatg
     7981 attcactaga aactgctgag ggctggaccg catctgggga ccatctgttc ttggccccga
     8041 gccggggcag gaactgctta ccacagatat cctgtttggc ccatcactca gctgtctcat
     8101 ctgttcttgg ccctgagccg gggcaggaac cgcttaccac agatatcctg tttggtattc
     8161 agctgtttct ttgttcctga ccttgatctg aacttttcta ttctcagtta tgtatttttc
     8221 catgccttgc aaagtggcgt tacttaagct agcttgccac ctacgggtgg ggtctttcaa
     8281 aagcaaagaa acggaagggc cgcggctttg gctccggtga gtgtggggaa tagggtagag
     8341 tggtctaaga ggcagaagcc cgcgaatttg ggagtgtgag ggggacacat ttggaggaca
     8401 gttttcggtg atctggtatg aattgctagc aattgctagc aattgctagc aattcttg
//
