LOCUS       sequence-396601-        5639 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expressing human E2F1 protein with FLAG-tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5639)
  AUTHORS   Wang ME, Chen J, Lu Y, Bawcom AR, Wu J, Ou J, Asara JM, Armstrong 
            AJ, Wang Q, Li L, Wang Y, Huang J, Chen M
  TITLE     RB1-deficient prostate tumor growth and metastasis are vulnerable to
            ferroptosis induction via the E2F/ACSL4 axis.
  JOURNAL   J Clin Invest. 2023 Mar 16:e166647. doi: 10.1172/JCI166647.
  PUBMED    36928314
REFERENCE   2  (bases 1 to 5639)
  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: "J Clin 
            Invest. 2023 Mar 16:e166647. doi: 10.1172/JCI166647."
COMMENT     Sequence Label: pCMV-Tag2B-E2F1
FEATURES             Location/Qualifiers
     source          1..5639
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        67..370
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        371..574
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     525..545
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     promoter        620..638
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     regulatory      673..682
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             682..705
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(2068..2084)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2069..2085)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     complement(2140..2159)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2141..2159)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     polyA_signal    2433..2554
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2470..2489)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2524..2543
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2561..3016)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2698..2719)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2910..2929
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3043..3147
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3149..3506
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3149..3340
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3172..3192)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3357..3492
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3541..4335
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             3541..4335
                     /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
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3595..3614)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4205..4224
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4523..4542)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4567..4614
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4943..5531
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5432..5451
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 atgcattagt tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga
       61 gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg
      121 cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg
      181 acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca
      241 tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc
      301 ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc
      361 tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc
      421 acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa
      481 tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag
      541 gcgtgtacgg tgggaggtct atataagcag agctggttta gtgaaccgtc agatccgcta
      601 gcgattacgc caagctcgaa attaaccctc actaaaggga acaaaagctg gagctccacc
      661 gcggtggcgg ccgccaccat ggattacaag gatgacgacg ataagagccc gggcggatcc
      721 catatggcct tggccggggc ccctgcgggc ggcccatgcg cgccggcgct ggaggccctg
      781 ctcggggccg gcgcgctgcg gctgctcgac tcctcgcaga tcgtcatcat ctccgccgcg
      841 caggacgcca gcgccccgcc ggctcccacc ggccccgcgg cgcccgccgc cggcccctgc
      901 gaccctgacc tgctgctctt cgccacaccg caggcgcccc ggcccacacc cagtgcgccg
      961 cggcccgcgc tcggccgccc gccggtgaag cggaggctgg acctggaaac tgaccatcag
     1021 tacctggccg agagcagtgg gccagctcgg ggcagaggcc gccatccagg aaaaggtgtg
     1081 aaatccccgg gggagaagtc acgctatgag acctcactga atctgaccac caagcgcttc
     1141 ctggagctgc tgagccactc ggctgacggt gtcgtcgacc tgaactgggc tgccgaggtg
     1201 ctgaaggtgc agaagcggcg catctatgac atcaccaacg tccttgaggg catccagctc
     1261 attgccaaga agtccaagaa ccacatccag tggctgggca gccacaccac agtgggcgtc
     1321 ggcggacggc ttgaggggtt gacccaggac ctccgacagc tgcaggagag cgagcagcag
     1381 ctggaccacc tgatgaatat ctgtactacg cagctgcgcc tgctctccga ggacactgac
     1441 agccagcgcc tggcctacgt gacgtgtcag gaccttcgta gcattgcaga ccctgcagag
     1501 cagatggtta tggtgatcaa agcccctcct gagacccagc tccaagccgt ggactcttcg
     1561 gagaactttc agatctccct taagagcaaa caaggcccga tcgatgtttt cctgtgccct
     1621 gaggagaccg taggtgggat cagccctggg aagaccccat cccaggaggt cacttctgag
     1681 gaggagaaca gggccactga ctctgccacc atagtgtcac caccaccatc atctcccccc
     1741 tcatccctca ccacagatcc cagccagtct ctactcagcc tggagcaaga accgctgttg
     1801 tcccggatgg gcagcctgcg ggctcccgtg gacgaggacc gcctgtcccc gctggtggcg
     1861 gccgactcgc tcctggagca tgtgcgggag gacttctccg gcctcctccc tgaggagttc
     1921 atcagccttt ccccacccca cgaggccctc gactaccact tcggcctcga ggagggcgag
     1981 ggcatcagag acctcttcga ctgtgacttt ggggacctca cccccctgga tttctgacag
     2041 gatgcatgaa ttcgatatca agcttatcga taccgtcgac ctcgaggggg ggcccggtac
     2101 cttaattaat taaggtacca ggtaagtgta cccaattcgc cctatagtga gtcgtattac
     2161 aattcactcg atcgcccttc ccaacagttg cgcagcctga atggcgaatg gagatccaat
     2221 ttttaagtgt ataatgtgtt aaactactga ttctaattgt ttgtgtattt tagattcaca
     2281 gtcccaaggc tcatttcagg cccctcagtc ctcacagtct gttcatgatc ataatcagcc
     2341 ataccacatt tgtagaggtt ttacttgctt taaaaaacct cccacacctc cccctgaacc
     2401 tgaaacataa aatgaatgca attgttgttg ttaacttgtt tattgcagct tataatggtt
     2461 acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta
     2521 gttgtggttt gtccaaactc atcaatgtat cttaacgcgt aaattgtaag cgttaatatt
     2581 ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa
     2641 atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca
     2701 gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc
     2761 gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg
     2821 aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg
     2881 ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg
     2941 gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg
     3001 ccgctacagg gcgcgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
     3061 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg
     3121 cttcaataat attgaaaaag gaagaatcct gaggcggaaa gaaccagctg tggaatgtgt
     3181 gtcagttagg gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc
     3241 atctcaatta gtcagcaacc aggtgtggaa agtccccagg ctccccagca ggcagaagta
     3301 tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc
     3361 cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta
     3421 tttatgcaga ggccgaggcc gcctcggcct ctgagctatt ccagaagtag tgaggaggct
     3481 tttttggagg cctaggcttt tgcaaagatc gatcaagaga caggatgagg atcgtttcgc
     3541 atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc
     3601 ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca
     3661 gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg
     3721 caagacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg
     3781 ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag
     3841 gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg
     3901 cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc
     3961 atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa
     4021 gaacatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgag catgcccgac
     4081 ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat
     4141 ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac
     4201 atagcgttgg ctacccgtga tattgctgaa gaacttggcg gcgaatgggc tgaccgcttc
     4261 ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt
     4321 gacgagttct tctgagcggg actctggggt tcgaaatgac cgaccaagcg acgcccaacc
     4381 tgccatcacg agatttcgat tccaccgccg ccttctatga aaggttgggc ttcggaatcg
     4441 ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg
     4501 cccaccctag ggggaggcta actgaaacac ggaaggagac aataccggaa ggaacccgcg
     4561 ctatgacggc aataaaaaga cagaataaaa cgcacggtgt tgggtcgttt gttcataaac
     4621 gcggggttcg gtcccagggc tggcactctg tcgatacccc accgagaccc cattggggcc
     4681 aatacgcccg cgtttcttcc ttttccccac cccacccccc aagttcgggt gaaggcccag
     4741 ggctcgcagc caacgtcggg gcggcaggcc ctgccatagc ctcaggttac tcatatatac
     4801 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg
     4861 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     4921 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     4981 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     5041 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt
     5101 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     5161 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     5221 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     5281 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     5341 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     5401 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     5461 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     5521 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     5581 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcc
//
