LOCUS       sequence-473154-        6715 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  transient overexpression of FBXL2 in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6715)
  AUTHORS   Kuchay S, Giorgi C, Simoneschi D, Pagan J, Missiroli S, Saraf A, 
            Florens L, Washburn MP, Collazo-Lorduy A, Castillo-Martin M, 
            Cordon-Cardo C, Sebti SM, Pinton P, Pagano M
  TITLE     PTEN counteracts FBXL2 to promote IP3R3- and Ca(2+)-mediated 
            apoptosis limiting tumour growth.
  JOURNAL   Nature. 2017 Jun 22;546(7659):554-558. doi: 10.1038/nature22965. 
            Epub 2017 Jun 14.
  PUBMED    28614300
REFERENCE   2  (bases 1 to 6715)
  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: 
            "10.1038/nature22965"; journalName: "Nature"; date: "2017-06-22- 
            22"; volume: "546"; issue: "7659"; pages: "554-558"
COMMENT     Sequence Label: pcDNA3 FLAG FBXL2
FEATURES             Location/Qualifiers
     source          1..6715
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     547..566
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(785..889)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(964..983)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        1155..1534
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1535..1738
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1689..1709
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1783..1802
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1783..1801
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1818..1841
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1861..1884
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     promoter        complement(3188..3206)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3189..3206)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3226..3243)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3232..3456
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3502..3930
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3589..3608)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3799..3820
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3939..3959)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3944..4273
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4124..4259
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4186..4205
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4340..5134
                     /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(4394..4413)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5004..5023
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5308..5429
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5345..5364)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5399..5418
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5478..5494)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5478..5494)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5491..5513)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5502..5518
                     /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(5526..5532,5533..5550,5551..5556))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5571..5592
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5709..5726)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5880..6468)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5960..5979)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6639..6715,1..715,716..784))
                     /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"
ORIGIN
        1 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
       61 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
      121 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
      181 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta
      241 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
      301 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
      361 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
      421 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
      481 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
      541 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
      601 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
      661 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
      721 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
      781 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
      841 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
      901 gaaaagtgcc acctgacgtc gacggatcgg gagatctccc gatcccctat ggtgcactct
      961 cagtacaatc tgctctgatg ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt
     1021 ggaggtcgct gagtagtgcg cgagcaaaat ttaagctaca acaaggcaag gcttgaccga
     1081 caattgcatg aagaatctgc ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc
     1141 cagatatacg cgttgacatt gattattgac tagttattaa tagtaatcaa ttacggggtc
     1201 attagttcat agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc
     1261 tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt
     1321 aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca
     1381 cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg
     1441 taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca
     1501 gtacatctac gtattagtca tcgctattac catggtgatg cggttttggc agtacatcaa
     1561 tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca ttgacgtcaa
     1621 tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta acaactccgc
     1681 cccattgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa gcagagctct
     1741 ctggctaact agagaaccca ctgcttactg gcttatcgaa attaatacga ctcactatag
     1801 ggagacccaa gcttatggac tacaaggacg acgatgacaa gctcgatgaa ttccaccatg
     1861 gactacaagg atgacgatga caaggttttc tcaaacaatg atgaaggcct tattaacaaa
     1921 aagttaccca aagaacttct gttaagaata ttttccttct tggatatagt aactttgtgc
     1981 cgatgtgcac agatttccaa ggcttggaac atcttagccc tggatggaag caactggcaa
     2041 agaatagatc tttttaactt tcaaacagat gtagagggtc gagtggtgga aaatatctcg
     2101 aagcgatgcg gtggattcct gaggaagctc agcttgcgag gctgcattgg tgttggggat
     2161 tcctccttga agacctttgc acagaactgc cgaaacattg aacatttgaa cctcaatgga
     2221 tgcacaaaaa tcactgacag cacgtgttat agccttagca gattctgttc caagctgaaa
     2281 catctggatc tgacctcctg tgtgtctatt acaaacagct ccttgaaggg gatcagtgag
     2341 ggctgccgaa acctggagta cctgaacctc tcttggtgtg atcagatcac gaaggatggc
     2401 atcgaggcac tggtgcgagg ttgtcgaggc ctgaaagccc tgctcctgag gggctgcaca
     2461 cagttagaag atgaagctct gaaacacatt cagaattact gccatgagct tgtgagcctc
     2521 aacttgcagt cctgctcacg tatcacggat gaaggtgtgg tgcagatatg caggggctgt
     2581 caccggctac aggctctctg cctttcgggt tgcagcaacc tcacagatgc ctctcttaca
     2641 gccctgggtt tgaactgtcc gcgactgcaa attttggagg ctgcccgatg ctcccatttg
     2701 actgacgcag gttttacact tttagctcgg aattgccacg aattggagaa gatggatctt
     2761 gaagaatgca tcctgataac cgacagcaca ctcatccagc tctccattca ctgtcctaaa
     2821 ctgcaagccc tgagcctgtc ccactgtgaa ctcatcacag atgatgggat cctgcatctg
     2881 agcaacagta cctgtggcca tgagaggctg cgggtactgg agttggacaa ctgcctcctc
     2941 atcactgatg tggccctgga acacctagag aactgccgag gcctggagcg cctcgagctg
     3001 tacgactgcc agcaggttac ccgtgcaggc atcaagcgga tgcgggctca gctccctcat
     3061 gtcaaagtcc acgcctactt tgctcccgtc accccaccga cagcagtggc aggaagtgga
     3121 cagcgactgt gcaggtgctg tgtcattctc tgagcggccg ctcgagcatg catctagagg
     3181 gccctattct atagtgtcac ctaaatgcta gagctcgctg atcagcctcg actgtgcctt
     3241 ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg
     3301 ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt
     3361 gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca
     3421 atagcaggca tgctggggat gcggtgggct ctatggcttc tgaggcggaa agaaccagct
     3481 ggggctctag ggggtatccc cacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
     3541 tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt
     3601 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
     3661 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg
     3721 gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg
     3781 agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct
     3841 cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg
     3901 agctgattta acaaaaattt aacgcgaatt aattctgtgg aatgtgtgtc agttagggtg
     3961 tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc
     4021 agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca
     4081 tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc
     4141 gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc
     4201 cgaggccgcc tctgcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct
     4261 aggcttttgc aaaaagctcc cgggagcttg tatatccatt ttcggatctg atcaagagac
     4321 aggatgagga tcgtttcgca tgattgaaca agatggattg cacgcaggtt ctccggccgc
     4381 ttgggtggag aggctattcg gctatgactg ggcacaacag acaatcggct gctctgatgc
     4441 cgccgtgttc cggctgtcag cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc
     4501 cggtgccctg aatgaactgc aggacgaggc agcgcggcta tcgtggctgg ccacgacggg
     4561 cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg ggaagggact ggctgctatt
     4621 gggcgaagtg ccggggcagg atctcctgtc atctcacctt gctcctgccg agaaagtatc
     4681 catcatggct gatgcaatgc ggcggctgca tacgcttgat ccggctacct gcccattcga
     4741 ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg atggaagccg gtcttgtcga
     4801 tcaggatgat ctggacgaag agcatcaggg gctcgcgcca gccgaactgt tcgccaggct
     4861 caaggcgcgc atgcccgacg gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc
     4921 gaatatcatg gtggaaaatg gccgcttttc tggattcatc gactgtggcc ggctgggtgt
     4981 ggcggaccgc tatcaggaca tagcgttggc tacccgtgat attgctgaag agcttggcgg
     5041 cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat
     5101 cgccttctat cgccttcttg acgagttctt ctgagcggga ctctggggtt cgaaatgacc
     5161 gaccaagcga cgcccaacct gccatcacga gatttcgatt ccaccgccgc cttctatgaa
     5221 aggttgggct tcggaatcgt tttccgggac gccggctgga tgatcctcca gcgcggggat
     5281 ctcatgctgg agttcttcgc ccaccccaac ttgtttattg cagcttataa tggttacaaa
     5341 taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt
     5401 ggtttgtcca aactcatcaa tgtatcttat catgtctgta taccgtcgac ctctagctag
     5461 agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt
     5521 ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc
     5581 taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     5641 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct
     5701 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     5761 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     5821 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     5881 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     5941 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     6001 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     6061 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     6121 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     6181 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     6241 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     6301 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc
     6361 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     6421 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     6481 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     6541 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     6601 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     6661 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccg
//
