LOCUS       sequence-473158-        7595 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  transient overexpression in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7595)
  AUTHORS   Kuchay S, Duan S, Schenkein E, Peschiaroli A, Saraf A, Florens L, 
            Washburn MP, Pagano M
  TITLE     FBXL2- and PTPL1-mediated degradation of p110-free p85beta 
            regulatory subunit controls the PI(3)K signalling cascade.
  JOURNAL   Nat Cell Biol. 2013 May;15(5):472-80. doi: 10.1038/ncb2731. Epub 
            2013 Apr 21.
  PUBMED    23604317
REFERENCE   2  (bases 1 to 7595)
  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/ncb2731"; 
            journalName: "Nat Cell Biol"; date: "2013-05"; volume: "15"; issue: 
            "5"; pages: "472-80"
COMMENT     Sequence Label: pcDNA FLAG p85beta
FEATURES             Location/Qualifiers
     source          1..7595
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     226..243
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    360..381
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(396..401,402..419,420..426)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    434..450
                     /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)."
     primer_bind     439..461
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     458..474
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     458..474
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    523..644
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(534..553)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     588..607
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(818..1612)
                     /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(929..948)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     1539..1558
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(1679..2008)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1693..1828
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(1747..1766)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     1993..2013
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(2022..2450)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2132..2153)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2344..2363
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    2496..2720
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     2709..2726
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     promoter        2746..2764
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     2746..2763
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             complement(4991..5014)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(5030..5049)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5031..5049)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(5094..5297)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(5123..5143)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        5298..5677
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     5849..5868
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        5943..6047
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6048..6116,6117..6908)
                     /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     complement(6266..6285)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(7079..7595,1..72)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7568..7587
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
       61 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
      121 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc
      181 tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc
      241 gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat
      301 taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt
      361 aatgtgagtt agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt
      421 atgttgtgtg gaattgtgag cggataacaa tttcacacag gaaacagcta tgaccatgat
      481 tacgccaagc tctagctaga ggtcgacggt atacagacat gataagatac attgatgagt
      541 ttggacaaac cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg
      601 ctattgcttt atttgtaacc attataagct gcaataaaca agttggggtg ggcgaagaac
      661 tccagcatga gatccccgcg ctggaggatc atccagccgg cgtcccggaa aacgattccg
      721 aagcccaacc tttcatagaa ggcggcggtg gaatcgaaat ctcgtgatgg caggttgggc
      781 gtcgcttggt cggtcatttc gaaccccaga gtcccgctca gaagaactcg tcaagaaggc
      841 gatagaaggc gatgcgctgc gaatcgggag cggcgatacc gtaaagcacg aggaagcggt
      901 cagcccattc gccgccaagc tcttcagcaa tatcacgggt agccaacgct atgtcctgat
      961 agcggtccgc cacacccagc cggccacagt cgatgaatcc agaaaagcgg ccattttcca
     1021 ccatgatatt cggcaagcag gcatcgccat gggtcacgac gagatcctcg ccgtcgggca
     1081 tgcgcgcctt gagcctggcg aacagttcgg ctggcgcgag cccctgatgc tcttcgtcca
     1141 gatcatcctg atcgacaaga ccggcttcca tccgagtacg tgctcgctcg atgcgatgtt
     1201 tcgcttggtg gtcgaatggg caggtagccg gatcaagcgt atgcagccgc cgcattgcat
     1261 cagccatgat ggatactttc tcggcaggag caaggtgaga tgacaggaga tcctgccccg
     1321 gcacttcgcc caatagcagc cagtcccttc ccgcttcagt gacaacgtcg agcacagctg
     1381 cgcaaggaac gcccgtcgtg gccagccacg atagccgcgc tgcctcgtcc tgcagttcat
     1441 tcagggcacc ggacaggtcg gtcttgacaa aaagaaccgg gcgcccctgc gctgacagcc
     1501 ggaacacggc ggcatcagag cagccgattg tctgttgtgc ccagtcatag ccgaatagcc
     1561 tctccaccca agcggccgga gaacctgcgt gcaatccatc ttgttcaatc atgcgaaacg
     1621 atcctcatcc tgtctcttga tcagatccga aaatggatat acaagctccc gggagctttt
     1681 tgcaaaagcc taggcctcca aaaaagcctc ctcactactt ctggaatagc tcagaggcag
     1741 aggcggcctc ggcctctgca taaataaaaa aaattagtca gccatggggc ggagaatggg
     1801 cggaactggg cggagttagg ggcgggatgg gcggagttag gggcgggact atggttgctg
     1861 actaattgag atgcatgctt tgcatacttc tgcctgctgg ggagcctggg gactttccac
     1921 acctggttgc tgactaattg agatgcatgc tttgcatact tctgcctgct ggggagcctg
     1981 gggactttcc acaccctaac tgacacacat tccacagaat taattcgcgt taaatttttg
     2041 ttaaatcagc tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa
     2101 agaatagacc gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa
     2161 gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg
     2221 tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa
     2281 ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa
     2341 ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct
     2401 gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt ggggataccc
     2461 cctagagccc cagctggttc tttccgcctc agaagccata gagcccaccg catccccagc
     2521 atgcctgcta ttgtcttccc aatcctcccc cttgctgtcc tgccccaccc caccccccag
     2581 aatagaatga cacctactca gacaatgcga tgcaatttcc tcattttatt aggaaaggac
     2641 agtgggagtg gcaccttcca gggtcaagga aggcacgggg gaggggcaaa caacagatgg
     2701 ctggcaacta gaaggcacag tcgaggctga tcagcgagct ctagcattta ggtgacacta
     2761 tagaataggg ccctctagat gcatgctcga gtcagcgggc ggcaggcggc gggccggggc
     2821 ccggggcgcg cactgggtgc gccagggtga cggtgagcgc gtcgttgtgc tgcaccagcg
     2881 aggcgtgctg gtagtgcagc accagctcct tcagcgaccc gtacaggttg tagggctccg
     2941 cgaagccgaa gccggtggcc gtgcggtaga tgacgcagtg cttggtgtcg ccgtccacta
     3001 ccacggagca ggcgtagcag ccccgctggc tgctctcgcg gatgaggaag gtgccatccc
     3061 gcttgccact cagcatctcc tctgcctgcg tgcggttgat cttgcccacg taccaagtgc
     3121 gttcctcgtg gtgcgggaga tcgtcctcgt cctccatgag tgcgtactgg tcctcagtct
     3181 catttttaat ccccagccac tcgttgattt tcttctgccg ggcgcctttc tgggtgagcc
     3241 acacgaggta ctggtctcgg atcttgcgca gctgcatgag gtccggcttg aggctgttca
     3301 tgcgcttgtc gatctctctg ttgtccgagg cctgggcccg cagctgctgc tccagcttcg
     3361 tgcggctctc atggatctcg gcaatgcggg acttgagccg ctcggagttc agcaggatcc
     3421 tttgcatctc tttctcgttg ccctcacgcc ggaagcgctc caggtattcc ttgctgcatt
     3481 tctcttgagt ctggccctgc tcttcaaaga tcttgatagt ctcattgaag gcctcaattg
     3541 cagtacgctt catctgcagc tcctgggagg tccgtgtgta ctcttcataa agctggtcat
     3601 actcgcggct cttgtcctgg tactgctggt gatagacctt aagctgggcg cccactgcct
     3661 ccacgctgtc ctccttgaca atctggtcct gctggtattt ggacacaggg tagaggagcc
     3721 gtgtgtccag cttggcattg tactgggcca gagactcgtg gcggtagtga ttgatgaggt
     3781 ccacaacgga gcagaaggtg agtggctctg agaagccata gtgcccatct cggtggaaga
     3841 ccttgatcag cttattgttc ccgcctttcc tgagggtcag cgtgtactcg ccctggatct
     3901 tgctagaagc atctcggact aggaaggtgc catcgggagt gtcccggagt ttctcgttca
     3961 cctcctccct tgaaatgtcc ccccagtacc actcagcatc ctgcagggag ggtgggctcc
     4021 ctccattggc caggactgtg ggggccggct ttgccttggg gggtttaggc ggcagcgctg
     4081 ggggcgcaac ctcctgctct tccaagtgtt cctgaagcag cttctccacc agcagcgccg
     4141 ggaagtcagg gctgggctca ctcccgtcgg gagcgccccc tggcggcggc gaggacggcg
     4201 gcggcggcgc gcgcagcagc agcggcccaa aggtggcgcc cagggcccgg accgcgggac
     4261 ccagggccgg ggcgcggcgg gccacgcggc ccaggtgctg gagcaggaag cgcagcgtga
     4321 gcgcgcggtg cagcggcagc gtcggtggct ccagcgccgg ccccacgggc cccgcggcct
     4381 cccgcagggc ccggcgcgcc tcggccgagg cctcgggggt cacgagcggc gcgggcagtg
     4441 ccagcaggaa gctcttaatg ccgtcagcca gggctgccgt gtcccactga tccacgtcgc
     4501 tcagggacca gtctgtacgc ggtgcgggca gctccgggcg gtagtgagat tcgctgtcca
     4561 gccctgtcct ttcaatggcc tccacaagct tcaccagaag agggggagcc acatcaggtg
     4621 gggagaactg ctcgggcaag tcggggagtg tgaggcctgg ctcaggggcc ccatcacggg
     4681 gcctggcggg cagtgggcgg gggccccgtg ggcgagggcc gggccgggcc agggccacgg
     4741 gccccaggaa ctccacatag gtgccaggga agtcacctcg ctgccgtgtg cgctcgttga
     4801 ggccgggcat ccagcccacg ctctgtgggc agcgctcgcc accctcggcc acgcccagcg
     4861 cctgcaaggc cgcccggctc actaccagca cgtcgccggg cagcagctcc aggtcctccg
     4921 gccgctcccg gcggaacggg tacagagcgc ggtactggaa gccctcaggg cccgccatga
     4981 attcatcgag cttgtcatcg tcgtccttgt agtccataag cttgggtctc cctatagtga
     5041 gtcgtattaa tttcgataag ccagtaagca gtgggttctc tagttagcca gagagctctg
     5101 cttatataga cctcccaccg tacacgccta ccgcccattt gcgtcaatgg ggcggagttg
     5161 ttacgacatt ttggaaagtc ccgttgattt tggtgccaaa acaaactccc attgacgtca
     5221 atggggtgga gacttggaaa tccccgtgag tcaaaccgct atccacgccc attgatgtac
     5281 tgccaaaacc gcatcaccat ggtaatagcg atgactaata cgtagatgta ctgccaagta
     5341 ggaaagtccc ataaggtcat gtactgggca taatgccagg cgggccattt accgtcattg
     5401 acgtcaatag ggggcgtact tggcatatga tacacttgat gtactgccaa gtgggcagtt
     5461 taccgtaaat actccaccca ttgacgtcaa tggaaagtcc ctattggcgt tactatggga
     5521 acatacgtca ttattgacgt caatgggcgg gggtcgttgg gcggtcagcc aggcgggcca
     5581 tttaccgtaa gttatgtaac gcggaactcc atatatgggc tatgaactaa tgaccccgta
     5641 attgattact attaataact agtcaataat caatgtcaac gcgtatatct ggcccgtaca
     5701 tcgcgaagca gcgcaaaacg cctaacccta agcagattct tcatgcaatt gtcggtcaag
     5761 ccttgccttg ttgtagctta aattttgctc gcgcactact cagcgacctc caacacacaa
     5821 gcagggagca gatactggct taactatgcg gcatcagagc agattgtact gagagtgcac
     5881 cataggggat cgggagatct cccgatccgt cgacgtcagg tggcactttt cggggaaatg
     5941 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     6001 gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac
     6061 atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc
     6121 cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca
     6181 tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc
     6241 caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg
     6301 ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac
     6361 cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca
     6421 taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg
     6481 agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac
     6541 cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg
     6601 caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat
     6661 taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg
     6721 ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg
     6781 cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc
     6841 aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc
     6901 attggtaact gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt
     6961 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt
     7021 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     7081 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     7141 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     7201 gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca
     7261 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     7321 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     7381 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     7441 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     7501 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     7561 ttccaggggg aaacgcctgg tatctttata gtcct
//
