LOCUS       sequence-473614-        5955 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 5955)
  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 5955)
  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: pEGFPc1 PTEN C124S
FEATURES             Location/Qualifiers
     source          1..5955
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     40..60
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      128..137
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(134..136,137..139,140..850)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(134..136,137..139,140..850)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(179..200)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(440..459)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     787..808
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2264..2385
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2301..2320)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2355..2374
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2392..2847)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2529..2550)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2741..2760
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2874..2978
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2980..3337
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2980..3171
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3003..3023)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3188..3323
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3372..4166
                     /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             3372..4166
                     /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(3426..3445)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4036..4055
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4354..4373)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4398..4445
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4774..5362
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5263..5282
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        5537..5840
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        join(5841..5955,1..89)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
ORIGIN
        1 gggactttcc aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg
       61 tacggtggga ggtctatata agcagagctg gtttagtgaa ccgtcagatc cgctagcgct
      121 accggtcgcc accatggtga gcaagggcga ggagctgttc accggggtgg tgcccatcct
      181 ggtcgagctg gacggcgacg taaacggcca caagttcagc gtgtccggcg agggcgaggg
      241 cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt
      301 gccctggccc accctcgtga ccaccctgac ctacggcgtg cagtgcttca gccgctaccc
      361 cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga
      421 gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga
      481 gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa
      541 catcctgggg cacaagctgg agtacaacta caacagccac aacgtctata tcatggccga
      601 caagcagaag aacggcatca aggtgaactt caagatccgc cacaacatcg aggacggcag
      661 cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct
      721 gcccgacaac cactacctga gcacccagtc cgccctgagc aaagacccca acgagaagcg
      781 cgatcacatg gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga
      841 gctgtacaag tccggccgga ctcagatctc gagctcaagc ttcgaattca tgacagccat
      901 catcaaagag atcgttagca gaaacaaaag gagatatcaa gaggatggat tcgacttaga
      961 cttgacctat atttatccaa acattattgc tatgggattt cctgcagaaa gacttgaagg
     1021 cgtatacagg aacaatattg atgatgtagt aaggtttttg gattcaaagc ataaaaacca
     1081 ttacaagata tacaatcttt gtgctgaaag acattatgac accgccaaat ttaattgcag
     1141 agttgcacaa tatccttttg aagaccataa cccaccacag ctagaactta tcaaaccctt
     1201 ttgtgaagat cttgaccaat ggctaagtga agatgacaat catgttgcag caattcactc
     1261 taaagctgga aagggacgaa ctggtgtaat gatatgtgca tatttattac atcggggcaa
     1321 atttttaaag gcacaagagg ccctagattt ctatggggaa gtaaggacca gagacaaaaa
     1381 gggagtaact attcccagtc agaggcgcta tgtgtattat tatagctacc tgttaaagaa
     1441 tcatctggat tatagaccag tggcactgtt gtttcacaag atgatgtttg aaactattcc
     1501 aatgttcagt ggcggaactt gcaatcctca gtttgtggtc tgccagctaa aggtgaagat
     1561 atattcctcc aattcaggac ccacacgacg ggaagacaag ttcatgtact ttgagttccc
     1621 tcagccgtta cctgtgtgtg gtgatatcaa agtagagttc ttccacaaac agaacaagat
     1681 gctaaaaaag gacaaaatgt ttcacttttg ggtaaataca ttcttcatac caggaccaga
     1741 ggaaacctca gaaaaagtag aaaatggaag tctgtgtgat caagaaatcg atagcatttg
     1801 cagtatagag cgtgcagata atgacaagga atatctagta cttactttaa caaaaaatga
     1861 tcttgacaaa gcaaataaag acaaagccaa ccgatacttt tctccaaatt ttaaggtgaa
     1921 gctgtacttc acaaaaacag tagaggagcc gtcaaatcca gaggctagca gttcaacttc
     1981 tgtaacacca gatgttagtg acaatgaacc tgatcattat agatattctg acaccactga
     2041 ctctgatcca gagaatgaac cttttgatga agatcagcat acacaaatta caaaagtctg
     2101 acgcggccgc tcgagcatgc atctagaggg cccgggatcc accggatcta gataactgat
     2161 cataatcagc cataccacat ttgtagaggt tttacttgct ttaaaaaacc tcccacacct
     2221 ccccctgaac ctgaaacata aaatgaatgc aattgttgtt gttaacttgt ttattgcagc
     2281 ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc
     2341 actgcattct agttgtggtt tgtccaaact catcaatgta tcttaacgcg taaattgtaa
     2401 gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc
     2461 aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga
     2521 gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag
     2581 ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc taatcaagtt
     2641 ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta
     2701 gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag
     2761 cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg
     2821 cgcttaatgc gccgctacag ggcgcgtcag gtggcacttt tcggggaaat gtgcgcggaa
     2881 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
     2941 cctgataaat gcttcaataa tattgaaaaa ggaagagtcc tgaggcggaa agaaccagct
     3001 gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat
     3061 gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc
     3121 aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac
     3181 tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact
     3241 aatttttttt atttatgcag aggccgaggc cgcctcggcc tctgagctat tccagaagta
     3301 gtgaggaggc ttttttggag gcctaggctt ttgcaaagat cgatcaagag acaggatgag
     3361 gatcgtttcg catgattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg
     3421 agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt
     3481 tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc
     3541 tgaatgaact gcaagacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt
     3601 gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag
     3661 tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg
     3721 ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag
     3781 cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg
     3841 atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcga
     3901 gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca
     3961 tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc
     4021 gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg
     4081 ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct
     4141 atcgccttct tgacgagttc ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc
     4201 gacgcccaac ctgccatcac gagatttcga ttccaccgcc gccttctatg aaaggttggg
     4261 cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct
     4321 ggagttcttc gcccacccta gggggaggct aactgaaaca cggaaggaga caataccgga
     4381 aggaacccgc gctatgacgg caataaaaag acagaataaa acgcacggtg ttgggtcgtt
     4441 tgttcataaa cgcggggttc ggtcccaggg ctggcactct gtcgataccc caccgagacc
     4501 ccattggggc caatacgccc gcgtttcttc cttttcccca ccccaccccc caagttcggg
     4561 tgaaggccca gggctcgcag ccaacgtcgg ggcggcaggc cctgccatag cctcaggtta
     4621 ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa
     4681 gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
     4741 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     4801 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     4861 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     4921 tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     4981 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     5041 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     5101 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     5161 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     5221 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     5281 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
     5341 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
     5401 ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg
     5461 tattaccgcc atgcattagt tattaatagt aatcaattac ggggtcatta gttcatagcc
     5521 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
     5581 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
     5641 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
     5701 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
     5761 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
     5821 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
     5881 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
     5941 ggcaccaaaa tcaac
//
