LOCUS       sequence-451278-        6614 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of the R-PTEN sensor with a 4A mutation.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6614)
  AUTHORS   Kagan T, Gabay M, Meenakshisundaram A, Levi Y, Eid S, Malchenko N, 
            Maman M, Nitzan A, Ravotto L, Zaidel-Bar R, Eickholt BJ, Gal M, 
            Laviv T
  TITLE     Genetically encoded biosensor for fluorescence lifetime imaging of 
            PTEN dynamics in the intact brain.
  JOURNAL   Nat Methods. 2025 Apr;22(4):764-777. doi: 
            10.1038/s41592-025-02610-9. Epub 2025 Feb 20.
  PUBMED    39979596
REFERENCE   2  (bases 1 to 6614)
  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/s41592-025-02610-9"; journalName: "Nat Methods"; date: 
            "2025-04"; volume: "22"; issue: "4"; pages: "764-777"
COMMENT     Sequence Label: CMV_R-PTEN_4A
FEATURES             Location/Qualifiers
     source          1..6614
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        61..364
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        365..568
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     519..539
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      607..616
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             613..1314
                     /codon_start=1
                     /product="monomeric cyan-excitable red fluorescent protein 
                     (Laviv et al., 2016)"
                     /label=mCyRFP1
                     /translation="MVSKGEELIKEEMTSKLYLEGSVNGHYFKCTHEGEGKPYEGKQTA
                     RIKVVEGGPLPFAFDILATHFMYGSKVFIKYPADIPDYFKQSFPEGFTWERVMVFEDGG
                     VLTTTQDTSLQDGVLIYNVKTRGVNFPANGPVMQKKTLGWEPSTETMYPADGGLEGRCD
                     KKLKLVGGGHLHVNFKTTYKSKKPVKMPGVHYVDRRLERIKEADNETYVEQYEHAVARY
                     SNLGGGMDELYK"
     CDS             2554..3288
                     /codon_start=1
                     /product="bright, rapidly maturing, monomeric far-red 
                     fluorescent protein (Bajar et al., 2016)"
                     /label=mMaroon1
                     /translation="MVSKGEELIKENMHTKLYLTGTVNNHYFECTAEGEGKPYEGTQTN
                     RIKVVRGGPLPFAFDILAPCFMYGSKTFINHPPDIPDYFKQSFPEGFTWERTTVYEDGG
                     TLTATQDTSLQDGCLIYNVQVRGENFPSNGPVMQKKTLGWEASTETLYPADGSLEGRLD
                     WALKLVGGGHLHCRLETTYRSKKPAKNLKMPGVYFIDRRLERIKEADNETYVEQHEVAV
                     ARYCDLPSKLGHKLNGMDELYK"
     polyA_signal    3402..3523
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3439..3458)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3493..3512
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3530..3985)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3667..3688)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3879..3898
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4012..4116
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4118..4475
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4118..4309
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4141..4161)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4326..4461
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4510..5304
                     /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             4510..5304
                     /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(4564..4583)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5174..5193
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5492..5511)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5536..5583
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5912..6500
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6401..6420
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
       61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      181 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      241 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      301 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      361 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      421 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      481 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      541 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta
      601 ccggtcgcca ccatggtgag caagggcgag gagctgatca aggaggaaat gaccagcaag
      661 ctgtacctgg aaggcagcgt gaacggccac tacttcaagt gcacccacga aggggagggc
      721 aagccctacg agggcaagca gaccgccagg atcaaggtgg tggagggagg ccccctgccg
      781 ttcgcattcg acatcctggc cacccacttt atgtacggga gcaaggtgtt catcaagtac
      841 cccgccgaca tccccgatta ttttaagcag tccttccctg agggcttcac atgggagaga
      901 gtcatggtgt tcgaagacgg gggcgtgctg accactaccc aggacaccag cctccaggac
      961 ggcgtgctca tctacaacgt caagaccaga ggggtgaact tcccagccaa cggccccgtg
     1021 atgcagaaga aaacactggg ctgggagccc agcaccgaga ccatgtaccc cgccgacggc
     1081 ggcctggaag gcagatgcga caagaagctg aagctcgtgg gcgggggcca cctgcacgtc
     1141 aacttcaaga ccacatacaa gtccaagaaa cccgtgaaga tgcccggcgt ccactacgtg
     1201 gacaggagac tggaaagaat caaggaggcc gacaacgaga cctacgtcga gcagtacgag
     1261 cacgctgtgg ccagatactc caacctgggc ggaggcatgg acgagctgta caagtccgga
     1321 ctcagatctc gagctatgac agccatcatc aaagagatcg ttagcagaaa caaaaggaga
     1381 tatcaagagg atggattcga cttagacttg acctatattt atccaaatat tattgctatg
     1441 ggatttcctg cagaaagact tgaaggtgta tacaggaaca atattgatga tgtagtaagg
     1501 tttttggatt caaagcataa aaaccattac aagatataca atctatgtgc tgagagacat
     1561 tatgacaccg ccaaatttaa ctgcagagtt gcacagtatc cttttgaaga ccataaccca
     1621 ccacagctag aacttatcaa acccttttgt gaagatcttg accaatggct aagtgaagac
     1681 gacaatcatg ttgcagcaat tcactgtaaa gctgggaaag gacggactgg tgtaatgatt
     1741 tgtgcatatt tattgcatcg gggcaagttt ttaaaggcac aagaggccct ggatttttat
     1801 ggggaagtaa ggaccagaga taaaaaggga gtaactattc ccagtcagag gcgctatgta
     1861 tattattata gctacctgtt aaagaatcac ctggattaca gaccagtggc actgttgttt
     1921 cacaagatga tgtttgaaac tattccaatg ttcagtggcg gaacttgcaa tccccagttt
     1981 gtggtctgcc agctaaaggt gaagatctac tcctccaact caggacccac gcggcgggag
     2041 gacaagctca tgtactttga gttccctcag ccattgcctg tgtgtggtga catcaaagta
     2101 gagttcttcc acaaacagaa caagatgctc aaaaaggaca aaatgtttca cttttgggta
     2161 aatacgttct tcataccagg accagaggaa acctcagaaa aagtggaaaa tggaagtctt
     2221 tgtgatcagg aaatcgatag catttgtagt atagagcgtg cggataatga caaggagtat
     2281 cttgtgctca ccctgacaaa aaatgatctt gacaaagcaa acaaagacaa ggccaaccga
     2341 tacttctctc caaattttaa ggtgaaatta tactttacaa aaacagtaga ggaaccatca
     2401 aatccagagg ctagcagttc aacttctgtg actccagacg ttagtgacaa tgaacctgat
     2461 cattatagat atgcagatgc agcagatgca gatccagaga atgaaccttt tgatgaagat
     2521 cagcattcac aaattacaaa agtccctacc cctatggtga gcaagggcga ggagctgatc
     2581 aaggagaaca tgcacaccaa gctgtacctg acaggcaccg tgaacaacca ctacttcgaa
     2641 tgcaccgccg aaggggaggg caagccctac gagggcaccc agaccaacag gatcaaggtt
     2701 gtgcgcggag gccccctgcc gttcgcattc gacatcctgg ccccctgctt tatgtacggg
     2761 agcaagacct tcattaacca tccacctgac attcccgatt attttaagca gtccttccct
     2821 gagggcttca catgggagag aaccaccgtt tacgaagacg ggggcacact cactgctaca
     2881 caggacacca gcctccagga cggctgcctg atctacaacg tccaagtcag aggggagaac
     2941 ttcccatcca acggccctgt gatgcagaag aaaacactcg gctgggaggc cagtaccgag
     3001 accctgtacc ccgctgacgg cagcctggaa ggcagactgg actgggccct gaagctcgtg
     3061 ggcgggggcc acctgcactg cagactggaa accacataca gatccaagaa acccgcaaag
     3121 aacctcaaga tgcccggcgt ctattttatt gacaggagac tggaaagaat caaggaggcc
     3181 gacaatgaga cctacgtcga gcagcacgag gtggctgtgg ccagatactg cgacctccct
     3241 agcaaactgg ggcacaaact taatggcatg gacgagctgt acaagtaaga taactgatca
     3301 taatcagcca taccacattt gtagaggttt tacttgcttt aaaaaacctc ccacacctcc
     3361 ccctgaacct gaaacataaa atgaatgcaa ttgttgttgt taacttgttt attgcagctt
     3421 ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac
     3481 tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttaacgcgta aattgtaagc
     3541 gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa
     3601 taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agaccgagat agggttgagt
     3661 gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa cgtcaaaggg
     3721 cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac catcacccta atcaagtttt
     3781 ttggggtcga ggtgccgtaa agcactaaat cggaacccta aagggagccc ccgatttaga
     3841 gcttgacggg gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg
     3901 ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg taaccaccac acccgccgcg
     3961 cttaatgcgc cgctacaggg cgcgtcaggt ggcacttttc ggggaaatgt gcgcggaacc
     4021 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc
     4081 tgataaatgc ttcaataata ttgaaaaagg aagagtcctg aggcggaaag aaccagctgt
     4141 ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc
     4201 aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag
     4261 gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc
     4321 cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa
     4381 ttttttttat ttatgcagag gccgaggccg cctcggcctc tgagctattc cagaagtagt
     4441 gaggaggctt ttttggaggc ctaggctttt gcaaagatcg atcaagagac aggatgagga
     4501 tcgtttcgca tgattgaaca agatggattg cacgcaggtt ctccggccgc ttgggtggag
     4561 aggctattcg gctatgactg ggcacaacag acaatcggct gctctgatgc cgccgtgttc
     4621 cggctgtcag cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc cggtgccctg
     4681 aatgaactgc aagacgaggc agcgcggcta tcgtggctgg ccacgacggg cgttccttgc
     4741 gcagctgtgc tcgacgttgt cactgaagcg ggaagggact ggctgctatt gggcgaagtg
     4801 ccggggcagg atctcctgtc atctcacctt gctcctgccg agaaagtatc catcatggct
     4861 gatgcaatgc ggcggctgca tacgcttgat ccggctacct gcccattcga ccaccaagcg
     4921 aaacatcgca tcgagcgagc acgtactcgg atggaagccg gtcttgtcga tcaggatgat
     4981 ctggacgaag agcatcaggg gctcgcgcca gccgaactgt tcgccaggct caaggcgagc
     5041 atgcccgacg gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg
     5101 gtggaaaatg gccgcttttc tggattcatc gactgtggcc ggctgggtgt ggcggaccgc
     5161 tatcaggaca tagcgttggc tacccgtgat attgctgaag agcttggcgg cgaatgggct
     5221 gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat cgccttctat
     5281 cgccttcttg acgagttctt ctgagcggga ctctggggtt cgaaatgacc gaccaagcga
     5341 cgcccaacct gccatcacga gatttcgatt ccaccgccgc cttctatgaa aggttgggct
     5401 tcggaatcgt tttccgggac gccggctgga tgatcctcca gcgcggggat ctcatgctgg
     5461 agttcttcgc ccaccctagg gggaggctaa ctgaaacacg gaaggagaca ataccggaag
     5521 gaacccgcgc tatgacggca ataaaaagac agaataaaac gcacggtgtt gggtcgtttg
     5581 ttcataaacg cggggttcgg tcccagggct ggcactctgt cgatacccca ccgagacccc
     5641 attggggcca atacgcccgc gtttcttcct tttccccacc ccacccccca agttcgggtg
     5701 aaggcccagg gctcgcagcc aacgtcgggg cggcaggccc tgccatagcc tcaggttact
     5761 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     5821 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     5881 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     5941 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     6001 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc
     6061 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     6121 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     6181 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     6241 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     6301 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     6361 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     6421 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     6481 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     6541 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     6601 ttaccgccat gcat
//
