LOCUS       sequence-477882-        6612 bp    DNA     circular SYN 08-JUL-2025
DEFINITION  Expresses the G-PTEN sensor under a CMV promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6612)
  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 6612)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jul 8, 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_G-PTEN
FEATURES             Location/Qualifiers
     source          1..6612
                     /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             join(613..615,616..618,619..1329)
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2569..2571,2572..3282)
                     /codon_start=1
                     /product="enhanced variant of YFP (Heikal et al., 2001)"
                     /label=Citrine
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     LICTTGKLPVPWPTLVTTFGYGLMCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNWNSVNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLLE
                     RVTAAGITLGMDELYK"
     polyA_signal    3400..3521
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3437..3456)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3491..3510
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3528..3983)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3665..3686)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3877..3896
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4010..4114
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4116..4473
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4116..4307
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4139..4159)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4324..4459
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4508..5302
                     /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             4508..5302
                     /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(4562..4581)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5172..5191
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5490..5509)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5534..5581
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5910..6498
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6399..6418
                     /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 gagctgttca ccggggtggt gcccatcctg
      661 gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc
      721 gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg
      781 ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc
      841 gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag
      901 cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag
      961 ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac
     1021 atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat catggccgac
     1081 aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc
     1141 gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg
     1201 cccgacaacc actacctgag cacccagtcc aagctgagca aagaccccaa cgagaagcgc
     1261 gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag
     1321 ctgtacaagt ccggactcag atctcgagct atgacagcca tcatcaaaga gatcgttagc
     1381 agaaacaaag ggagatatca agaggatgga ttcgacttag acttgaccta tatttatcca
     1441 aatattattg ctatgggatt tcctgcagaa agacttgaag gtgtatacag gaacaatatt
     1501 gatgatgtag taaggttttt ggattcaaag cataaaaacc attacaagat atacaatcta
     1561 tgtgctgaga gacattatga caccgccaaa tttaactgca gagttgcaca gtatcctttt
     1621 gaagaccata acccaccaca gctagaactt atcaaaccct tttgtgaaga tcttgaccaa
     1681 tggctaagtg aagacgacaa tcatgttgca gcaattcact gtaaagctgg gaaaggacgg
     1741 actggtgtaa tgatttgtgc atatttattg catcggggca agtttttaaa ggcacaagag
     1801 gccctggatt tttatgggga agtaaggacc agagataaaa agggagtaac tattcccagt
     1861 cagaggcgct atgtatatta ttatagctac ctgttaaaga atcacctgga ttacagacca
     1921 gtggcactgt tgtttcacaa gatgatgttt gaaactattc caatgttcag tggcggaact
     1981 tgcaatcccc agtttgtggt ctgccagcta aaggtgaaga tctactcctc caactcagga
     2041 cccacgcggc gggaggacaa gctcatgtac tttgagttcc ctcagccatt gcctgtgtgt
     2101 ggtgacatca aagtagagtt cttccacaaa cagaacaaga tgctcaaaaa ggacaaaatg
     2161 tttcactttt gggtaaatac gttcttcata ccaggaccag aggaaacctc agaaaaagtg
     2221 gaaaatggaa gtctttgtga tcaggaaatc gatagcattt gtagtataga gcgtgcggat
     2281 aatgacaagg agtatcttgt gctcaccctg acaaaaaatg atcttgacaa agcaaacaaa
     2341 gacaaggcca accgatactt ctctccaaat tttaaggtga aattatactt tacaaaaaca
     2401 gtagaggaac catcaaatcc agaggctagc agttcaactt ctgtgactcc agacgttagt
     2461 gacaatgaac ctgatcatta tagatattct gacaccactg actctgatcc agagaatgaa
     2521 ccttttgatg aagatcagca ttcacaaatt acaaaagtcc ctacccctgt gagcaagggc
     2581 gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc
     2641 cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa gctgaccctg
     2701 aagctcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccttc
     2761 ggctacggcc tgatgtgctt cgcccgctac cccgaccaca tgaagcagca cgacttcttc
     2821 aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa ggacgacggc
     2881 aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag
     2941 ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac
     3001 tggaacagcg tcaacgtcta tatcatggcc gacaagcaga agaacggcat caaggtgaac
     3061 ttcaagatcc gccacaacat cgaggacggc agcgtgcagc tcgccgacca ctaccagcag
     3121 aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct gagctaccag
     3181 tccgccctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct ggagagagtg
     3241 accgccgccg ggatcactct cggcatggac gagctgtaca agtgaagata actgatcata
     3301 atcagccata ccacatttgt agaggtttta cttgctttaa aaaacctccc acacctcccc
     3361 ctgaacctga aacataaaat gaatgcaatt gttgttgtta acttgtttat tgcagcttat
     3421 aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg
     3481 cattctagtt gtggtttgtc caaactcatc aatgtatctt aacgcgtaaa ttgtaagcgt
     3541 taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata
     3601 ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtgt
     3661 tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg
     3721 aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcaccctaat caagtttttt
     3781 ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc gatttagagc
     3841 ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga aaggagcggg
     3901 cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac ccgccgcgct
     3961 taatgcgccg ctacagggcg cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc
     4021 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg
     4081 ataaatgctt caataatatt gaaaaaggaa gagtcctgag gcggaaagaa ccagctgtgg
     4141 aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
     4201 agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc
     4261 agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg
     4321 cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt
     4381 ttttttattt atgcagaggc cgaggccgcc tcggcctctg agctattcca gaagtagtga
     4441 ggaggctttt ttggaggcct aggcttttgc aaagatcgat caagagacag gatgaggatc
     4501 gtttcgcatg attgaacaag atggattgca cgcaggttct ccggccgctt gggtggagag
     4561 gctattcggc tatgactggg cacaacagac aatcggctgc tctgatgccg ccgtgttccg
     4621 gctgtcagcg caggggcgcc cggttctttt tgtcaagacc gacctgtccg gtgccctgaa
     4681 tgaactgcaa gacgaggcag cgcggctatc gtggctggcc acgacgggcg ttccttgcgc
     4741 agctgtgctc gacgttgtca ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc
     4801 ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca tcatggctga
     4861 tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc ccattcgacc accaagcgaa
     4921 acatcgcatc gagcgagcac gtactcggat ggaagccggt cttgtcgatc aggatgatct
     4981 ggacgaagag catcaggggc tcgcgccagc cgaactgttc gccaggctca aggcgagcat
     5041 gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga atatcatggt
     5101 ggaaaatggc cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta
     5161 tcaggacata gcgttggcta cccgtgatat tgctgaagag cttggcggcg aatgggctga
     5221 ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg
     5281 ccttcttgac gagttcttct gagcgggact ctggggttcg aaatgaccga ccaagcgacg
     5341 cccaacctgc catcacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc
     5401 ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag
     5461 ttcttcgccc accctagggg gaggctaact gaaacacgga aggagacaat accggaagga
     5521 acccgcgcta tgacggcaat aaaaagacag aataaaacgc acggtgttgg gtcgtttgtt
     5581 cataaacgcg gggttcggtc ccagggctgg cactctgtcg ataccccacc gagaccccat
     5641 tggggccaat acgcccgcgt ttcttccttt tccccacccc accccccaag ttcgggtgaa
     5701 ggcccagggc tcgcagccaa cgtcggggcg gcaggccctg ccatagcctc aggttactca
     5761 tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc
     5821 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     5881 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     5941 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     6001 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt
     6061 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     6121 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     6181 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     6241 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     6301 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     6361 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     6421 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     6481 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     6541 tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt
     6601 accgccatgc at
//
