LOCUS       sequence-451256-        7062 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  An AAV backbone for expression of the G-PTEN sensor under the CAG 
            promoter, in a Cre dependent manner..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7062)
  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 7062)
  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: AAV-FLEX-CAG_G-PTEN
FEATURES             Location/Qualifiers
     source          1..7062
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(182..770)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(262..281)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(941..1732,1733..1801))
                     /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     1564..1583
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1802..1906)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(1932..2387)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2069..2090)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2281..2300
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   2459..2588
                     /label=AAV2 ITR
     promoter        2899..3176
                     /label=chicken beta-actin promoter
     primer_bind     3455..3477
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     3539..3558
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     protein_bind    complement(3615..3648)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    complement(3699..3732)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(join(5712..6422,6423..6425,6426..6428))
                     /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"
     primer_bind     complement(5754..5775)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     6103..6122
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     6362..6383
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      6425..6434
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    6451..6484
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    6535..6568
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     polyA_signal    6595..6716
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6606..6625)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     6660..6679
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   6878..7007
                     /label=AAV2 ITR
ORIGIN
        1 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
       61 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
      121 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
      181 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
      241 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
      301 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
      361 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
      421 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
      481 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
      541 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
      601 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
      661 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
      721 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
      781 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
      841 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
      901 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
      961 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     1021 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     1081 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     1141 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     1201 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     1261 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     1321 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     1381 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     1441 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     1501 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     1561 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     1621 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     1681 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     1741 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     1801 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     1861 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     1921 aagtgccacc taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta
     1981 aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga
     2041 atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa
     2101 cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga
     2161 accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc
     2221 taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga
     2281 agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg
     2341 cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtccc attcgccatt
     2401 caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat tacgccagct
     2461 gcgcgctcgc tcgctcactg aggccgcccg ggcaaagccc gggcgtcggg cgacctttgg
     2521 tcgcccggcc tcagtgagcg agcgagcgcg cagagaggga gtggccaact ccatcactag
     2581 gggttccttg tagttaatga ttaacccgcc atgctactta tctacgtagc catgctctag
     2641 gaagagtacc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt
     2701 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
     2761 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
     2821 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag
     2881 tcatcgctat taccatggtc gaggtgagcc ccacgttctg cttcactctc cccatctccc
     2941 ccccctcccc acccccaatt ttgtatttat ttatttttta attattttgt gcagcgatgg
     3001 gggcgggggg gggggggggg cgcgcgccrg gsggggsggg gsggggsgrg gggsggggsg
     3061 gggsgrggcg gagaggtgcg gcggcagcca atcagagcgg cgcgctccga aagtttcctt
     3121 ttatggcgag gcggcggcgg cggcggccct ataaaaagcg aagcgcgcgg cgggcgggag
     3181 tcgctgcgcg ctgccttcgc cccgtgcccc gctccgccgc cgcctcgcgc cgcccgcccc
     3241 ggctctgact gaccgcgtta ctcccacagg tgagcgggcg ggacggccct tctcctccgg
     3301 gctgtaatta gcgcttggtt taatgacggc ttgtttcttt tctgtggctg cgtgaaagcc
     3361 ttgaggggct ccgggagggc cctttgtgcg gggggagcgg ctcggggctg tccgcggggg
     3421 gacggctgcc ttcggggggg acggggcagg gcggggttcg gcttctggcg tgtgaccggc
     3481 ggctctagag cctctgctaa ccatgttcat gccttcttct ttttcctaca gctcctgggc
     3541 aacgtgctgg ttattgtgct gtctcatcat tttggcaaag aattggatcg ggatcctcta
     3601 gagtcgactc cggaataact tcgtatagga tactttatac gaagttatgc agaatggtag
     3661 ctggattgta gctgctatta gcaatatgaa acctcttaat aacttcgtat agcatacatt
     3721 atacgaagtt atgggctgat tatgatcagt tatcttcact tatagagttc atccataccc
     3781 agtgtaatac ctgcggctgt gactctttcg agcagaacca tgtggtctct tttttcatta
     3841 ggatctttac tcagagcact ttgataagac agatagtgat tatctgggag gagaacaggt
     3901 ccatcaccta ttggtgtatt ttgttgataa tgatctgcga gttgaacact tccgtcttct
     3961 atattgtgtc taattttgaa attaactttt ataccatttt tctgtttgtc tgccataata
     4021 taaacattga cactattcca attatattcg agtttatgac cgagtatatt tccatcttcc
     4081 ttaaaatcta tacctttcag ttctattcta ttaacgagtg tatctccctc aaatttaacc
     4141 tcagccctag ttttataatt accgtcatct ttaaaaaata tagttctttc ttgaacataa
     4201 ccttctggca ttgctgattt aaaaaaatca tgttgtttca tgtgatcagg atacctagca
     4261 aaacacatga gtccatatcc aaaagtagta accagagtag gccaagggac aggcagtttc
     4321 cctgtagtac atatcagttt gagtgtgagt ttaccataag tagcatctcc ttccccttct
     4381 ccactgactg agaatttatg tccattaaca tctccatcca gttcaaccag tataggaaca
     4441 actcctgtaa acagttcctc tcctttagaa acaggggtag ggacttttgt aatttgtgaa
     4501 tgctgatctt catcaaaagg ttcattctct ggatcagagt cagtggtgtc agaatatcta
     4561 taatgatcag gttcattgtc actaacgtct ggagtcacag aagttgaact gctagcctct
     4621 ggatttgatg gttcctctac tgtttttgta aagtataatt tcaccttaaa atttggagag
     4681 aagtatcggt tggccttgtc tttgtttgct ttgtcaagat cattttttgt cagggtgagc
     4741 acaagatact ccttgtcatt atccgcacgc tctatactac aaatgctatc gatttcctga
     4801 tcacaaagac ttccattttc cactttttct gaggtttcct ctggtcctgg tatgaagaac
     4861 gtatttaccc aaaagtgaaa cattttgtcc tttttgagca tcttgttctg tttgtggaag
     4921 aactctactt tgatgtcacc acacacaggc aatggctgag ggaactcaaa gtacatgagc
     4981 ttgtcctccc gccgcgtggg tcctgagttg gaggagtaga tcttcacctt tagctggcag
     5041 accacaaact ggggattgca agttccgcca ctgaacattg gaatagtttc aaacatcatc
     5101 ttgtgaaaca acagtgccac tggtctgtaa tccaggtgat tctttaacag gtagctataa
     5161 taatatacat agcgcctctg actgggaata gttactccct ttttatctct ggtccttact
     5221 tccccataaa aatccagggc ctcttgtgcc tttaaaaact tgccccgatg caataaatat
     5281 gcacaaatca ttacaccagt ccgtcctttc ccagctttac agtgaattgc tgcaacatga
     5341 ttgtcgtctt cacttagcca ttggtcaaga tcttcacaaa agggtttgat aagttctagc
     5401 tgtggtgggt tatggtcttc aaaaggatac tgtgcaactc tgcagttaaa tttggcggtg
     5461 tcataatgtc tctcagcaca tagattgtat atcttgtaat ggtttttatg ctttgaatcc
     5521 aaaaacctta ctacatcatc aatattgttc ctgtatacac cttcaagtct ttctgcagga
     5581 aatcccatag caataatatt tggataaata taggtcaagt ctaagtcgaa tccatcctct
     5641 tgatatctcc ctttgtttct gctaacgatc tctttgatga tggctgtcat agctcgagat
     5701 ctgagtccgg acttgtacag ctcgtccatg ccgagagtga tcccggcggc ggtcacgaac
     5761 tccagcagga ccatgtgatc gcgcttctcg ttggggtctt tgctcagctt ggactgggtg
     5821 ctcaggtagt ggttgtcggg cagcagcacg gggccgtcgc cgatgggggt gttctgctgg
     5881 tagtggtcgg cgagctgcac gctgccgtcc tcgatgttgt ggcggatctt gaagttcacc
     5941 ttgatgccgt tcttctgctt gtcggccatg atatagacgt tgtggctgtt gtagttgtac
     6001 tccagcttgt gccccaggat gttgccgtcc tccttgaagt cgatgccctt cagctcgatg
     6061 cggttcacca gggtgtcgcc ctcgaacttc acctcggcgc gggtcttgta gttgccgtcg
     6121 tccttgaaga agatggtgcg ctcctggacg tagccttcgg gcatggcgga cttgaagaag
     6181 tcgtgctgct tcatgtggtc ggggtagcgg ctgaagcact gcacgccgta ggtcagggtg
     6241 gtcacgaggg tgggccaggg cacgggcagc ttgccggtgg tgcagatgaa cttcagggtc
     6301 agcttgccgt aggtggcatc gccctcgccc tcgccggaca cgctgaactt gtggccgttt
     6361 acgtcgccgt ccagctcgac caggatgggc accaccccgg tgaacagctc ctcgcccttg
     6421 ctcaccatgg tggcgaccgg tagcgctagc ataacttcgt ataaagtatc ctatacgaag
     6481 ttatttgcct taacccagaa attatcactg ttattcttta gaatggtgca aagaataact
     6541 tcgtataatg tatgctatac gaagttatga attggccgct tcgagcagac atgataagat
     6601 acattgatga gtttggacaa accacaacta gaatgcagtg aaaaaaatgc tttatttgtg
     6661 aaatttgtga tgctattgct ttatttgtaa ccattataag ctgcaataaa caagttaaca
     6721 acaacaattg cattcatttt atgtttcagg ttcaggggga gatgtgggag gttttttaaa
     6781 gcaagtaaaa cctctacaaa tgtggtaaaa tcgataagga tcttcctaga gcatggctac
     6841 gtagataagt agcatggcgg gttaatcatt aactacaagg aacccctagt gatggagttg
     6901 gccactccct ctctgcgcgc tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga
     6961 cgcccgggct ttgcccgggc ggcctcagtg agcgagcgag cgcgcagctg cattaatgaa
     7021 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ct
//
