LOCUS       sequence-459854-        6687 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Ple155-driven EGFP, also encoding U6-driven sgRNAs to direct saCas9 
            to both sides of stop cassette in Ai14 mice. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6687)
  AUTHORS   Coughlin GM, Borsos M, Barcelona BH, Appling N, Mayfield AMH, Mackey
            ED, Eser RA, Jackson CR, Chen X, Kumar SR, Gradinaru V
  TITLE     Spatial genomics of AAV vectors reveals mechanism of transcriptional
            crosstalk that enables targeted delivery of large genetic cargo.
  JOURNAL   Nat Biotechnol. 2025 Mar 20. doi: 10.1038/s41587-025-02565-4.
  PUBMED    40113953
REFERENCE   2  (bases 1 to 6687)
  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"; journalName: "Nat 
            Biotechnol. 2025 Mar 20. doi: 10.1038/s41587-025-02565-4."
COMMENT     Sequence Label: pAAV.Ple155-CI-EGFP-W3SL_2x-U6-sasgAi14
FEATURES             Location/Qualifiers
     source          1..6687
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(5..27)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     127..146
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(163..618)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(300..321)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     512..531
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   693..833
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   704..833
                     /label=AAV2 ITR
     intron          2534..2666
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     regulatory      2673..2682
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2679..2681,2682..2684,2685..2687,2688..3401)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MAVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLT
                     LKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKD
                     DGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGI
                     KVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVL
                     LEFVTAAGITLGMDELYK"
     CDS             join(2679..2681,2682..2684,2685..2687,2688..3398)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MAVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLT
                     LKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKD
                     DGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGI
                     KVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVL
                     LEFVTAAGITLGMDELYK"
     primer_bind     complement(2727..2748)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2988..3007)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3335..3356
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3463..3483)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     promoter        3874..4122
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     promoter        4265..4513
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     repeat_region   4642..4782
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4642..4771
                     /label=AAV2 ITR
     rep_origin      complement(4844..5432)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4924..4943)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5603..6394,6395..6463))
                     /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     6226..6245
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6464..6568)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6636..6654
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 aaaacctctg acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg
       61 ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggctggctta
      121 actatgcggc atcagagcag attgtactga gagtgcacca taaaattgta aacgttaata
      181 ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac caatagaccg
      241 aaatcggcaa aatcccttat aaatcaaaag aatagcccga gatagagttg agtgttgttc
      301 cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa
      361 ccgtctatca gggcgatggc ccactacgtg aaccatcacc caaatcaagt tttttggggt
      421 cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac
      481 ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta
      541 aggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg
      601 cgccgctaca gggcgcgtac tatggttgct ttgacgtatg cggtgtgaaa taccgcacag
      661 atgcgtaagg agaaaatacc gcatcaggcg cccctgcagg cagctgcgcg ctcgctcgct
      721 cactgaggcc gcccgggcaa agcccgggcg tcgggcgacc tttggtcgcc cggcctcagt
      781 gagcgagcga gcgcgcagag agggagtggc caactccatc actaggggtt cctgcggccg
      841 cctcgagtca gcagattgaa gaagccctcc tggtctgggg agcccgcctg gggacagact
      901 cgctcagtct ggttggccct tcagcttggg ggcccctccc caaacttcct agccagcttg
      961 ctcacaccct gaccccgggg cctgccgtcc ccacttcctc agctctcacc atggtccccg
     1021 ccgatcctct ctgcagagct gttctgaatg agacatgagt ctccttccca atcccggccc
     1081 ccgccccagg ggccctggcc agcagtgcca cttcacgtgg taccgcttca agggacaggc
     1141 tccgatgcgt gtcccgccgt cctagattgg ggcctgatga gtgtggcctg ggagctggga
     1201 cacgaatcag ggaaacatgg cccaggagct acccccaggt cccagcatct cccatcaata
     1261 ggggtccaca cggagagccc tgccctctgc cctggggcct ggcactcaga cccccaagcc
     1321 caccagcccc tttctacagc cacaactggg tcagggggtc ctaggagact cactcgttaa
     1381 ttaggtgccc tacaaactaa ttagtcttgt caatcatggg ctctgagacc ttgagctggg
     1441 ggtggggtgg gggcagggcc ctctcacctc ggcacagggg cctgagcctt cctccgtctt
     1501 ctcctcctga tccggacact tcattggcat agagggagag agtgtgaact tggccctttg
     1561 tggaacagag gaggctcggg cagaggtggt gatagtgcag cccattcatt ctgagatgaa
     1621 acttccactg gtttccgtaa agacgtcttg gggagggaag ggaaggggat ggggacctcc
     1681 cagtggtatc ccctgcttgg gcactgaggg aaagccacag tggctcgggg taaaaggcag
     1741 ggacatcctc tccccgcctg cctctgtccc cagggagtct cgcctcctgt tcccacctgg
     1801 ggctagggtg atagaggaga ggagatagct caacctggca tttaggtggt gtgggaacag
     1861 gagaccccag actttcttgt tttggggtct ggggcaggca accaggctcc agggacagtg
     1921 agttgaagga agggtggctg ggagacccct tgacttgctg ccaaggagac agagctggag
     1981 ctagggtggc gggtggtgtc tgaggcaggt gcagagaggg agggagggaa ggggcctttg
     2041 actccaacct cctttttctt taccgactgc aggtggcagc tgcccttcca ggagccagtg
     2101 ggggaacctg ggtggctggg tggggacacc tgcaagtcct ccctaagcca gctaccaccc
     2161 tacactgttg gcctcccttc tccaactgtg gggacgctgc tcaggccttt tgtgacatca
     2221 cacctgagag tccctggggt ccagtcattg ctgctgggca cagcgaggtc caagctcagg
     2281 tcgccctgcc ccctacccac catgccagat ccagcatcgt tgtgggcaaa caattatctg
     2341 gatgatcttt atggggctta agcttgggtg ggagcagatg gggcatgagc tggggatttg
     2401 gggatggggg gaatccacac ccccacgtcc tggacgttta aaaggccctc tctggcactg
     2461 ggccggggca gaggccagca gaaaagtgac tggagtccag gcgcgccgaa gttggtcgtg
     2521 aggcactggg caggtaagta tcaaggttac aagacaggtt taaggagacc aatagaaact
     2581 gggcttgtcg agacagagaa gactcttgcg tttctgatag gcacctattg gtcttactga
     2641 catccacttt gcctttctct ccacagaccg gtgccaccat ggccgtgagc aagggcgagg
     2701 agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca
     2761 agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt
     2821 tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accctgacct
     2881 acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac ttcttcaagt
     2941 ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact
     3001 acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga
     3061 agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca
     3121 acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag gtgaacttca
     3181 agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca
     3241 cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc acccagtccg
     3301 ccctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg
     3361 ccgccgggat cactctcggc atggacgagc tgtacaagta ggaattcata atcaacctct
     3421 ggattacaaa atttgtgaaa gattgactgg tattcttaac tatgttgctc cttttacgct
     3481 atgtggatac gctgctttaa tgcctttgta tcatgctatt gcttcccgta tggctttcat
     3541 tttctcctcc ttgtataaat cctggttagt tcttgccacg gcggaactca tcgccgcctg
     3601 ccttgcccgc tgctggacag gggctcggct gttgggcact gacaattccg tggtgtttat
     3661 ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatc tagctttatt tgtgaaattt
     3721 gtgatgctat tgctttattt gtaaccatta taagctgcaa taaacaagtt aacaacaaca
     3781 attgcattca ttttatgttt caggttcagg gggagatgtg ggaggttttt taaacaacta
     3841 gtaggtgagg catcgcctga aatccagctg gtggagggcc tatttcccat gattccttca
     3901 tatttgcata tacgatacaa ggctgttaga gagataattg gaattaattt gactgtaaac
     3961 acaaagatat tagtacaaaa tacgtgacgt agaaagtaat aatttcttgg gtagtttgca
     4021 gttttaaaat tatgttttaa aatggactat catatgctta ccgtaacttg aaagtatttc
     4081 gatttcttgg ctttatatat cttgtggaaa ggacgaaaca ccctctagag tcgcagatcc
     4141 tcgttagagt actctggaaa cagaatctac tctaacaagg caaaatgccg tgtttatctc
     4201 gtcaacttgt tggcgagatt ttttgtttta cgcagcgtag gcatcgcctg aaatccagct
     4261 ggtggagggc ctatttccca tgattccttc atatttgcat atacgataca aggctgttag
     4321 agagataatt ggaattaatt tgactgtaaa cacaaagata ttagtacaaa atacgtgacg
     4381 tagaaagtaa taatttcttg ggtagtttgc agttttaaaa ttatgtttta aaatggacta
     4441 tcatatgctt accgtaactt gaaagtattt cgatttcttg gctttatata tcttgtggaa
     4501 aggacgaaac accacgaagt tatattaagg gttgttagag tactctggaa acagaatcta
     4561 ctctaacaag gcaaaatgcc gtgtttatct cgtcaacttg ttggcgagat tttttgtttt
     4621 acgcagggta ccttgggccg caggaacccc tagtgatgga gttggccact ccctctctgc
     4681 gcgctcgctc gctcactgag gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc
     4741 gagcggcctc agtgagcgag cgagcgcgca gctgcctgca ggacatgtga gcaaaaggcc
     4801 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     4861 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     4921 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     4981 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     5041 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     5101 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     5161 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     5221 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     5281 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     5341 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     5401 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     5461 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
     5521 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
     5581 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
     5641 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     5701 gggagggctt accatctggc cccagtgctg caatgatacc gcggcttcca cgctcaccgg
     5761 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     5821 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     5881 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
     5941 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     6001 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     6061 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     6121 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     6181 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
     6241 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     6301 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     6361 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     6421 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     6481 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     6541 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtct
     6601 aagaaaccat tattatcatg acattaacct ataaaaatag gcgtatcacg aggccctttc
     6661 gtctcgcgcg tttcggtgat gacggtg
//
