LOCUS       sequence-459845-        6299 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Ple155-driven EGFP, also encoding U6-driven sgRNA cassette, with 
            SapI sites to clone crRNA sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6299)
  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 6299)
  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.U6-sasgRNA(SapI)_Ple155-CI-EGFP-W3SL_BbsI...
FEATURES             Location/Qualifiers
     source          1..6299
                     /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
     promoter        842..1090
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     842..862
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1013..1032
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     intron          2890..3022
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     regulatory      3029..3038
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(3035..3037,3038..3040,3041..3043,3044..3757)
                     /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(3035..3037,3038..3040,3041..3043,3044..3754)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MAVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLT
                     LKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKD
                     DGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGI
                     KVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVL
                     LEFVTAAGITLGMDELYK"
     primer_bind     complement(3083..3104)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3344..3363)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3691..3712
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3819..3839)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     repeat_region   4254..4394
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4254..4383
                     /label=AAV2 ITR
     rep_origin      complement(4456..5044)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4536..4555)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5215..6006,6007..6075))
                     /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     5838..5857
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6076..6180)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6248..6266
                     /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 cgagggccta tttcccatga ttccttcata tttgcatata cgatacaagg ctgttagaga
      901 gataattgga attaatttga ctgtaaacac aaagatatta gtacaaaata cgtgacgtag
      961 aaagtaataa tttcttgggt agtttgcagt tttaaaatta tgttttaaaa tggactatca
     1021 tatgcttacc gtaacttgaa agtatttcga tttcttggct ttatatatct tgtggaaagg
     1081 acgaaacacc ggaagagcga gctcttctgt tagagtactc tggaaacaga atctactcta
     1141 acaaggcaaa atgccgtgtt tatctcgtca acttgttggc gagatttttt tggccgcctc
     1201 gagtcagcag attgaagaag ccctcctggt ctggggagcc cgcctgggga cagactcgct
     1261 cagtctggtt ggcccttcag cttgggggcc cctccccaaa cttcctagcc agcttgctca
     1321 caccctgacc ccggggcctg ccgtccccac ttcctcagct ctcaccatgg tccccgccga
     1381 tcctctctgc agagctgttc tgaatgagac atgagtctcc ttcccaatcc cggcccccgc
     1441 cccaggggcc ctggccagca gtgccacttc acgtggtacc gcttcaaggg acaggctccg
     1501 atgcgtgtcc cgccgtccta gattggggcc tgatgagtgt ggcctgggag ctgggacacg
     1561 aatcagggaa acatggccca ggagctaccc ccaggtccca gcatctccca tcaatagggg
     1621 tccacacgga gagccctgcc ctctgccctg gggcctggca ctcagacccc caagcccacc
     1681 agcccctttc tacagccaca actgggtcag ggggtcctag gagactcact cgttaattag
     1741 gtgccctaca aactaattag tcttgtcaat catgggctct gagaccttga gctgggggtg
     1801 gggtgggggc agggccctct cacctcggca caggggcctg agccttcctc cgtcttctcc
     1861 tcctgatccg gacacttcat tggcatagag ggagagagtg tgaacttggc cctttgtgga
     1921 acagaggagg ctcgggcaga ggtggtgata gtgcagccca ttcattctga gatgaaactt
     1981 ccactggttt ccgtaaagac gtcttgggga gggaagggaa ggggatgggg acctcccagt
     2041 ggtatcccct gcttgggcac tgagggaaag ccacagtggc tcggggtaaa aggcagggac
     2101 atcctctccc cgcctgcctc tgtccccagg gagtctcgcc tcctgttccc acctggggct
     2161 agggtgatag aggagaggag atagctcaac ctggcattta ggtggtgtgg gaacaggaga
     2221 ccccagactt tcttgttttg gggtctgggg caggcaacca ggctccaggg acagtgagtt
     2281 gaaggaaggg tggctgggag accccttgac ttgctgccaa ggagacagag ctggagctag
     2341 ggtggcgggt ggtgtctgag gcaggtgcag agagggaggg agggaagggg cctttgactc
     2401 caacctcctt tttctttacc gactgcaggt ggcagctgcc cttccaggag ccagtggggg
     2461 aacctgggtg gctgggtggg gacacctgca agtcctccct aagccagcta ccaccctaca
     2521 ctgttggcct cccttctcca actgtgggga cgctgctcag gccttttgtg acatcacacc
     2581 tgagagtccc tggggtccag tcattgctgc tgggcacagc gaggtccaag ctcaggtcgc
     2641 cctgccccct acccaccatg ccagatccag catcgttgtg ggcaaacaat tatctggatg
     2701 atctttatgg ggcttaagct tgggtgggag cagatggggc atgagctggg gatttgggga
     2761 tggggggaat ccacaccccc acgtcctgga cgtttaaaag gccctctctg gcactgggcc
     2821 ggggcagagg ccagcagaaa agtgactgga gtccaggcgc gccgaagttg gtcgtgaggc
     2881 actgggcagg taagtatcaa ggttacaaga caggtttaag gagaccaata gaaactgggc
     2941 ttgtcgagac agagaagact cttgcgtttc tgataggcac ctattggtct tactgacatc
     3001 cactttgcct ttctctccac agaccggtgc caccatggcc gtgagcaagg gcgaggagct
     3061 gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt
     3121 cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc aagctgaccc tgaagttcat
     3181 ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg
     3241 cgtgcagtgc ttcagccgct accccgacca catgaagcag cacgacttct tcaagtccgc
     3301 catgcccgaa ggctacgtcc aggagcgcac catcttcttc aaggacgacg gcaactacaa
     3361 gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg
     3421 catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca actacaacag
     3481 ccacaacgtc tatatcatgg ccgacaagca gaagaacggc atcaaggtga acttcaagat
     3541 ccgccacaac atcgaggacg gcagcgtgca gctcgccgac cactaccagc agaacacccc
     3601 catcggcgac ggccccgtgc tgctgcccga caaccactac ctgagcaccc agtccgccct
     3661 gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg tgaccgccgc
     3721 cgggatcact ctcggcatgg acgagctgta caagtaggaa ttcataatca acctctggat
     3781 tacaaaattt gtgaaagatt gactggtatt cttaactatg ttgctccttt tacgctatgt
     3841 ggatacgctg ctttaatgcc tttgtatcat gctattgctt cccgtatggc tttcattttc
     3901 tcctccttgt ataaatcctg gttagttctt gccacggcgg aactcatcgc cgcctgcctt
     3961 gcccgctgct ggacaggggc tcggctgttg ggcactgaca attccgtggt gtttatttgt
     4021 gaaatttgtg atgctattgc tttatttgta accatctagc tttatttgtg aaatttgtga
     4081 tgctattgct ttatttgtaa ccattataag ctgcaataaa caagttaaca acaacaattg
     4141 cattcatttt atgtttcagg ttcaggggga gatgtgggag gttttttaaa caagtgtctg
     4201 gtgagggtct tcggtaccaa gaagacctag tcacgtcagg taccttgggc cgcaggaacc
     4261 cctagtgatg gagttggcca ctccctctct gcgcgctcgc tcgctcactg aggccgggcg
     4321 accaaaggtc gcccgacgcc cgggctttgc ccgagcggcc tcagtgagcg agcgagcgcg
     4381 cagctgcctg caggacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     4441 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     4501 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     4561 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     4621 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
     4681 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     4741 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     4801 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     4861 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct
     4921 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     4981 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     5041 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     5101 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
     5161 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
     5221 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     5281 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
     5341 tgcaatgata ccgcggcttc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
     5401 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
     5461 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
     5521 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
     5581 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
     5641 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
     5701 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
     5761 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
     5821 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
     5881 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
     5941 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
     6001 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
     6061 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg
     6121 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
     6181 cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc attattatca tgacattaac
     6241 ctataaaaat aggcgtatca cgaggccctt tcgtctcgcg cgtttcggtg atgacggtg
//
