LOCUS       sequence-459726-        6430 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Single stranded AAV genome with concatemerization-dependent 
            barcodes, for tracking AAV concatemers via SpECTr. Also expresses 
            EGFP from Ple155 element, active in Purkinje cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6430)
  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 6430)
  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.BC(p1-10)-Ple155-CI-EGFP-W3SL-BC(p1-10)
FEATURES             Location/Qualifiers
     source          1..6430
                     /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          2804..2936
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     regulatory      2943..2952
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2949..2951,2952..2954,2955..2957,2958..3671)
                     /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(2949..2951,2952..2954,2955..2957,2958..3668)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MAVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLT
                     LKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKD
                     DGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGI
                     KVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVL
                     LEFVTAAGITLGMDELYK"
     primer_bind     complement(2997..3018)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3258..3277)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3605..3626
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3733..3753)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     repeat_region   4385..4525
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4385..4514
                     /label=AAV2 ITR
     rep_origin      complement(4587..5175)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4667..4686)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5346..6137,6138..6206))
                     /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     5969..5988
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6207..6311)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6379..6397
                     /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 cctgctgaca
      841 ggaggattct ctcatcactg aaacaaaaga agctgttcca atccacgaac ccgaacctga
      901 atcactttct ttgaattttt tgcgactggt gttcaagctg aatttaagtt cccggtatta
      961 gacaaattcc tggccatgcc ttttatttta gaatgacgac atttccgcct tgtaccttat
     1021 tctgggttgt ttcacaagtg gggttattac accatgtgat atgccaacgt caattcttgc
     1081 tgtgtacctg catggtaatg tgatgggccg cctcgagtca gcagattgaa gaagccctcc
     1141 tggtctgggg agcccgcctg gggacagact cgctcagtct ggttggccct tcagcttggg
     1201 ggcccctccc caaacttcct agccagcttg ctcacaccct gaccccgggg cctgccgtcc
     1261 ccacttcctc agctctcacc atggtccccg ccgatcctct ctgcagagct gttctgaatg
     1321 agacatgagt ctccttccca atcccggccc ccgccccagg ggccctggcc agcagtgcca
     1381 cttcacgtgg taccgcttca agggacaggc tccgatgcgt gtcccgccgt cctagattgg
     1441 ggcctgatga gtgtggcctg ggagctggga cacgaatcag ggaaacatgg cccaggagct
     1501 acccccaggt cccagcatct cccatcaata ggggtccaca cggagagccc tgccctctgc
     1561 cctggggcct ggcactcaga cccccaagcc caccagcccc tttctacagc cacaactggg
     1621 tcagggggtc ctaggagact cactcgttaa ttaggtgccc tacaaactaa ttagtcttgt
     1681 caatcatggg ctctgagacc ttgagctggg ggtggggtgg gggcagggcc ctctcacctc
     1741 ggcacagggg cctgagcctt cctccgtctt ctcctcctga tccggacact tcattggcat
     1801 agagggagag agtgtgaact tggccctttg tggaacagag gaggctcggg cagaggtggt
     1861 gatagtgcag cccattcatt ctgagatgaa acttccactg gtttccgtaa agacgtcttg
     1921 gggagggaag ggaaggggat ggggacctcc cagtggtatc ccctgcttgg gcactgaggg
     1981 aaagccacag tggctcgggg taaaaggcag ggacatcctc tccccgcctg cctctgtccc
     2041 cagggagtct cgcctcctgt tcccacctgg ggctagggtg atagaggaga ggagatagct
     2101 caacctggca tttaggtggt gtgggaacag gagaccccag actttcttgt tttggggtct
     2161 ggggcaggca accaggctcc agggacagtg agttgaagga agggtggctg ggagacccct
     2221 tgacttgctg ccaaggagac agagctggag ctagggtggc gggtggtgtc tgaggcaggt
     2281 gcagagaggg agggagggaa ggggcctttg actccaacct cctttttctt taccgactgc
     2341 aggtggcagc tgcccttcca ggagccagtg ggggaacctg ggtggctggg tggggacacc
     2401 tgcaagtcct ccctaagcca gctaccaccc tacactgttg gcctcccttc tccaactgtg
     2461 gggacgctgc tcaggccttt tgtgacatca cacctgagag tccctggggt ccagtcattg
     2521 ctgctgggca cagcgaggtc caagctcagg tcgccctgcc ccctacccac catgccagat
     2581 ccagcatcgt tgtgggcaaa caattatctg gatgatcttt atggggctta agcttgggtg
     2641 ggagcagatg gggcatgagc tggggatttg gggatggggg gaatccacac ccccacgtcc
     2701 tggacgttta aaaggccctc tctggcactg ggccggggca gaggccagca gaaaagtgac
     2761 tggagtccag gcgcgccgaa gttggtcgtg aggcactggg caggtaagta tcaaggttac
     2821 aagacaggtt taaggagacc aatagaaact gggcttgtcg agacagagaa gactcttgcg
     2881 tttctgatag gcacctattg gtcttactga catccacttt gcctttctct ccacagaccg
     2941 gtgccaccat ggccgtgagc aagggcgagg agctgttcac cggggtggtg cccatcctgg
     3001 tcgagctgga cggcgacgta aacggccaca agttcagcgt gtccggcgag ggcgagggcg
     3061 atgccaccta cggcaagctg accctgaagt tcatctgcac caccggcaag ctgcccgtgc
     3121 cctggcccac cctcgtgacc accctgacct acggcgtgca gtgcttcagc cgctaccccg
     3181 accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc
     3241 gcaccatctt cttcaaggac gacggcaact acaagacccg cgccgaggtg aagttcgagg
     3301 gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca
     3361 tcctggggca caagctggag tacaactaca acagccacaa cgtctatatc atggccgaca
     3421 agcagaagaa cggcatcaag gtgaacttca agatccgcca caacatcgag gacggcagcg
     3481 tgcagctcgc cgaccactac cagcagaaca cccccatcgg cgacggcccc gtgctgctgc
     3541 ccgacaacca ctacctgagc acccagtccg ccctgagcaa agaccccaac gagaagcgcg
     3601 atcacatggt cctgctggag ttcgtgaccg ccgccgggat cactctcggc atggacgagc
     3661 tgtacaagta ggaattcata atcaacctct ggattacaaa atttgtgaaa gattgactgg
     3721 tattcttaac tatgttgctc cttttacgct atgtggatac gctgctttaa tgcctttgta
     3781 tcatgctatt gcttcccgta tggctttcat tttctcctcc ttgtataaat cctggttagt
     3841 tcttgccacg gcggaactca tcgccgcctg ccttgcccgc tgctggacag gggctcggct
     3901 gttgggcact gacaattccg tggtgtttat ttgtgaaatt tgtgatgcta ttgctttatt
     3961 tgtaaccatc tagctttatt tgtgaaattt gtgatgctat tgctttattt gtaaccatta
     4021 taagctgcaa taaacaagtt aacaacaaca attgcattca ttttatgttt caggttcagg
     4081 gggagatgtg ggaggttttt taaacaaggt accatcacat taccatgcag gtacacagca
     4141 agaattgacg ttggcatatc acatggtgta ataaccccac ttgtgaaaca acccagaata
     4201 aggtacaagg cggaaatgtc gtcattctaa aataaaaggc atggccagga atttgtctaa
     4261 taccgggaac ttaaattcag cttgaacacc agtcgcaaaa aattcaaaga aagtgattca
     4321 ggttcgggtt cgtggattgg aacagcttct tttgtttcag tgatgagaga atcctcctgt
     4381 cagcaggaac ccctagtgat ggagttggcc actccctctc tgcgcgctcg ctcgctcact
     4441 gaggccgggc gaccaaaggt cgcccgacgc ccgggctttg cccgagcggc ctcagtgagc
     4501 gagcgagcgc gcagctgcct gcaggacatg tgagcaaaag gccagcaaaa ggccaggaac
     4561 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     4621 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     4681 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     4741 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     4801 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     4861 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     4921 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     4981 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
     5041 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     5101 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     5161 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     5221 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     5281 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     5341 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     5401 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
     5461 ggccccagtg ctgcaatgat accgcggctt ccacgctcac cggctccaga tttatcagca
     5521 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     5581 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     5641 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     5701 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
     5761 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
     5821 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
     5881 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
     5941 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
     6001 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
     6061 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
     6121 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
     6181 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
     6241 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     6301 ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac cattattatc
     6361 atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtctcgc gcgtttcggt
     6421 gatgacggtg
//
