LOCUS       sequence-459858-        5241 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 the minBG promoter with hDLXI56i enhancer..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5241)
  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 5241)
  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)-hDLXI56i-minBG-CI-EGFP-W3SL-BC(...
FEATURES             Location/Qualifiers
     source          1..5241
                     /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
     regulatory      1754..1763
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1760..1762,1763..1765,1766..1768,1769..2482)
                     /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(1760..1762,1763..1765,1766..1768,1769..2479)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MAVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLT
                     LKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKD
                     DGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGI
                     KVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVL
                     LEFVTAAGITLGMDELYK"
     primer_bind     complement(1808..1829)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2069..2088)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2416..2437
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2544..2564)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     repeat_region   3196..3336
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3196..3325
                     /label=AAV2 ITR
     rep_origin      complement(3398..3986)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3478..3497)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4157..4948,4949..5017))
                     /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     4780..4799
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5018..5122)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5190..5208
                     /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 cctcgagata ggtaccgagc tctatgcact
     1141 cacagtggtt tggcatgcat ctggtgaatt ttttttaacg aaaaattagt gttggtttcg
     1201 atgtatggta gcattctccc taacgtaatt tgaataattc agcaaagccc cactaccagc
     1261 tgtacttctg cagcctcttc cattcttttc agcattataa ttttggttaa ttttcaattt
     1321 taggtcctac gtctctgcaa tttgtgtatg aataacagaa taatttccct cttttgtttc
     1381 gcctttcctg ttcctgaatc taaataaaga tggcttttta gtattaaaag tggaagaaaa
     1441 ttacaggtaa ttatctttga cggtaaaaac gctgtaatca gcgggctaca tgaaaaatta
     1501 ctctaattat ggctgcattt aagagaatgg aaaaaaacct tcttgtggat aaaaacctta
     1561 aattgtcccc aatgtctgct tcaaattgga tggcactgca gctggaggct ttgttcagaa
     1621 ttgatcctgg ggagctacga acccaaagtt tcacagtagg gagctcgggc tgggcataaa
     1681 agtcagggca gagccatcta ttgcttacat ttgcttctgg gatccagatc tttcgaagct
     1741 agcgctaacc ggtgccacca tggccgtgag caagggcgag gagctgttca ccggggtggt
     1801 gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga
     1861 gggcgagggc gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa
     1921 gctgcccgtg ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag
     1981 ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta
     2041 cgtccaggag cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt
     2101 gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga
     2161 ggacggcaac atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat
     2221 catggccgac aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga
     2281 ggacggcagc gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc
     2341 cgtgctgctg cccgacaacc actacctgag cacccagtcc gccctgagca aagaccccaa
     2401 cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg
     2461 catggacgag ctgtacaagt aggaattcat aatcaacctc tggattacaa aatttgtgaa
     2521 agattgactg gtattcttaa ctatgttgct ccttttacgc tatgtggata cgctgcttta
     2581 atgcctttgt atcatgctat tgcttcccgt atggctttca ttttctcctc cttgtataaa
     2641 tcctggttag ttcttgccac ggcggaactc atcgccgcct gccttgcccg ctgctggaca
     2701 ggggctcggc tgttgggcac tgacaattcc gtggtgttta tttgtgaaat ttgtgatgct
     2761 attgctttat ttgtaaccat ctagctttat ttgtgaaatt tgtgatgcta ttgctttatt
     2821 tgtaaccatt ataagctgca ataaacaagt taacaacaac aattgcattc attttatgtt
     2881 tcaggttcag ggggagatgt gggaggtttt ttaaacaagg taccatcaca ttaccatgca
     2941 ggtacacagc aagaattgac gttggcatat cacatggtgt aataacccca cttgtgaaac
     3001 aacccagaat aaggtacaag gcggaaatgt cgtcattcta aaataaaagg catggccagg
     3061 aatttgtcta ataccgggaa cttaaattca gcttgaacac cagtcgcaaa aaattcaaag
     3121 aaagtgattc aggttcgggt tcgtggattg gaacagcttc ttttgtttca gtgatgagag
     3181 aatcctcctg tcagcaggaa cccctagtga tggagttggc cactccctct ctgcgcgctc
     3241 gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt gcccgagcgg
     3301 cctcagtgag cgagcgagcg cgcagctgcc tgcaggacat gtgagcaaaa ggccagcaaa
     3361 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     3421 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     3481 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     3541 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     3601 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     3661 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     3721 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     3781 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa
     3841 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     3901 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     3961 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     4021 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     4081 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     4141 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     4201 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     4261 gcttaccatc tggccccagt gctgcaatga taccgcggct tccacgctca ccggctccag
     4321 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     4381 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     4441 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
     4501 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     4561 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     4621 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     4681 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     4741 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
     4801 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     4861 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     4921 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     4981 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
     5041 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     5101 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtctaagaaa
     5161 ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc tttcgtctcg
     5221 cgcgtttcgg tgatgacggt g
//
