LOCUS       sequence-459852-        5730 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 CAG promoter. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5730)
  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 5730)
  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)-CAG-EGFP-W3SL-BC(p1-10)
FEATURES             Location/Qualifiers
     source          1..5730
                     /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
     enhancer        1118..1497
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1499..1776
                     /label=chicken beta-actin promoter
     primer_bind     2106..2128
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2190..2209
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      2243..2252
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2249..2251,2252..2254,2255..2257,2258..2971)
                     /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(2249..2251,2252..2254,2255..2257,2258..2968)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MAVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLT
                     LKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKD
                     DGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGI
                     KVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVL
                     LEFVTAAGITLGMDELYK"
     primer_bind     complement(2297..2318)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2558..2577)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2905..2926
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3033..3053)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     repeat_region   3685..3825
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3685..3814
                     /label=AAV2 ITR
     rep_origin      complement(3887..4475)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3967..3986)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4646..5437,5438..5506))
                     /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     5269..5288
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5507..5611)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5679..5697
                     /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 cctcgaggac attgattatt gactagttat
     1141 taatagtaat caattacggg gtcattagtt catagcccat atatggagtt ccgcgttaca
     1201 taacttacgg taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca
     1261 ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg
     1321 gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg
     1381 ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc
     1441 ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtc
     1501 gaggtgagcc ccacgttctg cttcactctc cccatctccc ccccctcccc acccccaatt
     1561 ttgtatttat ttatttttta attattttgt gcagcgatgg gggcgggggg gggggggggg
     1621 cgcgcgccag gcggggcggg gcggggcgag gggcggggcg gggcgaggcg gagaggtgcg
     1681 gcggcagcca atcagagcgg cgcgctccga aagtttcctt ttatggcgag gcggcggcgg
     1741 cggcggccct ataaaaagcg aagcgcgcgg cgggcgggag tcgctgcgcg ctgccttcgc
     1801 cccgtgcccc gctccgccgc cgcctcgcgc cgcccgcccc ggctctgact gaccgcgtta
     1861 ctcccacagg tgagcgggcg ggacggccct tctcctccgg gctgtaatta gcgcttggtt
     1921 taatgacggc ttgtttcttt tctgtggctg cgtgaaagcc ttgaggggct ccgggagcgc
     1981 cggcaggaag gaaatgggcg gggagggcct tcgtgcgtcg ccgcgccgcc gtccccttct
     2041 ccctctccag cctcggggct gtccgcgggg ggacggctgc cttcgggggg gacggggcag
     2101 ggcggggttc ggcttctggc gtgtgaccgg cggctctaga gcctctgcta accatgttca
     2161 tgccttcttc tttttcctac agctcctggg caacgtgctg gttattgtgc tgtctcatca
     2221 ttttggcaaa gaattgaccg gtgccaccat ggccgtgagc aagggcgagg agctgttcac
     2281 cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt
     2341 gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac
     2401 caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accctgacct acggcgtgca
     2461 gtgcttcagc cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc
     2521 cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg
     2581 cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga
     2641 cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca acagccacaa
     2701 cgtctatatc atggccgaca agcagaagaa cggcatcaag gtgaacttca agatccgcca
     2761 caacatcgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg
     2821 cgacggcccc gtgctgctgc ccgacaacca ctacctgagc acccagtccg ccctgagcaa
     2881 agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat
     2941 cactctcggc atggacgagc tgtacaagta ggaattcata atcaacctct ggattacaaa
     3001 atttgtgaaa gattgactgg tattcttaac tatgttgctc cttttacgct atgtggatac
     3061 gctgctttaa tgcctttgta tcatgctatt gcttcccgta tggctttcat tttctcctcc
     3121 ttgtataaat cctggttagt tcttgccacg gcggaactca tcgccgcctg ccttgcccgc
     3181 tgctggacag gggctcggct gttgggcact gacaattccg tggtgtttat ttgtgaaatt
     3241 tgtgatgcta ttgctttatt tgtaaccatc tagctttatt tgtgaaattt gtgatgctat
     3301 tgctttattt gtaaccatta taagctgcaa taaacaagtt aacaacaaca attgcattca
     3361 ttttatgttt caggttcagg gggagatgtg ggaggttttt taaacaaggt accatcacat
     3421 taccatgcag gtacacagca agaattgacg ttggcatatc acatggtgta ataaccccac
     3481 ttgtgaaaca acccagaata aggtacaagg cggaaatgtc gtcattctaa aataaaaggc
     3541 atggccagga atttgtctaa taccgggaac ttaaattcag cttgaacacc agtcgcaaaa
     3601 aattcaaaga aagtgattca ggttcgggtt cgtggattgg aacagcttct tttgtttcag
     3661 tgatgagaga atcctcctgt cagcaggaac ccctagtgat ggagttggcc actccctctc
     3721 tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc ccgggctttg
     3781 cccgagcggc ctcagtgagc gagcgagcgc gcagctgcct gcaggacatg tgagcaaaag
     3841 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     3901 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     3961 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     4021 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     4081 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     4141 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     4201 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     4261 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     4321 ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     4381 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     4441 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     4501 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     4561 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     4621 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
     4681 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
     4741 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcggctt ccacgctcac
     4801 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
     4861 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
     4921 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
     4981 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
     5041 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
     5101 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
     5161 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
     5221 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
     5281 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
     5341 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
     5401 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
     5461 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
     5521 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     5581 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg
     5641 tctaagaaac cattattatc atgacattaa cctataaaaa taggcgtatc acgaggccct
     5701 ttcgtctcgc gcgtttcggt gatgacggtg
//
