LOCUS       sequence-459709-        5256 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 mscRE4 enhancer..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5256)
  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 5256)
  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)-mscRE4-minBG-CI-EGFP-W3SL-BC(p1...
FEATURES             Location/Qualifiers
     source          1..5256
                     /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      1769..1778
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1775..1777,1778..1780,1781..1783,1784..2497)
                     /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(1775..1777,1778..1780,1781..1783,1784..2494)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MAVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLT
                     LKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKD
                     DGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGI
                     KVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVL
                     LEFVTAAGITLGMDELYK"
     primer_bind     complement(1823..1844)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2084..2103)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2431..2452
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2559..2579)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     repeat_region   3211..3351
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3211..3340
                     /label=AAV2 ITR
     rep_origin      complement(3413..4001)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3493..3512)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4172..4963,4964..5032))
                     /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     4795..4814
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5033..5137)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5205..5223
                     /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 agccttgggg gcaatcaaac
     1141 tattacattg agtccttgga tttgctacaa attacatttt aaatgcaatc attttataaa
     1201 agcttcaaca ctcacacttg gaagcgttac cctgttgaat atcactgact cactaacttg
     1261 cattgccatg ctaacttgct ttcagagaga tctcagaaca catcatcttc tgctatttca
     1321 atacatgcac attaatttcc tatcaacgtg tgctgatcag gaactctgta atctggcacc
     1381 ggtgtttatt tttattcctg tctattcctg ttggctcacg aaaagattgt ttgagcaagt
     1441 gttttatggt gagttgtatc atatgtacat tgatttaatc tgcccacatt cagttctaca
     1501 agcggagcca aaaaaataga gacaagcata attttcattc aacatgagcc cctcaatgca
     1561 agccaagtac ctcatctggt gctcagctaa agcaacagca atctgttcca ccctggagac
     1621 acaactggcc acagaaaact tagtgaaaag aggcaatgct atgcacagga caaatgagct
     1681 cgggctgggc ataaaagtca gggcagagcc atctattgct tacatttgct tctgggatcc
     1741 agatctttcg aagctagcgc taaccggtgc caccatggcc gtgagcaagg gcgaggagct
     1801 gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt
     1861 cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc aagctgaccc tgaagttcat
     1921 ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg
     1981 cgtgcagtgc ttcagccgct accccgacca catgaagcag cacgacttct tcaagtccgc
     2041 catgcccgaa ggctacgtcc aggagcgcac catcttcttc aaggacgacg gcaactacaa
     2101 gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg
     2161 catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca actacaacag
     2221 ccacaacgtc tatatcatgg ccgacaagca gaagaacggc atcaaggtga acttcaagat
     2281 ccgccacaac atcgaggacg gcagcgtgca gctcgccgac cactaccagc agaacacccc
     2341 catcggcgac ggccccgtgc tgctgcccga caaccactac ctgagcaccc agtccgccct
     2401 gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg tgaccgccgc
     2461 cgggatcact ctcggcatgg acgagctgta caagtaggaa ttcataatca acctctggat
     2521 tacaaaattt gtgaaagatt gactggtatt cttaactatg ttgctccttt tacgctatgt
     2581 ggatacgctg ctttaatgcc tttgtatcat gctattgctt cccgtatggc tttcattttc
     2641 tcctccttgt ataaatcctg gttagttctt gccacggcgg aactcatcgc cgcctgcctt
     2701 gcccgctgct ggacaggggc tcggctgttg ggcactgaca attccgtggt gtttatttgt
     2761 gaaatttgtg atgctattgc tttatttgta accatctagc tttatttgtg aaatttgtga
     2821 tgctattgct ttatttgtaa ccattataag ctgcaataaa caagttaaca acaacaattg
     2881 cattcatttt atgtttcagg ttcaggggga gatgtgggag gttttttaaa caaggtacca
     2941 tcacattacc atgcaggtac acagcaagaa ttgacgttgg catatcacat ggtgtaataa
     3001 ccccacttgt gaaacaaccc agaataaggt acaaggcgga aatgtcgtca ttctaaaata
     3061 aaaggcatgg ccaggaattt gtctaatacc gggaacttaa attcagcttg aacaccagtc
     3121 gcaaaaaatt caaagaaagt gattcaggtt cgggttcgtg gattggaaca gcttcttttg
     3181 tttcagtgat gagagaatcc tcctgtcagc aggaacccct agtgatggag ttggccactc
     3241 cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg
     3301 gctttgcccg agcggcctca gtgagcgagc gagcgcgcag ctgcctgcag gacatgtgag
     3361 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     3421 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     3481 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     3541 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     3601 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     3661 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     3721 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     3781 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     3841 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     3901 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     3961 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     4021 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     4081 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     4141 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     4201 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     4261 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cggcttccac
     4321 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     4381 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     4441 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     4501 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     4561 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     4621 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     4681 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     4741 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     4801 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     4861 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     4921 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     4981 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     5041 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     5101 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
     5161 ctgacgtcta agaaaccatt attatcatga cattaaccta taaaaatagg cgtatcacga
     5221 ggccctttcg tctcgcgcgt ttcggtgatg acggtg
//
