LOCUS       sequence-459725-        5467 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Single stranded AAV genome with barcodeless T7 RNA polymerase 
            promoter and SP6-driven barcode, 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 5467)
  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 5467)
  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.T7-SP6-BC(p11-14)-CAG-EGFP-W3SL-T7
FEATURES             Location/Qualifiers
     source          1..5467
                     /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        complement(844..862)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     1272..1292
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(join(1354..2067,2068..2070,2071..2073,
                     2074..2076))
                     /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             complement(join(1357..2067,2068..2070,2071..2073,
                     2074..2076))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MAVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLT
                     LKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKD
                     DGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGI
                     KVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVL
                     LEFVTAAGITLGMDELYK"
     primer_bind     complement(1399..1420)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1748..1767
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2007..2028
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      2073..2082
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(2116..2135)
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     primer_bind     complement(2197..2219)
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     promoter        complement(2549..2824)
                     /label=chicken beta-actin promoter
     enhancer        2826..3205
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        complement(3332..3350)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3333..3350)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     promoter        3401..3419
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     repeat_region   3422..3562
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3422..3551
                     /label=AAV2 ITR
     rep_origin      complement(3624..4212)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3704..3723)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4383..5174,5175..5243))
                     /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     5006..5025
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5244..5348)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5416..5434
                     /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 cctgcggccc
      841 aagcctatag tgagtcgtat tacccggcca ggaaccgtaa aaaggacttg cccgcgttgc
      901 tggcgtttaa cggtaccttg tttaaaaaac ctcccacatc tccccctgaa cctgaaacat
      961 aaaatgaatg caattgttgt tgttaacttg tttattgcag cttataatgg ttacaaataa
     1021 agcaatagca tcacaaattt cacaaataaa gctagatggt tacaaataaa gcaatagcat
     1081 cacaaatttc acaaataaac accacggaat tgtcagtgcc caacagccga gcccctgtcc
     1141 agcagcgggc aaggcaggcg gcgatgagtt ccgccgtggc aagaactaac caggatttat
     1201 acaaggagga gaaaatgaaa gccatacggg aagcaatagc atgatacaaa ggcattaaag
     1261 cagcgtatcc acatagcgta aaaggagcaa catagttaag aataccagtc aatctttcac
     1321 aaattttgta atccagaggt tgattatgaa ttcctacttg tacagctcgt ccatgccgag
     1381 agtgatcccg gcggcggtca cgaactccag caggaccatg tgatcgcgct tctcgttggg
     1441 gtctttgctc agggcggact gggtgctcag gtagtggttg tcgggcagca gcacggggcc
     1501 gtcgccgatg ggggtgttct gctggtagtg gtcggcgagc tgcacgctgc cgtcctcgat
     1561 gttgtggcgg atcttgaagt tcaccttgat gccgttcttc tgcttgtcgg ccatgatata
     1621 gacgttgtgg ctgttgtagt tgtactccag cttgtgcccc aggatgttgc cgtcctcctt
     1681 gaagtcgatg cccttcagct cgatgcggtt caccagggtg tcgccctcga acttcacctc
     1741 ggcgcgggtc ttgtagttgc cgtcgtcctt gaagaagatg gtgcgctcct ggacgtagcc
     1801 ttcgggcatg gcggacttga agaagtcgtg ctgcttcatg tggtcggggt agcggctgaa
     1861 gcactgcacg ccgtaggtca gggtggtcac gagggtgggc cagggcacgg gcagcttgcc
     1921 ggtggtgcag atgaacttca gggtcagctt gccgtaggtg gcatcgccct cgccctcgcc
     1981 ggacacgctg aacttgtggc cgtttacgtc gccgtccagc tcgaccagga tgggcaccac
     2041 cccggtgaac agctcctcgc ccttgctcac ggccatggtg gcaccggtca attctttgcc
     2101 aaaatgatga gacagcacaa taaccagcac gttgcccagg agctgtagga aaaagaagaa
     2161 ggcatgaaca tggttagcag aggctctaga gccgccggtc acacgccaga agccgaaccc
     2221 cgccctgccc cgtccccccc gaaggcagcc gtccccccgc ggacagcccc gaggctggag
     2281 agggagaagg ggacggcggc gcggcgacgc acgaaggccc tccccgccca tttccttcct
     2341 gccggcgctc ccggagcccc tcaaggcttt cacgcagcca cagaaaagaa acaagccgtc
     2401 attaaaccaa gcgctaatta cagcccggag gagaagggcc gtcccgcccg ctcacctgtg
     2461 ggagtaacgc ggtcagtcag agccggggcg ggcggcgcga ggcggcggcg gagcggggca
     2521 cggggcgaag gcagcgcgca gcgactcccg cccgccgcgc gcttcgcttt ttatagggcc
     2581 gccgccgccg ccgcctcgcc ataaaaggaa actttcggag cgcgccgctc tgattggctg
     2641 ccgccgcacc tctccgcctc gccccgcccc gcccctcgcc ccgccccgcc ccgcctggcg
     2701 cgcgcccccc ccccccccgc ccccatcgct gcacaaaata attaaaaaat aaataaatac
     2761 aaaattgggg gtggggaggg gggggagatg gggagagtga agcagaacgt ggggctcacc
     2821 tcgaccatgg taatagcgat gactaatacg tagatgtact gccaagtagg aaagtcccat
     2881 aaggtcatgt actgggcata atgccaggcg ggccatttac cgtcattgac gtcaataggg
     2941 ggcgtacttg gcatatgata cacttgatgt actgccaagt gggcagttta ccgtaaatac
     3001 tccacccatt gacgtcaatg gaaagtccct attggcgtta ctatgggaac atacgtcatt
     3061 attgacgtca atgggcgggg gtcgttgggc ggtcagccag gcgggccatt taccgtaagt
     3121 tatgtaacgc ggaactccat atatgggcta tgaactaatg accccgtaat tgattactat
     3181 taataactag tcaataatca atgtcctcga ggcggccttg tctctccaga tgctcttggg
     3241 ccatcttcca catcgtccgt agcagccttg gcaatttgcc atcactggca aatacacata
     3301 aatccaatga atacggttac cacccgtgta ttctatagtg tcacctaaat cgttaaacgc
     3361 cagcaacgcg ggcaagtcct ttttacggtt cctggccggg taatacgact cactataggg
     3421 caggaacccc tagtgatgga gttggccact ccctctctgc gcgctcgctc gctcactgag
     3481 gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc gagcggcctc agtgagcgag
     3541 cgagcgcgca gctgcctgca ggacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     3601 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     3661 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     3721 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     3781 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     3841 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     3901 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     3961 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     4021 acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc
     4081 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     4141 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     4201 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     4261 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
     4321 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
     4381 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
     4441 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
     4501 cccagtgctg caatgatacc gcggcttcca cgctcaccgg ctccagattt atcagcaata
     4561 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
     4621 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
     4681 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
     4741 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa
     4801 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca
     4861 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt
     4921 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
     4981 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg
     5041 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
     5101 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
     5161 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
     5221 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag
     5281 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
     5341 gttccgcgca catttccccg aaaagtgcca cctgacgtct aagaaaccat tattatcatg
     5401 acattaacct ataaaaatag gcgtatcacg aggccctttc gtctcgcgcg tttcggtgat
     5461 gacggtg
//
