LOCUS       sequence-459839-        5296 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 tdTomato from SCP1 promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5296)
  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 5296)
  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)-SCP1-tdTomato-W3SL-T7
FEATURES             Location/Qualifiers
     source          1..5296
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   651..791
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   662..791
                     /label=AAV2 ITR
     promoter        complement(794..812)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        863..881
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     863..880
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     intron          1112..1244
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     regulatory      1251..1260
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(2750..2770)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     promoter        3180..3198
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     repeat_region   3209..3349
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3209..3338
                     /label=AAV2 ITR
     rep_origin      3424..3879
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3511..3530)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3721..3742
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3896..3915)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4015..4037
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4075..4093)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4161..4265
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4266..4334,4335..5126)
                     /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     complement(4484..4503)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt cctgcaggca
      661 gctgcgcgct cgctcgctca ctgaggccgc tcgggcaaag cccgggcgtc gggcgacctt
      721 tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca actccatcac
      781 taggggttcc tgccctatag tgagtcgtat tacccggcca ggaaccgtaa aaaggacttg
      841 cccgcgttgc tggcgtttaa cgatttaggt gacactatag aatacacggg tggtaaccgt
      901 attcattgga tttatgtgta tttgccagtg atggcaaatt gccaaggctg ctacggacga
      961 tgtggaagat ggcccaagag catctggaga gacaaggccg cctcgagctt gatatcgaat
     1021 tcctgcgtac ttatataagg gggtgggggc gcgttcgtcc tcagtcgcga tcgaacactc
     1081 gagccgagca gacgtgccta cggaccgaca ggtaagtatc aaggttacaa gacaggttta
     1141 aggagaccaa tagaaactgg gcttgtcgag acagagaaga ctcttgcgtt tctgataggc
     1201 acctattggt cttactgaca tccactttgc ctttctctcc acagaccggt gccaccatgg
     1261 tgagtaaggg cgaggaagtg atcaaagagt tcatgcggtt taaggtgaga atggaaggaa
     1321 gcatgaacgg ccacgagttc gaaattgagg gagaaggaga gggacggccc tacgagggca
     1381 cccagacagc caagctgaaa gtgacaaagg gcgggcctct gccattcgct tgggacatcc
     1441 tgagcccaca gtttatgtac ggctccaagg cctatgtgaa acatccagct gacattcccg
     1501 attataagaa actgagcttc cccgaggggt ttaagtggga aagagtgatg aacttcgagg
     1561 acggaggcct ggtgactgtg acccaggaca gctccctgca ggatgggacc ctgatctaca
     1621 aggtgaaaat gagagggaca aattttcccc ctgatggacc tgtgatgcag aagaaaacta
     1681 tgggatggga ggcctccacc gaaaggctgt atccacgcga cggggtgctg aaaggagaaa
     1741 tccaccaggc tctgaagctg aaagatgggg gacattacct ggtggagttc aagacaatct
     1801 acatggccaa gaaacctgtg cagctgccag gctactatta cgtggacaca aaactggata
     1861 tcacttcaca caacgaggac tacactattg tggagcagta tgaacggagc gaggggagac
     1921 accatctgtt cctgggccat gggactggaa gtaccggctc agggtctagt ggaaccgcct
     1981 caagcgagga taacaatatg gctgtgatca aagagttcat gaggtttaag gtgcgcatgg
     2041 agggcagcat gaatgggcac gaatttgaga ttgaaggaga gggcgaaggg aggccttacg
     2101 agggcacaca gactgccaag ctgaaagtga ccaagggagg accactgcct ttcgcttggg
     2161 atatcctgtc tcctcagttt atgtacggaa gtaaggccta tgtcaagcat cccgctgaca
     2221 ttcctgatta caagaaactg tctttcccag agggctttaa gtgggagaga gtgatgaatt
     2281 ttgaagatgg aggcctggtg accgtgacac aggactcctc tctgcaggat ggcactctga
     2341 tctacaaagt caaaatgcgc ggcaccaatt ttccacccga tgggcccgtg atgcagaaga
     2401 aaacaatggg gtgggaggcc agcactgaac ggctgtatcc tagagacgga gtgctgaagg
     2461 gcgaaatcca ccaggccctg aagctgaaag acggcggcca ctacctggtg gagttcaaaa
     2521 ccatctacat ggccaagaaa ccagtgcagc tgcccggcta ttactatgtg gacaccaagc
     2581 tggatatcac atcccacaat gaagactaca ccattgtgga acagtatgag aggtctgaag
     2641 gacgccacca tctgtttctg tacggcatgg atgagctgta taagtaaacg cgtcataatc
     2701 aacctctgga ttacaaaatt tgtgaaagat tgactggtat tcttaactat gttgctcctt
     2761 ttacgctatg tggatacgct gctttaatgc ctttgtatca tgctattgct tcccgtatgg
     2821 ctttcatttt ctcctccttg tataaatcct ggttagttct tgccacggcg gaactcatcg
     2881 ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt gggcactgac aattccgtgg
     2941 tgtttatttg tgaaatttgt gatgctattg ctttatttgt aaccatctag ctttatttgt
     3001 gaaatttgtg atgctattgc tttatttgta accattataa gctgcaataa acaagttaac
     3061 aacaacaatt gcattcattt tatgtttcag gttcaggggg agatgtggga ggttttttaa
     3121 acaaggtacc gttaaacgcc agcaacgcgg gcaagtcctt tttacggttc ctggccgggt
     3181 aatacgactc actataggct tgggccgcag gaacccctag tgatggagtt ggccactccc
     3241 tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc
     3301 tttgcccggg cggcctcagt gagcgagcga gcgcgcagct gcctgcaggg gcgcctgatg
     3361 cggtattttc tccttacgca tctgtgcggt atttcacacc gcatacgtca aagcaaccat
     3421 agtacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
     3481 ccgctacact tgccagcgcc ttagcgcccg ctcctttcgc tttcttccct tcctttctcg
     3541 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
     3601 ttagtgcttt acggcacctc gaccccaaaa aacttgattt gggtgatggt tcacgtagtg
     3661 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
     3721 gtggactctt gttccaaact ggaacaacac tcaactctat ctcgggctat tcttttgatt
     3781 tataagggat tttgccgatt tcggtctatt ggttaaaaaa tgagctgatt taacaaaaat
     3841 ttaacgcgaa ttttaacaaa atattaacgt ttacaatttt atggtgcact ctcagtacaa
     3901 tctgctctga tgccgcatag ttaagccagc cccgacaccc gccaacaccc gctgacgcgc
     3961 cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga
     4021 gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgagacga aagggcctcg
     4081 tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg
     4141 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
     4201 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
     4261 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc
     4321 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg
     4381 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
     4441 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat
     4501 tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg
     4561 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag
     4621 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa
     4681 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc
     4741 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca
     4801 cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc
     4861 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc
     4921 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg
     4981 gaagccgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta
     5041 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag
     5101 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga
     5161 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc
     5221 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     5281 agatcaaagg atcttc
//
