LOCUS       sequence-459833-        4635 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 mRuby2 from minBG promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4635)
  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 4635)
  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)-minBG-CI-mRuby2-W3SL-T7
FEATURES             Location/Qualifiers
     source          1..4635
                     /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          1168..1300
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     regulatory      1307..1316
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1313..2023
                     /codon_start=1
                     /product="monomeric red fluorescent protein derived from 
                     mRuby, with improved photophysical properties (Lam et al., 
                     2012)"
                     /label=mRuby2
                     /translation="MVSKGEELIKENMRMKVVMEGSVNGHQFKCTGEGEGNPYMGTQTM
                     RIKVIEGGPLPFAFDILATSFMYGSRTFIKYPKGIPDFFKQSFPEGFTWERVTRYEDGG
                     VVTVMQDTSLEDGCLVYHVQVRGVNFPSNGPVMQKKTKGWEPNTEMMYPADGGLRGYTH
                     MALKVDGGGHLSCSFVTTYRSKKTVGNIKMPGIHAVDHRLERLEESDNEMFVVQREHAV
                     AKFAGLGGGMDELYK"
     primer_bind     complement(2089..2109)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     promoter        2519..2537
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     repeat_region   2548..2688
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2548..2677
                     /label=AAV2 ITR
     rep_origin      2763..3218
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2850..2869)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3060..3081
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3235..3254)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3354..3376
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3414..3432)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3500..3604
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3605..3673,3674..4465)
                     /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(3823..3842)
                     /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 tcctgcagcc cgggctgggc ataaaagtca gggcagagcc atctattgct tacatttgct
     1081 tctagcctgc aggtcgagga gcgcagcctt ccagaagcag agcgcggcgc cttaagctgc
     1141 agaagttggt cgtgaggcac tgggcaggta agtatcaagg ttacaagaca ggtttaagga
     1201 gaccaataga aactgggctt gtcgagacag agaagactct tgcgtttctg ataggcacct
     1261 attggtctta ctgacatcca ctttgccttt ctctccacag accggtgcca ccatggtgag
     1321 caagggagag gaactcatca aggagaacat gcgcatgaag gtagtcatgg aagggtctgt
     1381 gaatgggcat cagttcaagt gcacagggga aggtgaaggg aatccctaca tgggtactca
     1441 gaccatgagg atcaaggtta ttgaaggtgg ccctctcccg tttgccttcg acattcttgc
     1501 cactagcttc atgtatggca gtcggacctt catcaagtac cccaaaggca ttccagactt
     1561 cttcaaacag tcctttccag agggcttcac atgggaaagg gtgacccgtt acgaggatgg
     1621 tggtgtggtc acagtcatgc aggacactag tctggaggac ggatgtctgg tctatcacgt
     1681 gcaagtgaga ggggtgaact ttccctccaa tgggcctgtg atgcagaaga aaaccaaagg
     1741 gtgggagcca aacacagaga tgatgtatcc tgcagatgga ggactgagag gctatacgca
     1801 catggctctg aaggttgacg gaggaggcca cttgtcatgc tcatttgtga ccacgtacag
     1861 gagcaagaaa actgtcggca acatcaaaat gcctggcata catgcggttg atcaccgctt
     1921 ggaacgactg gaggagtctg acaacgagat gtttgtggta caacgggaac atgcagttgc
     1981 caagtttgct ggccttggcg gaggaatgga tgagctgtac aaatgaacgc gtcataatca
     2041 acctctggat tacaaaattt gtgaaagatt gactggtatt cttaactatg ttgctccttt
     2101 tacgctatgt ggatacgctg ctttaatgcc tttgtatcat gctattgctt cccgtatggc
     2161 tttcattttc tcctccttgt ataaatcctg gttagttctt gccacggcgg aactcatcgc
     2221 cgcctgcctt gcccgctgct ggacaggggc tcggctgttg ggcactgaca attccgtggt
     2281 gtttatttgt gaaatttgtg atgctattgc tttatttgta accatctagc tttatttgtg
     2341 aaatttgtga tgctattgct ttatttgtaa ccattataag ctgcaataaa caagttaaca
     2401 acaacaattg cattcatttt atgtttcagg ttcaggggga gatgtgggag gttttttaaa
     2461 caaggtaccg ttaaacgcca gcaacgcggg caagtccttt ttacggttcc tggccgggta
     2521 atacgactca ctataggctt gggccgcagg aacccctagt gatggagttg gccactccct
     2581 ctctgcgcgc tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct
     2641 ttgcccgggc ggcctcagtg agcgagcgag cgcgcagctg cctgcagggg cgcctgatgc
     2701 ggtattttct ccttacgcat ctgtgcggta tttcacaccg catacgtcaa agcaaccata
     2761 gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
     2821 cgctacactt gccagcgcct tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
     2881 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
     2941 tagtgcttta cggcacctcg accccaaaaa acttgatttg ggtgatggtt cacgtagtgg
     3001 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag
     3061 tggactcttg ttccaaactg gaacaacact caactctatc tcgggctatt cttttgattt
     3121 ataagggatt ttgccgattt cggtctattg gttaaaaaat gagctgattt aacaaaaatt
     3181 taacgcgaat tttaacaaaa tattaacgtt tacaatttta tggtgcactc tcagtacaat
     3241 ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg ctgacgcgcc
     3301 ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag
     3361 ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa agggcctcgt
     3421 gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg
     3481 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
     3541 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa
     3601 gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct
     3661 tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg
     3721 tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg
     3781 ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt
     3841 atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga
     3901 cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga
     3961 attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac
     4021 gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg
     4081 ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac
     4141 gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct
     4201 agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct
     4261 gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg
     4321 aagccgcggt atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat
     4381 ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg
     4441 tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat
     4501 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct
     4561 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
     4621 gatcaaagga tcttc
//
