LOCUS       sequence-459844-        4710 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 Ef1s promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4710)
  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 4710)
  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)-Ef1s-CI-mRuby2-W3SL-T7
FEATURES             Location/Qualifiers
     source          1..4710
                     /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"
     promoter        1027..1238
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     intron          1243..1375
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     regulatory      1382..1391
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1388..2098
                     /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(2164..2184)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     promoter        2594..2612
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     repeat_region   2623..2763
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2623..2752
                     /label=AAV2 ITR
     rep_origin      2838..3293
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2925..2944)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3135..3156
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3310..3329)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3429..3451
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3489..3507)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3575..3679
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3680..3748,3749..4540)
                     /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(3898..3917)
                     /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 tcctgcgggc agagcgcaca tcgcccacag tccccgagaa gttgggggga ggggtcggca
     1081 attgaacggg tgcctagaga aggtggcgcg gggtaaactg ggaaagtgat gtcgtgtact
     1141 ggctccgcct ttttcccgag ggtgggggag aaccgtatat aagtgcagta gtcgccgtga
     1201 acgttctttt tcgcaacggg tttgccgcca gaacacagac aggtaagtat caaggttaca
     1261 agacaggttt aaggagacca atagaaactg ggcttgtcga gacagagaag actcttgcgt
     1321 ttctgatagg cacctattgg tcttactgac atccactttg cctttctctc cacagaccgg
     1381 tgccaccatg gtgagcaagg gagaggaact catcaaggag aacatgcgca tgaaggtagt
     1441 catggaaggg tctgtgaatg ggcatcagtt caagtgcaca ggggaaggtg aagggaatcc
     1501 ctacatgggt actcagacca tgaggatcaa ggttattgaa ggtggccctc tcccgtttgc
     1561 cttcgacatt cttgccacta gcttcatgta tggcagtcgg accttcatca agtaccccaa
     1621 aggcattcca gacttcttca aacagtcctt tccagagggc ttcacatggg aaagggtgac
     1681 ccgttacgag gatggtggtg tggtcacagt catgcaggac actagtctgg aggacggatg
     1741 tctggtctat cacgtgcaag tgagaggggt gaactttccc tccaatgggc ctgtgatgca
     1801 gaagaaaacc aaagggtggg agccaaacac agagatgatg tatcctgcag atggaggact
     1861 gagaggctat acgcacatgg ctctgaaggt tgacggagga ggccacttgt catgctcatt
     1921 tgtgaccacg tacaggagca agaaaactgt cggcaacatc aaaatgcctg gcatacatgc
     1981 ggttgatcac cgcttggaac gactggagga gtctgacaac gagatgtttg tggtacaacg
     2041 ggaacatgca gttgccaagt ttgctggcct tggcggagga atggatgagc tgtacaaatg
     2101 aacgcgtcat aatcaacctc tggattacaa aatttgtgaa agattgactg gtattcttaa
     2161 ctatgttgct ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgctat
     2221 tgcttcccgt atggctttca ttttctcctc cttgtataaa tcctggttag ttcttgccac
     2281 ggcggaactc atcgccgcct gccttgcccg ctgctggaca ggggctcggc tgttgggcac
     2341 tgacaattcc gtggtgttta tttgtgaaat ttgtgatgct attgctttat ttgtaaccat
     2401 ctagctttat ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt ataagctgca
     2461 ataaacaagt taacaacaac aattgcattc attttatgtt tcaggttcag ggggagatgt
     2521 gggaggtttt ttaaacaagg taccgttaaa cgccagcaac gcgggcaagt cctttttacg
     2581 gttcctggcc gggtaatacg actcactata ggcttgggcc gcaggaaccc ctagtgatgg
     2641 agttggccac tccctctctg cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg
     2701 cccgacgccc gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc agctgcctgc
     2761 aggggcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatac
     2821 gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
     2881 tacgcgcagc gtgaccgcta cacttgccag cgccttagcg cccgctcctt tcgctttctt
     2941 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
     3001 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg atttgggtga
     3061 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
     3121 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaact ctatctcggg
     3181 ctattctttt gatttataag ggattttgcc gatttcggtc tattggttaa aaaatgagct
     3241 gatttaacaa aaatttaacg cgaattttaa caaaatatta acgtttacaa ttttatggtg
     3301 cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac acccgccaac
     3361 acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt
     3421 gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag
     3481 acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc
     3541 ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt
     3601 ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata
     3661 atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt
     3721 tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc
     3781 tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat
     3841 ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct
     3901 atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca
     3961 ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg
     4021 catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa
     4081 cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg
     4141 ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga
     4201 cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg
     4261 cgaactactt actctagctt cccggcaaca attaatagac tggatggagg cggataaagt
     4321 tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg
     4381 agccggtgag cgtggaagcc gcggtatcat tgcagcactg gggccagatg gtaagccctc
     4441 ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca
     4501 gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc
     4561 atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat
     4621 cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     4681 agaccccgta gaaaagatca aaggatcttc
//
