LOCUS       sequence-459729-        5165 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 with mDLX 
            enhancer..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5165)
  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 5165)
  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)-mDLX-minBG-CI-mRuby2-W3...
FEATURES             Location/Qualifiers
     source          1..5165
                     /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          1698..1830
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     regulatory      1837..1846
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1843..2553
                     /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(2619..2639)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     promoter        3049..3067
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     repeat_region   3078..3218
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3078..3207
                     /label=AAV2 ITR
     rep_origin      3293..3748
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3380..3399)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3590..3611
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3765..3784)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3884..3906
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3944..3962)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4030..4134
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4135..4203,4204..4995)
                     /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(4353..4372)
                     /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 cctcgagtat acactcacag
     1021 tggtttggca tatatttggt gaaatttttt aaggaaaaat tagtgttggt ttcgatatat
     1081 ggtagctttt tctctaacat aatttgaata attcagcaaa gccctactac cagctgtact
     1141 tctgcagcct cttccattct ttccagcatt ataattttgg ttaattttca attttaggtc
     1201 ctacgtctct gcaatttgtg tatgaataac agaataattt ccctcttttg tttcgccttt
     1261 cctgttcctg aatctaaata aagatggctt tttagtatta aaagtggaag aaaattacag
     1321 gtaattatct ttgacggtaa aaacgctgta atcagcgggc tacatgaaaa attactctaa
     1381 ttatggctgc atttaagaga atggaaaaaa accttcttgt ggataaaaac cttaaattgt
     1441 ccccaatgtc tgcttcaaat tggatggcac tgcagctgga ggctttgttc agaattgatc
     1501 ctggggagct acgaacccaa agtttcacag taggaagctt gatatcgaat tcctgcagcc
     1561 cgggctgggc ataaaagtca gggcagagcc atctattgct tacatttgct tctagcctgc
     1621 aggtcgagga gcgcagcctt ccagaagcag agcgcggcgc cttaagctgc agaagttggt
     1681 cgtgaggcac tgggcaggta agtatcaagg ttacaagaca ggtttaagga gaccaataga
     1741 aactgggctt gtcgagacag agaagactct tgcgtttctg ataggcacct attggtctta
     1801 ctgacatcca ctttgccttt ctctccacag accggtgcca ccatggtgag caagggagag
     1861 gaactcatca aggagaacat gcgcatgaag gtagtcatgg aagggtctgt gaatgggcat
     1921 cagttcaagt gcacagggga aggtgaaggg aatccctaca tgggtactca gaccatgagg
     1981 atcaaggtta ttgaaggtgg ccctctcccg tttgccttcg acattcttgc cactagcttc
     2041 atgtatggca gtcggacctt catcaagtac cccaaaggca ttccagactt cttcaaacag
     2101 tcctttccag agggcttcac atgggaaagg gtgacccgtt acgaggatgg tggtgtggtc
     2161 acagtcatgc aggacactag tctggaggac ggatgtctgg tctatcacgt gcaagtgaga
     2221 ggggtgaact ttccctccaa tgggcctgtg atgcagaaga aaaccaaagg gtgggagcca
     2281 aacacagaga tgatgtatcc tgcagatgga ggactgagag gctatacgca catggctctg
     2341 aaggttgacg gaggaggcca cttgtcatgc tcatttgtga ccacgtacag gagcaagaaa
     2401 actgtcggca acatcaaaat gcctggcata catgcggttg atcaccgctt ggaacgactg
     2461 gaggagtctg acaacgagat gtttgtggta caacgggaac atgcagttgc caagtttgct
     2521 ggccttggcg gaggaatgga tgagctgtac aaatgaacgc gtcataatca acctctggat
     2581 tacaaaattt gtgaaagatt gactggtatt cttaactatg ttgctccttt tacgctatgt
     2641 ggatacgctg ctttaatgcc tttgtatcat gctattgctt cccgtatggc tttcattttc
     2701 tcctccttgt ataaatcctg gttagttctt gccacggcgg aactcatcgc cgcctgcctt
     2761 gcccgctgct ggacaggggc tcggctgttg ggcactgaca attccgtggt gtttatttgt
     2821 gaaatttgtg atgctattgc tttatttgta accatctagc tttatttgtg aaatttgtga
     2881 tgctattgct ttatttgtaa ccattataag ctgcaataaa caagttaaca acaacaattg
     2941 cattcatttt atgtttcagg ttcaggggga gatgtgggag gttttttaaa caaggtaccg
     3001 ttaaacgcca gcaacgcggg caagtccttt ttacggttcc tggccgggta atacgactca
     3061 ctataggctt gggccgcagg aacccctagt gatggagttg gccactccct ctctgcgcgc
     3121 tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc
     3181 ggcctcagtg agcgagcgag cgcgcagctg cctgcagggg cgcctgatgc ggtattttct
     3241 ccttacgcat ctgtgcggta tttcacaccg catacgtcaa agcaaccata gtacgcgccc
     3301 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt
     3361 gccagcgcct tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc
     3421 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta
     3481 cggcacctcg accccaaaaa acttgatttg ggtgatggtt cacgtagtgg gccatcgccc
     3541 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg
     3601 ttccaaactg gaacaacact caactctatc tcgggctatt cttttgattt ataagggatt
     3661 ttgccgattt cggtctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat
     3721 tttaacaaaa tattaacgtt tacaatttta tggtgcactc tcagtacaat ctgctctgat
     3781 gccgcatagt taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct
     3841 tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt
     3901 cagaggtttt caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta
     3961 tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg
     4021 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     4081 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
     4141 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
     4201 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
     4261 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
     4321 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt
     4381 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
     4441 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
     4501 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
     4561 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
     4621 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta
     4681 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
     4741 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
     4801 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg aagccgcggt
     4861 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
     4921 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
     4981 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
     5041 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     5101 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     5161 tcttc
//
