LOCUS       sequence-459843-        4579 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 SCP1 promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4579)
  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 4579)
  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-CI-mRuby2-W3SL-T7
FEATURES             Location/Qualifiers
     source          1..4579
                     /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"
     CDS             1257..1967
                     /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(2033..2053)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     promoter        2463..2481
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     repeat_region   2492..2632
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2492..2621
                     /label=AAV2 ITR
     rep_origin      2707..3162
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2794..2813)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3004..3025
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3179..3198)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3298..3320
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3358..3376)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3444..3548
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3549..3617,3618..4409)
                     /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(3767..3786)
                     /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 tgagcaaggg agaggaactc atcaaggaga acatgcgcat gaaggtagtc atggaagggt
     1321 ctgtgaatgg gcatcagttc aagtgcacag gggaaggtga agggaatccc tacatgggta
     1381 ctcagaccat gaggatcaag gttattgaag gtggccctct cccgtttgcc ttcgacattc
     1441 ttgccactag cttcatgtat ggcagtcgga ccttcatcaa gtaccccaaa ggcattccag
     1501 acttcttcaa acagtccttt ccagagggct tcacatggga aagggtgacc cgttacgagg
     1561 atggtggtgt ggtcacagtc atgcaggaca ctagtctgga ggacggatgt ctggtctatc
     1621 acgtgcaagt gagaggggtg aactttccct ccaatgggcc tgtgatgcag aagaaaacca
     1681 aagggtggga gccaaacaca gagatgatgt atcctgcaga tggaggactg agaggctata
     1741 cgcacatggc tctgaaggtt gacggaggag gccacttgtc atgctcattt gtgaccacgt
     1801 acaggagcaa gaaaactgtc ggcaacatca aaatgcctgg catacatgcg gttgatcacc
     1861 gcttggaacg actggaggag tctgacaacg agatgtttgt ggtacaacgg gaacatgcag
     1921 ttgccaagtt tgctggcctt ggcggaggaa tggatgagct gtacaaatga acgcgtcata
     1981 atcaacctct ggattacaaa atttgtgaaa gattgactgg tattcttaac tatgttgctc
     2041 cttttacgct atgtggatac gctgctttaa tgcctttgta tcatgctatt gcttcccgta
     2101 tggctttcat tttctcctcc ttgtataaat cctggttagt tcttgccacg gcggaactca
     2161 tcgccgcctg ccttgcccgc tgctggacag gggctcggct gttgggcact gacaattccg
     2221 tggtgtttat ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatc tagctttatt
     2281 tgtgaaattt gtgatgctat tgctttattt gtaaccatta taagctgcaa taaacaagtt
     2341 aacaacaaca attgcattca ttttatgttt caggttcagg gggagatgtg ggaggttttt
     2401 taaacaaggt accgttaaac gccagcaacg cgggcaagtc ctttttacgg ttcctggccg
     2461 ggtaatacga ctcactatag gcttgggccg caggaacccc tagtgatgga gttggccact
     2521 ccctctctgc gcgctcgctc gctcactgag gccgggcgac caaaggtcgc ccgacgcccg
     2581 ggctttgccc gggcggcctc agtgagcgag cgagcgcgca gctgcctgca ggggcgcctg
     2641 atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatacg tcaaagcaac
     2701 catagtacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
     2761 tgaccgctac acttgccagc gccttagcgc ccgctccttt cgctttcttc ccttcctttc
     2821 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
     2881 gatttagtgc tttacggcac ctcgacccca aaaaacttga tttgggtgat ggttcacgta
     2941 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
     3001 atagtggact cttgttccaa actggaacaa cactcaactc tatctcgggc tattcttttg
     3061 atttataagg gattttgccg atttcggtct attggttaaa aaatgagctg atttaacaaa
     3121 aatttaacgc gaattttaac aaaatattaa cgtttacaat tttatggtgc actctcagta
     3181 caatctgctc tgatgccgca tagttaagcc agccccgaca cccgccaaca cccgctgacg
     3241 cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg
     3301 ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc
     3361 tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct tagacgtcag
     3421 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
     3481 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa
     3541 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt
     3601 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt
     3661 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt
     3721 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg
     3781 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga
     3841 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa
     3901 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga
     3961 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa
     4021 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca
     4081 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta
     4141 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac
     4201 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc
     4261 gtggaagccg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag
     4321 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga
     4381 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt
     4441 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
     4501 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     4561 aaaagatcaa aggatcttc
//
