LOCUS       sequence-459853-        5075 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Self complementary AAV genome with barcodeless T7 RNA polymerase 
            promoter and SP6-driven barcode, for tracking AAV concatemers via 
            SpECTr. Also expresses EGFP from CAG promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5075)
  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 5075)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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: pscAAV.T7-SP6-BC(p11-14)-CAG-EGFP-SV40pA-T7
FEATURES             Location/Qualifiers
     source          1..5075
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(164..182)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        233..251
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     233..250
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     promoter        619..894
                     /label=chicken beta-actin promoter
     primer_bind     1173..1195
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1257..1276
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      1316..1325
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1322..1324,1325..1327,1328..2041)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1322..1324,1325..1327,1328..2038)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1367..1388)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1628..1647)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1975..1996
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     2085..2104
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        2263..2281
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2430..2447)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2430..2446)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2439..2461)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2655..2674)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2774..2796
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2834..2852)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2920..3024
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3025..3093,3094..3885)
                     /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(3243..3262)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4056..4644
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4545..4564
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    4932..4953
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4968..4973,4974..4991,4992..4998)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5006..5022
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     5011..5033
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5030..5046
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5030..5046
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 aaagcttccc ggggggatct gggccactcc ctctctgcgc gctcgctcgc tcactgaggc
       61 cgggcgacca aaggtcgccc ggcctcagtg agcgagcgag cgcgcagaga gggagtggcc
      121 aactccatca ctaggggttc ctggaggggt ggagtcgtga cctcctatag tgagtcgtat
      181 tacccggcca ggaaccgtaa aaaggacttg cccgcgttgc tggcgtttaa cgatttaggt
      241 gacactatag aatacacggg tggtaaccgt attcattgga tttatgtgta tttgccagtg
      301 atggcaaatt gccaaggctg ctacggacga tgtggaagat ggcccaagag catctggaga
      361 gacaaaggcc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt
      421 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
      481 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
      541 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag
      601 tcatcgctat taccatggtc gaggtgagcc ccacgttctg cttcactctc cccatctccc
      661 ccccctcccc acccccaatt ttgtatttat ttatttttta attattttgt gcagcgatgg
      721 gggcgggggg ggggggggcg cgcgcgaggc ggggcggggc ggggcgaggg gcggggcggg
      781 gcgaggcgga gaggtgcggc ggcagccaat cagagcggcg cgctccgaaa gtttcctttt
      841 atggcgaggc ggcggcggcg gcggccctat aaaaagcgaa gcgcgcggcg ggcgggagtc
      901 gctgcgcgct gccttcgccc cgtgccccgc tccgccgccg cctcgcgccg cccgccccgg
      961 ctctgactga ccgcgttact cccacaggtg agcgggcggg acggcccttc tcctccgggc
     1021 tgtaattagc gcttggttta atgacggctt gtttcttttc tgtggctgcg tgaaagcctt
     1081 gaggggctcc gggagggccc tttgtgcggg gggagcggct cggggctgtc cgcgggggga
     1141 cggctgcctt cgggggggac ggggcagggc ggggttcggc ttctggcgtg tgaccggcgg
     1201 ctctagagcc tctgctaacc atgttcatgc cttcttcttt ttcctacagc tcctgggcaa
     1261 cgtgctggtt attgtgctgt ctcatcattt tggcaaagaa ttggatccac cggtcgccac
     1321 catggtgagc aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga
     1381 cggcgacgta aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta
     1441 cggcaagctg accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac
     1501 cctcgtgacc accctgacct acggcgtgca gtgcttcagc cgctaccccg accacatgaa
     1561 gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt
     1621 cttcaaggac gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct
     1681 ggtgaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca
     1741 caagctggag tacaactaca acagccacaa cgtctatatc atggccgaca agcagaagaa
     1801 cggcatcaag gtgaacttca agatccgcca caacatcgag gacggcagcg tgcagctcgc
     1861 cgaccactac cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca
     1921 ctacctgagc acccagtccg ccctgagcaa agaccccaac gagaagcgcg atcacatggt
     1981 cctgctggag ttcgtgaccg ccgccgggat cactctcggc atggacgagc tgtacaagta
     2041 aagcggccgc taggcctcac ctgcgatctc gatgctttat ttgtgaaatt tgtgatgcta
     2101 ttgctttatt tgtaaccatt ataagctgca ataaacaagt taacaacaac aattgcattc
     2161 attttatgtt tcaggttcag ggggaggtgt gggaggtttt ttaaatcact gacgttaaac
     2221 gccagcaacg cgggcaagtc ctttttacgg ttcctggccg ggtaatacga ctcactatag
     2281 gctagtccac tccctctctg cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg
     2341 cccgacgccc gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc agagagggac
     2401 agatccgggc ccgcatgcgt cgacaattca ctggccgtcg ttttacaacg tcgtgactgg
     2461 gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg
     2521 cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc
     2581 gaatggcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata
     2641 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg
     2701 ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa
     2761 gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc
     2821 gcgagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg
     2881 gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta
     2941 tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt
     3001 caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc
     3061 ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa
     3121 gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt
     3181 aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt
     3241 ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc
     3301 atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg
     3361 gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg
     3421 gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac
     3481 atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca
     3541 aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta
     3601 actggcgaac tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat
     3661 aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa
     3721 tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag
     3781 ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat
     3841 agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt
     3901 tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg
     3961 aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga
     4021 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     4081 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     4141 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     4201 gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     4261 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     4321 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     4381 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     4441 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     4501 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     4561 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     4621 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     4681 ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac
     4741 cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc
     4801 gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt
     4861 tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag
     4921 cgcaacgcaa ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg
     4981 cttccggctc gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc
     5041 tatgaccatg attacgccaa gctctcgaga tctag
//
