LOCUS       sequence-459910-        5146 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Self complementary AAV genome with concatemerization-dependent 
            barcodes, 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 5146)
  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 5146)
  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: pscAAV.BC(p1-6)-CAG-EGFP-SV40pA-BC(p1-6)
FEATURES             Location/Qualifiers
     source          1..5146
                     /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"
     protein_bind    877..898
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(913..918,919..936,937..943)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    951..967
                     /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     956..978
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     975..991
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     975..991
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        1624..1901
                     /label=chicken beta-actin promoter
     primer_bind     2180..2202
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2264..2283
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      2323..2332
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2329..2331,2332..2334,2335..3048)
                     /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(2329..2331,2332..2334,2335..3045)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2374..2395)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2635..2654)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2982..3003
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     3092..3111
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(3521..3538)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3521..3537)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3530..3552)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(3746..3765)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3865..3887
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3925..3943)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4011..4115
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4116..4184,4185..4976)
                     /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(4334..4353)
                     /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 tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat
      781 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt
      841 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta
      901 ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg
      961 ataacaattt cacacaggaa acagctatga ccatgattac gccaagctct cgagatctag
     1021 aaagcttccc ggggggatct gggccactcc ctctctgcgc gctcgctcgc tcactgaggc
     1081 cgggcgacca aaggtcgccc ggcctcagtg agcgagcgag cgcgcagaga gggagtggcc
     1141 aactccatca ctaggggttc ctggaggggt ggagtcgtga ccttgacagg aggattctct
     1201 catcactgaa acaaaagaag ctgttccaat ccacgaaccc gaacctgaat cactttcttt
     1261 gaattttttg cgactggtgt tcaagctgaa tttaagttcc cggtattaga caaattcctg
     1321 gccatgcctt ttattttaga atgacgacat ttccgccttg taccttattc aggccattga
     1381 cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat
     1441 gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa
     1501 gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca
     1561 tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc gctattacca
     1621 tggtcgaggt gagccccacg ttctgcttca ctctccccat ctcccccccc tccccacccc
     1681 caattttgta tttatttatt ttttaattat tttgtgcagc gatgggggcg gggggggggg
     1741 gggggcgcgc gccaggcggg gcggggcggg gcgaggggcg gggcggggcg aggcggagag
     1801 gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt tccttttatg gcgaggcggc
     1861 ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc gggagtcgct gcgcgctgcc
     1921 ttcgccccgt gccccgctcc gccgccgcct cgcgccgccc gccccggctc tgactgaccg
     1981 cgttactccc acaggtgagc gggcgggacg gcccttctcc tccgggctgt aattagcgct
     2041 tggtttaatg acggcttgtt tcttttctgt ggctgcgtga aagccttgag gggctccggg
     2101 agggcccttt gtgcgggggg agcggctcgg ggctgtccgc ggggggacgg ctgccttcgg
     2161 gggggacggg gcagggcggg gttcggcttc tggcgtgtga ccggcggctc tagagcctct
     2221 gctaaccatg ttcatgcctt cttctttttc ctacagctcc tgggcaacgt gctggttatt
     2281 gtgctgtctc atcattttgg caaagaattg gatccaccgg tcgccaccat ggtgagcaag
     2341 ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac
     2401 ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc
     2461 ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc
     2521 ctgacctacg gcgtgcagtg cttcagccgc taccccgacc acatgaagca gcacgacttc
     2581 ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac
     2641 ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc
     2701 gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac
     2761 aactacaaca gccacaacgt ctatatcatg gccgacaagc agaagaacgg catcaaggtg
     2821 aacttcaaga tccgccacaa catcgaggac ggcagcgtgc agctcgccga ccactaccag
     2881 cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagcacc
     2941 cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc
     3001 gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaagtaaag cggccgctag
     3061 gcctcacctg cgatctcgat gctttatttg tgaaatttgt gatgctattg ctttatttgt
     3121 aaccattata agctgcaata aacaagttaa caacaacaat tgcattcatt ttatgtttca
     3181 ggttcagggg gaggtgtggg aggtttttta aacattctaa aataaaaggc atggccagga
     3241 atttgtctaa taccgggaac ttaaattcag cttgaacacc agtcgcaaaa aattcaaaga
     3301 aagtgattca ggttcgggtt cgtggattgg aacagcttct tttgtttcag tgatgagaga
     3361 atcctcctgt cactagtcca ctccctctct gcgcgctcgc tcgctcactg aggccgggcg
     3421 accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg
     3481 cagagaggga cagatccggg cccgcatgcg tcgacaattc actggccgtc gttttacaac
     3541 gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt
     3601 tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca
     3661 gcctgaatgg cgaatggcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt
     3721 cacaccgcat atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagc
     3781 cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg
     3841 cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat
     3901 caccgaaacg cgcgagacga aagggcctcg tgatacgcct atttttatag gttaatgtca
     3961 tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc
     4021 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct
     4081 gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg
     4141 cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg
     4201 tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc
     4261 tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca
     4321 cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac
     4381 tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa
     4441 agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg
     4501 ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt
     4561 ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg
     4621 aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc
     4681 gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga
     4741 tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta
     4801 ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc
     4861 cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg
     4921 atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt
     4981 cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa
     5041 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt
     5101 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttc
//
