LOCUS       sequence-459765-        5233 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Self complementary AAV genome with components for tracking 
            self-complementary AAV genomes via AAV-Zombie. Also expresses EGFP 
            from CAG promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5233)
  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 5233)
  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.CAG-EGFP-SV40pA-T7-T3-BC(p1-14)
FEATURES             Location/Qualifiers
     source          1..5233
                     /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        1437..1715
                     /label=chicken beta-actin promoter
     primer_bind     1994..2016
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2078..2097
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      2137..2146
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2143..2145,2146..2148,2149..2862)
                     /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(2143..2145,2146..2148,2149..2859)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2188..2209)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2449..2468)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2796..2817
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     2906..2925
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        complement(3411..3429)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3439..3458)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3440..3458)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3608..3625)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3608..3624)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3617..3639)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(3833..3852)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3952..3974
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4012..4030)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4098..4202
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4203..4271,4272..5063)
                     /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(4421..4440)
                     /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 cgggcgacct ttggtcgccc ggcctcagtg agcgagcgag cgcgcagaga gggagtggcc
     1141 aactccatca ctaggggttc ctggaggggt ggagtcgtga cctaggccat tgacgtcaat
     1201 aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga
     1261 gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc
     1321 ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt
     1381 atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtcga
     1441 ggtgagcccc acgttctgct tcactctccc catctccccc ccctccccac ccccaatttt
     1501 gtatttattt attttttaat tattttgtgc agcgatgggg gcgggggggg gggggggggc
     1561 gcgcgccagg cggggcgggg cggggcgagg ggcggggcgg ggcgaggcgg agaggtgcgg
     1621 cggcagccaa tcagagcggc gcgctccgaa agtttccttt tatggcgagg cggcggcggc
     1681 ggcggcccta taaaaagcga agcgcgcggc gggcgggagt cgctgcgcgc tgccttcgcc
     1741 ccgtgccccg ctccgccgcc gcctcgcgcc gcccgccccg gctctgactg accgcgttac
     1801 tcccacaggt gagcgggcgg gacggccctt ctcctccggg ctgtaattag cgcttggttt
     1861 aatgacggct tgtttctttt ctgtggctgc gtgaaagcct tgaggggctc cgggagggcc
     1921 ctttgtgcgg ggggagcggc tcggggctgt ccgcgggggg acggctgcct tcggggggga
     1981 cggggcaggg cggggttcgg cttctggcgt gtgaccggcg gctctagagc ctctgctaac
     2041 catgttcatg ccttcttctt tttcctacag ctcctgggca acgtgctggt tattgtgctg
     2101 tctcatcatt ttggcaaaga attggatcca ccggtcgcca ccatggtgag caagggcgag
     2161 gagctgttca ccggggtggt gcccatcctg gtcgagctgg acggcgacgt aaacggccac
     2221 aagttcagcg tgtccggcga gggcgagggc gatgccacct acggcaagct gaccctgaag
     2281 ttcatctgca ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac caccctgacc
     2341 tacggcgtgc agtgcttcag ccgctacccc gaccacatga agcagcacga cttcttcaag
     2401 tccgccatgc ccgaaggcta cgtccaggag cgcaccatct tcttcaagga cgacggcaac
     2461 tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg catcgagctg
     2521 aagggcatcg acttcaagga ggacggcaac atcctggggc acaagctgga gtacaactac
     2581 aacagccaca acgtctatat catggccgac aagcagaaga acggcatcaa ggtgaacttc
     2641 aagatccgcc acaacatcga ggacggcagc gtgcagctcg ccgaccacta ccagcagaac
     2701 acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag cacccagtcc
     2761 gccctgagca aagaccccaa cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc
     2821 gccgccggga tcactctcgg catggacgag ctgtacaagt aaagcggccg ctaggcctca
     2881 cctgcgatct cgatgcttta tttgtgaaat ttgtgatgct attgctttat ttgtaaccat
     2941 tataagctgc aataaacaag ttaacaacaa caattgcatt cattttatgt ttcaggttca
     3001 gggggaggtg tgggaggttt tttaaacgac agcttgtctc tccagatgct cttgggccat
     3061 cttccacatc gtccgtagca gccttggcaa tttgccatca ctggcaaata cacataaatc
     3121 caatgaatac ggttaccacc atcacattac catgcaggta cacagcaaga attgacgttg
     3181 gcatatcaca tggtgtaata accccacttg tgaaacaacc cagaataagg tacaaggcgg
     3241 aaatgtcgtc attctaaaat aaaaggcatg gccaggaatt tgtctaatac cgggaactta
     3301 aattcagctt gaacaccagt cgcaaaaaat tcaaagaaag tgattcaggt tcgggttcgt
     3361 ggattggaac agcttctttt gtttcagtga tgagagaatc ctcctgtcac cctttagtga
     3421 gggttaattc tcgagtctcc ctatagtgag tcgtattaat tcctccactc cctctctgcg
     3481 cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg
     3541 ggcggcctca gtgagcgagc gagcgcgcag agagggacag atccgggccc gcatgcgtcg
     3601 acaattcact ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaac
     3661 ttaatcgcct tgcagcacat ccccctttcg ccagctggcg taatagcgaa gaggcccgca
     3721 ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga atggcgcctg atgcggtatt
     3781 ttctccttac gcatctgtgc ggtatttcac accgcatatg gtgcactctc agtacaatct
     3841 gctctgatgc cgcatagtta agccagcccc gacacccgcc aacacccgct gacgcgccct
     3901 gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct
     3961 gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga
     4021 tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca
     4081 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
     4141 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga
     4201 gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc
     4261 ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg
     4321 cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc
     4381 ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat
     4441 cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact
     4501 tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat
     4561 tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga
     4621 tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc
     4681 ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga
     4741 tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag
     4801 cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc
     4861 gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt
     4921 ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct
     4981 acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg
     5041 cctcactgat taagcattgg taactgtcag accaagttta ctcatatata ctttagattg
     5101 atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca
     5161 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     5221 tcaaaggatc ttc
//
