LOCUS       sequence-461305-        5419 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Single stranded AAV genome with components for tracking AAV genome 
            via AAV-Zombie. Also expresses a nuclear localized EGFP from CAG 
            promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5419)
  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 5419)
  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.CAG-NLS-EGFP-WPRE-SV40pA-T7-T3-BC(p1-14)
FEATURES             Location/Qualifiers
     source          1..5419
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     435..454
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        838..1141
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1143..1420
                     /label=chicken beta-actin promoter
     primer_bind     1750..1772
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1834..1853
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      1886..1895
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1901..1921
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(1922..1924,1925..1927,1928..2638)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1922..1924,1925..1927,1928..2638)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1967..1988)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2228..2247)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2575..2596
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2645..2665
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     misc_feature    2676..3264
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2729..2749)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3147..3158)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3288..3409
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3299..3318)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3353..3372
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        complement(3893..3911)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3921..3940)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3922..3940)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     repeat_region   3949..4078
                     /label=AAV2 ITR
     primer_bind     complement(4143..4161)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4229..4333
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4334..4402,4403..5194)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(4552..4571)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(5365..5419,1..534)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
       61 ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac
      121 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
      181 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
      241 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
      301 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
      361 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
      421 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
      481 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
      541 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgtaataa
      601 acacacacac accaacaacc gtggttggtt gttgtgttgg tttattctcg agttggccac
      661 tccctctctg cgcgctcgct cgctcactga ggccgcccgg gcaaagcccg ggcgtcctca
      721 gtgagcgagc gagcgcgcag agagggagtg gccaactcca tcactagggg ttcctactag
      781 ttattaatag taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcgt
      841 tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac
      901 gtcaataatg acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg
      961 ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag
     1021 tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat
     1081 gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat
     1141 ggtcgaggtg agccccacgt tctgcttcac tctccccatc tcccccccct ccccaccccc
     1201 aattttgtat ttatttattt tttaattatt ttgtgcagcg atgggggcgg gggggggggg
     1261 ggggcgcgcg ccaggcgggg cggggcgggg cgaggggcgg ggcggggcga ggcggagagg
     1321 tgcggcggca gccaatcaga gcggcgcgct ccgaaagttt ccttttatgg cgaggcggcg
     1381 gcggcggcgg ccctataaaa agcgaagcgc gcggcgggcg ggagtcgctg cgcgctgcct
     1441 tcgccccgtg ccccgctccg ccgccgcctc gcgccgcccg ccccggctct gactgaccgc
     1501 gttactccca caggtgagcg ggcgggacgg cccttctcct ccgggctgta attagcgctt
     1561 ggtttaatga cggcttgttt cttttctgtg gctgcgtgaa agccttgagg ggctccggga
     1621 gcgccggcag gaaggaaatg ggcggggagg gccttcgtgc gtcgccgcgc cgccgtcccc
     1681 ttctccctct ccagcctcgg ggctgtccgc ggggggacgg ctgccttcgg gggggacggg
     1741 gcagggcggg gttcggcttc tggcgtgtga ccggcggctc tagagcctct gctaaccatg
     1801 ttcatgcctt cttctttttc ctacagctcc tgggcaacgt gctggttatt gtgctgtctc
     1861 atcattttgg caaagaattg cggccgccac catgggaagc ccaaagaaga agcgtaaggt
     1921 aatggtgagc aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga
     1981 cggcgacgta aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta
     2041 cggcaagctg accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac
     2101 cctcgtgacc accctgacct acggcgtgca gtgcttcagc cgctaccccg accacatgaa
     2161 gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt
     2221 cttcaaggac gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct
     2281 ggtgaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca
     2341 caagctggag tacaactaca acagccacaa cgtctatatc atggccgaca agcagaagaa
     2401 cggcatcaag gtgaacttca agatccgcca caacatcgag gacggcagcg tgcagctcgc
     2461 cgaccactac cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca
     2521 ctacctgagc acccagtccg ccctgagcaa agaccccaac gagaagcgcg atcacatggt
     2581 cctgctggag ttcgtgaccg ccgccgggat cactctcggc atggacgagc tgtacaaggg
     2641 aagccccaag aaaaagcgga aggtgtaagg atcctaatca acctctggat tacaaaattt
     2701 gtgaaagatt gactggtatt cttaactatg ttgctccttt tacgctatgt ggatacgctg
     2761 ctttaatgcc tttgtatcat gctattgctt cccgtatggc tttcattttc tcctccttgt
     2821 ataaatcctg gttgctgtct ctttatgagg agttgtggcc cgttgtcagg caacgtggcg
     2881 tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg gggcattgcc accacctgtc
     2941 agctcctttc cgggactttc gctttccccc tccctattgc cacggcggaa ctcatcgccg
     3001 cctgccttgc ccgctgctgg acaggggcaa ggctgttggg cactgacaat tccgtggtgt
     3061 tgtcggggaa atcatcgtcc tttccttggc tgctcgcctg tgttgccacc tggattctgc
     3121 gcgggacgtc cttctgctac gtcccttcgg ccctcaatcc agcggacctt ccttcccgcg
     3181 gcctgctgcc ggctctgcgg cctcttccgc gtcttcgcct tcgccctcag acgagtcgga
     3241 tctccctttg ggccgcctcc ccgcctggta ccttcgagca gacatgataa gatacattga
     3301 tgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg
     3361 tgatgctatt gctttatttg taaccattat aagctgcaat aaacaagtta acaacaacaa
     3421 ttgcattcat tttatgtttc aggttcaggg ggagatgtgg gaggtttttt aaagcaagta
     3481 aaacctctac aaatgtggta aaatcaagcg acagcttgtc tctccagatg ctcttgggcc
     3541 atcttccaca tcgtccgtag cagccttggc aatttgccat cactggcaaa tacacataaa
     3601 tccaatgaat acggttacca ccatcacatt accatgcagg tacacagcaa gaattgacgt
     3661 tggcatatca catggtgtaa taaccccact tgtgaaacaa cccagaataa ggtacaaggc
     3721 ggaaatgtcg tcattctaaa ataaaaggca tggccaggaa tttgtctaat accgggaact
     3781 taaattcagc ttgaacacca gtcgcaaaaa attcaaagaa agtgattcag gttcgggttc
     3841 gtggattgga acagcttctt ttgtttcagt gatgagagaa tcctcctgtc accctttagt
     3901 gagggttaat tctcgagtct ccctatagtg agtcgtatta attcccttag gaacccctag
     3961 tgatggagtt ggccactccc tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa
     4021 aggtcgcccg acgcccgggc tttgcccggg cggcctcagt gagcgagcga gcgcgcagag
     4081 agggagtggc caagctagcg ggcgattaag gaaagggcta gatcattctt gaagacgaaa
     4141 gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagac
     4201 gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat
     4261 acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg
     4321 aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc
     4381 attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga
     4441 tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga
     4501 gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg
     4561 cgcggtatta tcccgtgttg acgccgggca agagcaactc ggtcgccgca tacactattc
     4621 tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac
     4681 agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact
     4741 tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca
     4801 tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg
     4861 tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact
     4921 acttactcta gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg
     4981 accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg
     5041 tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat
     5101 cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc
     5161 tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat
     5221 actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt
     5281 tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
     5341 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     5401 gcaaacaaaa aaaccaccg
//
