LOCUS       sequence-464561-        3845 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV EGFP donor.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3845)
  AUTHORS   Fell CW, Schmitt-Ulms C, Tagliaferri DV, Gootenberg JS, Abudayyeh OO
  TITLE     Precise kilobase-scale genomic insertions in mammalian cells using 
            PASTE.
  JOURNAL   Nat Protoc. 2024 Dec 15. doi: 10.1038/s41596-024-01090-z.
  PUBMED    39676077
REFERENCE   2  (bases 1 to 3845)
  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 Protoc.
            2024 Dec 15. doi: 10.1038/s41596-024-01090-z."
COMMENT     Sequence Label: AAV EGFP donor pDY0404
FEATURES             Location/Qualifiers
     source          1..3845
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     16..35
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(254..358)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(384..839)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(521..542)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     733..752
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   911..1040
                     /label=AAV2 ITR
     protein_bind    1222..1269
                     /label=Bxb1 attP
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="Minimal attP site for the Mycopbacteriophage Bxb1 
                     integrase (Voutev and Mann, 2017)"
     CDS             1288..1953
                     /codon_start=1
                     /product="green fluorescent protein 2 from Pontellina 
                     plumata, also known as ppluGFP2 (Shagin et al., 2004)"
                     /label=CopGFP
                     /translation="MPAMKIECRITGTLNGVEFELVGGGEGTPEQGRMTNKMKSTKGAL
                     TFSPYLLSHVMGYGFYHFGTYPSGYENPFLHAINNGGYTNTRIEKYEDGGVLHVSFSYR
                     YEAGRVIGDFKVVGTGFPEDSVIFTDKIIRSNATVEHLHPMGDNVLVGSFARTFSLRDG
                     GYYSFVVDSHMHFKSAIHPSILQNGGPMFAFRRVEELHSNTELGIVEYQHAFKTPIAFA
                     "
     CDS             1288..1953
                     /codon_start=1
                     /product="green fluorescent protein 2 from Pontellina 
                     plumata, also known as ppluGFP2 (Shagin et al., 2004)"
                     /label=CopGFP
                     /translation="MPAMKIECRITGTLNGVEFELVGGGEGTPEQGRMTNKMKSTKGAL
                     TFSPYLLSHVMGYGFYHFGTYPSGYENPFLHAINNGGYTNTRIEKYEDGGVLHVSFSYR
                     YEAGRVIGDFKVVGTGFPEDSVIFTDKIIRSNATVEHLHPMGDNVLVGSFARTFSLRDG
                     GYYSFVVDSHMHFKSAIHPSILQNGGPMFAFRRVEELHSNTELGIVEYQHAFKTPIAFA
                     "
     primer_bind     complement(1967..1986)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2021..2040
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   2117..2246
                     /label=AAV2 ITR
     rep_origin      complement(2479..3067)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2559..2578)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3238..3845,1..184,185..253))
                     /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"
ORIGIN
        1 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
       61 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
      121 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
      181 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
      241 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
      301 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
      361 catttccccg aaaagtgcca cctaaattgt aagcgttaat attttgttaa aattcgcgtt
      421 aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta
      481 taaatcaaaa gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc
      541 actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg
      601 cccactacgt gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact
      661 aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt
      721 ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc
      781 ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc
      841 ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg cctcttcgct
      901 attacgccag ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg
      961 ggcgaccttt ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa
     1021 ctccatcact aggggttcct tgtagttaat gattaacccg ccatgctact tatctacgta
     1081 gccatgctct aggaagagta ccattgacgt caataatgac gtatgttccc atagtaacgc
     1141 caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg
     1201 cagtacatca agtgtatcag tggtttgtct ggtcaaccac cgcggtctca gtggtgtacg
     1261 gtacaaaccc agctaccggt cgccaccatg cccgccatga agatcgagtg ccgcatcacc
     1321 ggcaccctga acggcgtgga gttcgagctg gtgggcggcg gagagggcac ccccgagcag
     1381 ggccgcatga ccaacaagat gaagagcacc aaaggcgccc tgaccttcag cccctacctg
     1441 ctgagccacg tgatgggcta cggcttctac cacttcggca cctaccccag cggctacgag
     1501 aaccccttcc tgcacgccat caacaacggc ggctacacca acacccgcat cgagaagtac
     1561 gaggacggcg gcgtgctgca cgtgagcttc agctaccgct acgaggccgg ccgcgtgatc
     1621 ggcgacttca aggtggtggg caccggcttc cccgaggaca gcgtgatctt caccgacaag
     1681 atcatccgca gcaacgccac cgtggagcac ctgcacccca tgggcgataa cgtgctggtg
     1741 ggcagcttcg cccgcacctt cagcctgcgc gacggcggct actacagctt cgtggtggac
     1801 agccacatgc acttcaagag cgccatccac cccagcatcc tgcagaacgg gggccccatg
     1861 ttcgccttcc gccgcgtgga ggagctgcac agcaacaccg agctgggcat cgtggagtac
     1921 cagcacgcct tcaagacccc catcgccttc gccagatctc gagctcgatg agtttggaca
     1981 aaccacaact agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg atgctattgc
     2041 tttatttgtg ggcccgggat cttcctagag catggctacg tagataagta gcatggcggg
     2101 ttaatcatta actacaagga acccctagtg atggagttgg ccactccctc tctgcgcgct
     2161 cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg
     2221 gcctcagtga gcgagcgagc gcgcagctgc attaatgaat cggccaacgc gcggggagag
     2281 gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
     2341 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
     2401 caggggataa cgcaggaaag aatgagcaaa aggccagcaa aaggccagga accgtaaaaa
     2461 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     2521 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     2581 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     2641 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
     2701 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     2761 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     2821 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     2881 gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc
     2941 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     3001 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     3061 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
     3121 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
     3181 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
     3241 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
     3301 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
     3361 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca
     3421 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
     3481 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
     3541 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
     3601 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
     3661 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
     3721 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
     3781 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
     3841 ttgcc
//
