LOCUS       sequence-414824-        6765 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV packaging vector containing a EGFP-P2A expression cassette, 
            APOBEC1-YTHmut cassette..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6765)
  AUTHORS   Feng S, Tellaetxe-Abete M, Zhang Y, Peng Y, Zhou H, Dong M, Larrea 
            E, Xue L, Zhang L, Koziol MJ
  TITLE     Single-cell discovery of m(6)A RNA modifications in the hippocampus.
  JOURNAL   Genome Res. 2024 Jul 23;34(6):822-836. doi: 10.1101/gr.278424.123.
  PUBMED    39009472
REFERENCE   2  (bases 1 to 6765)
  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"; doi: "10.1101/gr.278424";
            journalName: "Genome Res"; date: "2024-07-23- 23"; volume: "34"; 
            issue: "6"; pages: "822-836"
COMMENT     Sequence Label: AAV-EGFP-APOBEC1-YTHmut
FEATURES             Location/Qualifiers
     source          1..6765
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(182..770)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(262..281)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(941..1732,1733..1801))
                     /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     1564..1583
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1802..1906)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(1932..2387)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2069..2090)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2281..2300
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   2459..2588
                     /label=AAV2 ITR
     promoter        2899..3176
                     /label=chicken beta-actin promoter
     primer_bind     3455..3477
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     3539..3558
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      3597..3606
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(3603..3605,3606..3608,3609..4319)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(3603..3605,3606..3608,3609..4319)
                     /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(3648..3669)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3909..3928)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4256..4277
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             4320..4376
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             4377..5063
                     /codon_start=1
                     /product="cytidine deaminase (C to U editing enzyme) from 
                     rat"
                     /label=APOBEC-1
                     /note="can use ssDNA as a substrate (Komor et al., 2016)"
                     /translation="MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWG
                     GRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFL
                     SRYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSN
                     EAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHI
                     LWATGLK"
     CDS             5625..5651
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /note="This is a variant of the standard HA tag, which has 
                     sequence YPYDVPDYA. Some anti-HA antibodies may not 
                     recognize this variant."
                     /translation="YPYDVPDYA"
     CDS             5625..5651
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     misc_feature    5667..6255
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5720..5740)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(6138..6149)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    6298..6419
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6309..6328)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     6363..6382
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   6581..6710
                     /label=AAV2 ITR
ORIGIN
        1 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
       61 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
      121 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
      181 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
      241 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
      301 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
      361 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
      421 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
      481 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
      541 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
      601 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
      661 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
      721 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
      781 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
      841 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
      901 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
      961 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     1021 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     1081 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     1141 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     1201 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     1261 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     1321 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     1381 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     1441 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     1501 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     1561 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     1621 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     1681 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     1741 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     1801 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     1861 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     1921 aagtgccacc taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta
     1981 aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga
     2041 atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa
     2101 cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga
     2161 accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc
     2221 taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga
     2281 agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg
     2341 cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtccc attcgccatt
     2401 caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat tacgccagct
     2461 gcgcgctcgc tcgctcactg aggccgcccg ggcaaagccc gggcgtcggg cgacctttgg
     2521 tcgcccggcc tcagtgagcg agcgagcgcg cagagaggga gtggccaact ccatcactag
     2581 gggttccttg tagttaatga ttaacccgcc atgctactta tctacgtagc catgctctag
     2641 gaagagtacc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt
     2701 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
     2761 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
     2821 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag
     2881 tcatcgctat taccatggtc gaggtgagcc ccacgttctg cttcactctc cccatctccc
     2941 ccccctcccc acccccaatt ttgtatttat ttatttttta attattttgt gcagcgatgg
     3001 gggcgggggg gggggggggg cgcgcgccag gcggggcggg gcggggcgag gggcggggcg
     3061 gggcgaggcg gagaggtgcg gcggcagcca atcagagcgg cgcgctccga aagtttcctt
     3121 ttatggcgag gcggcggcgg cggcggccct ataaaaagcg aagcgcgcgg cgggcgggag
     3181 tcgctgcgcg ctgccttcgc cccgtgcccc gctccgccgc cgcctcgcgc cgcccgcccc
     3241 ggctctgact gaccgcgtta ctcccacagg tgagcgggcg ggacggccct tctcctccgg
     3301 gctgtaatta gcgcttggtt taatgacggc ttgtttcttt tctgtggctg cgtgaaagcc
     3361 ttgaggggct ccgggagggc cctttgtgcg gggggagcgg ctcggggctg tccgcggggg
     3421 gacggctgcc ttcggggggg acggggcagg gcggggttcg gcttctggcg tgtgaccggc
     3481 ggctctagag cctctgctaa ccatgttcat gccttcttct ttttcctaca gctcctgggc
     3541 aacgtgctgg ttattgtgct gtctcatcat tttggcaaag aattggatcc ggtaccgcca
     3601 ccatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg
     3661 acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct
     3721 acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca
     3781 ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc gaccacatga
     3841 agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct
     3901 tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc
     3961 tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc
     4021 acaagctgga gtacaactac aacagccaca acgtctatat catggccgac aagcagaaga
     4081 acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc gtgcagctcg
     4141 ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc
     4201 actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc gatcacatgg
     4261 tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaagg
     4321 ccactaactt ctccctgttg aaacaagcag gggatgtcga agagaatccc gggccaatga
     4381 gctcagagac tggcccagtg gctgtggacc ccacattgag acggcggatc gagccccatg
     4441 agtttgaggt attcttcgat ccgagagagc tccgcaagga gacctgcctg ctttacgaaa
     4501 ttaattgggg gggccggcac tccatttggc gacatacatc acagaacact aacaagcacg
     4561 tcgaagtcaa cttcatcgag aagttcacga cagaaagata tttctgtccg aacacaaggt
     4621 gcagcattac ctggtttctc agctggagcc catgcggcga atgtagtagg gccatcactg
     4681 aattcctgtc aaggtatccc cacgtcactc tgtttattta catcgcaagg ctgtaccacc
     4741 acgctgaccc ccgcaatcga caaggcctgc gggatttgat ctcttcaggt gtgactatcc
     4801 aaattatgac tgagcaggag tcaggatact gctggagaaa ctttgtgaat tatagcccga
     4861 gtaatgaagc ccactggcct aggtatcccc atctgtgggt acgactgtac gttcttgaac
     4921 tgtactgcat catactgggc ctgcctcctt gtctcaacat tctgagaagg aagcagccac
     4981 agctgacatt ctttaccatc gctcttcagt cttgtcatta ccagcgactg cccccacaca
     5041 ttctctgggc caccgggttg aaaagcggca gcgagactcc cgggacctca gagtccgcca
     5101 caccagaagg ccgggttttc atcattaaga gctactctga ggacgatatt caccgttcca
     5161 ttaagtataa tatttggtgc agcacagagc atggtaacaa gagactggat gctgcttatc
     5221 gttccatgaa cgggaaaggc cccgtttact tacttttcag tgtcaacggc agtggacact
     5281 tctgtggcgt ggcagaaatg aaatctgctg tggactacaa cacatgtgca ggtgtgtggt
     5341 cccaggacaa atggaagggt cgttttgatg tcaggtggat ttttgtgaag gacgttccca
     5401 atagccaact gcgacacatt cgcctagaga acaacgagaa taaaccagtg accaactcta
     5461 gggacactca ggaagtgcct ctggaaaagg ctaagcaggt gttgaaaatt atagccagct
     5521 acaagcacac cacttccatt tttgatgact tctcacacta tgagaaacgc caagaggaag
     5581 aagaaagtgt taaaaaggaa cgtcaaggtc gtgggaaact cgagtacccc tacgacgtgc
     5641 ccgactacgc ctaaaagctt atcgataatc aacctctgga ttacaaaatt tgtgaaagat
     5701 tgactggtat tcttaactat gttgctcctt ttacgctatg tggatacgct gctttaatgc
     5761 ctttgtatca tgctattgct tcccgtatgg ctttcatttt ctcctccttg tataaatcct
     5821 ggttgctgtc tctttatgag gagttgtggc ccgttgtcag gcaacgtggc gtggtgtgca
     5881 ctgtgtttgc tgacgcaacc cccactggtt ggggcattgc caccacctgt cagctccttt
     5941 ccgggacttt cgctttcccc ctccctattg ccacggcgga actcatcgcc gcctgccttg
     6001 cccgctgctg gacaggggct cggctgttgg gcactgacaa ttccgtggtg ttgtcgggga
     6061 aatcatcgtc ctttccttgg ctgctcgcct gtgttgccac ctggattctg cgcgggacgt
     6121 ccttctgcta cgtcccttcg gccctcaatc cagcggacct tccttcccgc ggcctgctgc
     6181 cggctctgcg gcctcttccg cgtcttcgcc ttcgccctca gacgagtcgg atctcccttt
     6241 gggccgcctc cccgcatcga taccgtcgac ccgggcggcc gcttcgagca gacatgataa
     6301 gatacattga tgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt
     6361 gtgaaatttg tgatgctatt gctttatttg taaccattat aagctgcaat aaacaagtta
     6421 acaacaacaa ttgcattcat tttatgtttc aggttcaggg ggagatgtgg gaggtttttt
     6481 aaagcaagta aaacctctac aaatgtggta aaatcgataa ggatcttcct agagcatggc
     6541 tacgtagata agtagcatgg cgggttaatc attaactaca aggaacccct agtgatggag
     6601 ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc
     6661 cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag ctgcattaat
     6721 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgct
//
