LOCUS       sequence-486526-        4488 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Used to construct AAV-based targeting vectors that carry an 
            IVS-2Aneo cassette for promoter-trapping.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4488)
  AUTHORS   Karnan S, Ota A, Konishi Y, Wahiduzzaman M, Hosokawa Y, Konishi H
  TITLE     Improved methods of AAV-mediated gene targeting for human cell lines
            using ribosome-skipping 2A peptide.
  JOURNAL   Nucleic Acids Res. 2015 Dec 10. pii: gkv1338.
  PUBMED    26657635
REFERENCE   2  (bases 1 to 4488)
  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: "Nucleic 
            Acids Res. 2015 Dec 10. pii: gkv1338."
COMMENT     Sequence Label: pAAV-2Aneo v2
FEATURES             Location/Qualifiers
     source          1..4488
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     primer_bind     161..177
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     161..177
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     protein_bind    complement(179..212)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     intron          255..484
                     /label=chimeric intron
                     /note="chimera between introns from adenovirus and 
                     immunoglobulin heavy chain genes"
     primer_bind     467..486
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     CDS             537..590
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             594..1388
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MAEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(648..667)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1258..1277
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    1410..1544
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1434..1453)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1488..1507
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     protein_bind    complement(1641..1674)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(1727..1743)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1728..1744)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     repeat_region   1751..1891
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   1751..1880
                     /label=AAV2 ITR
     rep_origin      1966..2421
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2053..2072)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2263..2284
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2438..2457)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2557..2579
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2617..2635)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2703..2807
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2808..2876,2877..3668)
                     /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(3026..3045)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3839..4427
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4328..4347
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gcggccgagc tccaccgcgg tggcggccgc tctagaacta gtggatccat
      181 aacttcgtat agcatacatt atacgaagtt atggaattta cccaattcgc tgtctgcgag
      241 ggccagctgt tggggtgagt actccctctc aaaagcgggc atgacttctg cgctaagatt
      301 gtcagtttcc aaaaacgagg aggatttgat attcacctgg cccgcggtga tgcctttgag
      361 ggtggccgcg tccatctggt cagaaaagac aatctttttg ttgtcaagct tgaggtgtgg
      421 caggcttgag atctggccat acacttgagt gacaatgaca tccactttgc ctttctctcc
      481 acaggtgtcc actcccaggt ccaactgcat tccggatgta cagtccggac tgtacagagg
      541 gcagaggaag tcttctaaca tgcggtgacg tggaggagaa tcccggccca accatggctg
      601 aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg
      661 actgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg
      721 ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaggacg
      781 aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg
      841 ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc
      901 tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc
      961 tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc
     1021 gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc
     1081 aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gcgcatgccc gacggcgagg
     1141 atctcgtcgt gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct
     1201 tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt
     1261 tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc
     1321 tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt
     1381 tcttctgagc gggactctgg ggttcggggg atccagacat gataagatac attgatgagt
     1441 ttggacaaac cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg
     1501 ctattgcttt atttgtaacc attataagct gcaataaaca agttaacaac aacaattgca
     1561 ttcattttat gtttcaggtt cagggggagg tgtgggaggt tttttctagg gggccccggc
     1621 gaggccggga tgagttggga ataacttcgt atagcataca ttatacgaag ttatccaaat
     1681 tttaaggtac cactgtgcat atgtatgaat tcgatatcaa gcttatcgat accgtcgacc
     1741 tcgaggccgc aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg
     1801 ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca
     1861 gtgagcgagc gagcgcgcag ctgcctgcag gggcgcctga tgcggtattt tctccttacg
     1921 catctgtgcg gtatttcaca ccgcatacgt caaagcaacc atagtacgcg ccctgtagcg
     1981 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg
     2041 ccttagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc
     2101 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc
     2161 tcgaccccaa aaaacttgat ttgggtgatg gttcacgtag tgggccatcg ccctgataga
     2221 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa
     2281 ctggaacaac actcaactct atctcgggct attcttttga tttataaggg attttgccga
     2341 tttcggtcta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca
     2401 aaatattaac gtttacaatt ttatggtgca ctctcagtac aatctgctct gatgccgcat
     2461 agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc
     2521 tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt
     2581 tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat
     2641 aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg
     2701 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     2761 gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac
     2821 atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc
     2881 cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca
     2941 tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc
     3001 caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg
     3061 ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac
     3121 cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca
     3181 taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg
     3241 agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac
     3301 cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg
     3361 caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat
     3421 taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg
     3481 ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg
     3541 cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc
     3601 aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc
     3661 attggtaact gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt
     3721 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt
     3781 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     3841 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     3901 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     3961 gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca
     4021 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     4081 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     4141 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     4201 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     4261 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     4321 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     4381 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     4441 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgt
//
