LOCUS Exported 4499 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Used to construct AAV-based targeting vectors that carry an IVS-2Aneo cassette for promoter-trapping. ACCESSION . VERSION . KEYWORDS pAAV-2Aneo v2 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 4499) 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 4499) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..4499 /organism="synthetic DNA construct" /mol_type="other DNA" repeat_region 1..141 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 12..141 /label=AAV2 ITR primer_bind 172..188 /label=SK primer /note="common sequencing primer, one of multiple similar variants" primer_bind 172..188 /label=pBluescriptSK /note="For pBluescript vector" protein_bind 190..223 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." intron 266..495 /label=chimeric intron /note="chimera between introns from adenovirus and immunoglobulin heavy chain genes" primer_bind 478..497 /label=pMT2-F /note="Synthetic intron, forward primer" CDS 548..601 /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 605..1399 /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(659..678) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 1269..1288 /label=Neo-F /note="Neomycin resistance gene, forward primer" polyA_signal 1421..1555 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(1445..1464) /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind 1499..1518 /label=SV40pA-R /note="SV40 polyA, reverse primer" protein_bind 1652..1685 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." primer_bind complement(1738..1754) /label=KS primer /note="common sequencing primer, one of multiple similar variants" primer_bind complement(1739..1755) /label=pBluescriptKS /note="For pBluescript vector" repeat_region 1762..1902 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 1762..1891 /label=AAV2 ITR rep_origin 1977..2432 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(2064..2083) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 2274..2295 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(2449..2468) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 2568..2590 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(2628..2646) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 2714..2818 /gene="bla" /label=AmpR promoter CDS 2819..3679 /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(3037..3056) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 3850..4438 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 4339..4358 /label=pBR322ori-F /note="pBR322 origin, forward primer" ORIGIN 1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc 61 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca 121 actccatcac taggggttcc tgcggccgag ctccaccgcg gtggcggccg ctctagaact 181 agtggatcca taacttcgta tagcatacat tatacgaagt tatggaattt acccaattcg 241 ctgtctgcga gggccagctg ttggggtgag tactccctct caaaagcggg catgacttct 301 gcgctaagat tgtcagtttc caaaaacgag gaggatttga tattcacctg gcccgcggtg 361 atgcctttga gggtggccgc gtccatctgg tcagaaaaga caatcttttt gttgtcaagc 421 ttgaggtgtg gcaggcttga gatctggcca tacacttgag tgacaatgac atccactttg 481 cctttctctc cacaggtgtc cactcccagg tccaactgca ttccggatgt acagtccgga 541 ctgtacagag ggcagaggaa gtcttctaac atgcggtgac gtggaggaga atcccggccc 601 aaccatggct gaacaagatg gattgcacgc aggttctccg gccgcttggg tggagaggct 661 attcggctat gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct 721 gtcagcgcag gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga 781 actgcaggac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc 841 tgtgctcgac gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg 901 gcaggatctc ctgtcatctc accttgctcc tgccgagaaa gtatccatca tggctgatgc 961 aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc aagcgaaaca 1021 tcgcatcgag cgagcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga 1081 cgaagagcat caggggctcg cgccagccga actgttcgcc aggctcaagg cgcgcatgcc 1141 cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata tcatggtgga 1201 aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca 1261 ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg 1321 cttcctcgtg ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct 1381 tcttgacgag ttcttctgag cgggactctg gggttcgggg gatccagaca tgataagata 1441 cattgatgag tttggacaaa ccacaactag aatgcagtga aaaaaatgct ttatttgtga 1501 aatttgtgat gctattgctt tatttgtaac cattataagc tgcaataaac aagttaacaa 1561 caacaattgc attcatttta tgtttcaggt tcagggggag gtgtgggagg ttttttctag 1621 ggggccccgg cgaggccggg atgagttggg aataacttcg tatagcatac attatacgaa 1681 gttatccaaa ttttaaggta ccactgtgca tatgtatgaa ttcgatatca agcttatcga 1741 taccgtcgac ctcgaggccg caggaacccc tagtgatgga gttggccact ccctctctgc 1801 gcgctcgctc gctcactgag gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc 1861 gggcggcctc agtgagcgag cgagcgcgca gctgcctgca ggggcgcctg atgcggtatt 1921 ttctccttac gcatctgtgc ggtatttcac accgcatacg tcaaagcaac catagtacgc 1981 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac 2041 acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt 2101 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc 2161 tttacggcac ctcgacccca aaaaacttga tttgggtgat ggttcacgta gtgggccatc 2221 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact 2281 cttgttccaa actggaacaa cactcaaccc tatctcgggc tattcttttg atttataagg 2341 gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc 2401 gaattttaac aaaatattaa cgtttacaat tttatggtgc actctcagta caatctgctc 2461 tgatgccgca tagttaagcc agccccgaca cccgccaaca cccgctgacg cgccctgacg 2521 ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat 2581 gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg 2641 cctattttta taggttaatg tcatgataat aatggtttct tagacgtcag gtggcacttt 2701 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta 2761 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat 2821 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt 2881 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg 2941 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga 3001 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg 3061 tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt 3121 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg 3181 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg 3241 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga 3301 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc 3361 tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc 3421 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc 3481 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg 3541 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac 3601 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc 3661 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt 3721 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac 3781 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa 3841 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc 3901 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt 3961 aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg 4021 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc 4081 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt 4141 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga 4201 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct 4261 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg 4321 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca 4381 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa 4441 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgt //