LOCUS       Exported                4507 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
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4507)
  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 4507)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..4507
                     /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             556..609
                     /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             613..1407
                     /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(667..686)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1277..1296
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    1429..1563
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1453..1472)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1507..1526
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     protein_bind    1660..1693
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     primer_bind     complement(1746..1762)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1747..1763)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     repeat_region   1770..1910
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   1770..1899
                     /label=AAV2 ITR
     rep_origin      1985..2440
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2072..2091)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2282..2303
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2457..2476)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2576..2598
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2636..2654)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2722..2826
                     /gene="bla"
                     /label=AmpR promoter
     CDS             2827..3687
                     /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(3045..3064)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3858..4446
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4347..4366
                     /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 ttccggaacg cgttgtacat
      541 ccggacgcgt gtacagaggg cagaggaagt cttctaacat gcggtgacgt ggaggagaat
      601 cccggcccaa ccatggctga acaagatgga ttgcacgcag gttctccggc cgcttgggtg
      661 gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga tgccgccgtg
      721 ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct gtccggtgcc
      781 ctgaatgaac tgcaggacga ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct
      841 tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct attgggcgaa
      901 gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt atccatcatg
      961 gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt cgaccaccaa
     1021 gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt cgatcaggat
     1081 gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg
     1141 cgcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt gccgaatatc
     1201 atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg tgtggcggac
     1261 cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg cggcgaatgg
     1321 gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg catcgccttc
     1381 tatcgccttc ttgacgagtt cttctgagcg ggactctggg gttcggggga tccagacatg
     1441 ataagataca ttgatgagtt tggacaaacc acaactagaa tgcagtgaaa aaaatgcttt
     1501 atttgtgaaa tttgtgatgc tattgcttta tttgtaacca ttataagctg caataaacaa
     1561 gttaacaaca acaattgcat tcattttatg tttcaggttc agggggaggt gtgggaggtt
     1621 ttttctaggg ggccccggcg aggccgggat gagttgggaa taacttcgta tagcatacat
     1681 tatacgaagt tatccaaatt ttaaggtacc actgtgcata tgtatgaatt cgatatcaag
     1741 cttatcgata ccgtcgacct cgaggccgca ggaaccccta gtgatggagt tggccactcc
     1801 ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca aaggtcgccc gacgcccggg
     1861 ctttgcccgg gcggcctcag tgagcgagcg agcgcgcagc tgcctgcagg ggcgcctgat
     1921 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatacgtc aaagcaacca
     1981 tagtacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
     2041 accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc
     2101 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
     2161 tttagtgctt tacggcacct cgaccccaaa aaacttgatt tgggtgatgg ttcacgtagt
     2221 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat
     2281 agtggactct tgttccaaac tggaacaaca ctcaacccta tctcgggcta ttcttttgat
     2341 ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa
     2401 tttaacgcga attttaacaa aatattaacg tttacaattt tatggtgcac tctcagtaca
     2461 atctgctctg atgccgcata gttaagccag ccccgacacc cgccaacacc cgctgacgcg
     2521 ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg
     2581 agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc
     2641 gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt
     2701 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca
     2761 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg
     2821 aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc
     2881 cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg
     2941 ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt
     3001 cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta
     3061 ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat
     3121 gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga
     3181 gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca
     3241 acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact
     3301 cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc
     3361 acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact
     3421 ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt
     3481 ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt
     3541 gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt
     3601 atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata
     3661 ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag
     3721 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat
     3781 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     3841 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     3901 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     3961 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg
     4021 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     4081 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     4141 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     4201 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     4261 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     4321 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     4381 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     4441 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     4501 cacatgt
//
