LOCUS       sequence-163087-        4890 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  rAAV-based template for genome engineering of protein C-termini 
            containing PQS2 and 3xHA tags and a selection cassette.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4890)
  AUTHORS   Vandemoortele G, Staes A, Gonnelli G, Samyn N, De Sutter D, 
            Vandermarliere E, Timmerman E, Gevaert K, Martens L, Eyckerman S
  TITLE     An extra dimension in protein tagging by quantifying universal 
            proteotypic peptides using targeted proteomics.
  JOURNAL   Sci Rep. 2016 Jun 6;6:27220. doi: 10.1038/srep27220.
  PUBMED    27264994
REFERENCE   2  (bases 1 to 4890)
  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: "Sci Rep."; 
            date: "2016-06-6"; volume: "6:27220. doi"; pages: " 
            10.1038/srep27220"
COMMENT     Sequence Label: pAav-MCS-PQS2-3xHA
FEATURES             Location/Qualifiers
     source          1..4890
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..141
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   12..141
                     /label=AAV2 ITR
     protein_bind    178..225
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             299..388
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     protein_bind    complement(416..449)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        461..961
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(496..519)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     894..913
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             975..1778
                     /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="MGSAIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQ
                     GRPVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQD
                     LLSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVNQDDLDE
                     EHQGLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQ
                     DIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(1038..1057)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1648..1667
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(1810..1827)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1816..2040
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    complement(2065..2098)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     repeat_region   2153..2293
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2153..2282
                     /label=AAV2 ITR
     rep_origin      2368..2823
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2455..2474)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2665..2686
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2840..2859)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2959..2981
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3019..3037)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3105..3209
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3210..3278,3279..4070)
                     /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(3428..3447)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4241..4829
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4730..4749
                     /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 tgcggccgca aacgcgtaaa gggcccagcg aattcgggaa
      181 gttcctattc cgaagttcct attctctaga aagtatagga acttcagtgg ccgaggctta
      241 tagaggcctg ggagcttctc caggaatcgg tgctggaaga ttcctggaga cagaaaacta
      301 cccatacgat gttccagatt acgctggcta tccctatgac gtcccggact atgcaggaag
      361 ctatccctat gacgttccag attacgctgg cgagcccgag gcttgaaagc ttcggataac
      421 ttcgtatagc atacattata cgaagttatc gcgccctacc gggtagggga ggcgcttttc
      481 ccaaggcagt ctggagcatg cgctttagca gccccgctgg gcacttggcg ctacacaagt
      541 ggcctctggc ctcgcacaca ttccacatcc accggtaggc gccaaccggc tccgttcttt
      601 ggtggcccct tcgcgccacc ttctactcct cccctagtca ggaagttccc ccccgccccg
      661 cagctcgcgt cgtgcaggac gtgacaaatg gaagtagcac gtctcactag tctcgtgcag
      721 atggacagca ccgctgagca atggaagcgg gtaggccttt ggggcagcgg ccaatagcag
      781 ctttggctcc ttcgctttct gggctcagag gctgggaagg ggtgggtccg ggggcgggct
      841 caggggcggg ctcaggggcg gggcgggcgc ccgaaggtcc tccggaagcc cggcattctg
      901 cacgcttcaa aagcgcacgt ctgccgcgct gttctcctct tcctcatctc cgggcctttc
      961 gacctgcagc caatatggga tcggccattg aacaagatgg attgcacgca ggttctccgg
     1021 ccgcttgggt ggagaggcta ttcggctatg actgggcaca acagacaatc ggctgctctg
     1081 atgccgccgt gttccggctg tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc
     1141 tgtccggtgc cctgaatgaa ctgcaggacg aggcagcgcg gctatcgtgg ctggccacga
     1201 cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg gactggctgc
     1261 tattgggcga agtgccgggg caggatctcc tgtcatctca ccttgctcct gccgagaaag
     1321 tatccatcat ggctgatgca atgcggcggc tgcatacgct tgatccggct acctgcccat
     1381 tcgaccacca agcgaaacat cgcatcgagc gagcacgtac tcggatggaa gccggtcttg
     1441 tcaatcagga tgatctggac gaagagcatc aggggctcgc gccagccgaa ctgttcgcca
     1501 ggctcaaggc gcgcatgccc gacggcgagg atctcgtcgt gacccatggc gatgcctgct
     1561 tgccgaatat catggtggaa aatggccgct tttctggatt catcgactgt ggccggctgg
     1621 gtgtggcgga tcgctatcag gacatagcgt tggctacccg tgatattgct gaagagcttg
     1681 gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc gattcgcagc
     1741 gcatcgcctt ctatcgcctt cttgacgagt tcttctgagg ggatcaattc tctagagctc
     1801 gctgatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg
     1861 tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa
     1921 ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca
     1981 gcaaggggga ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg
     2041 cttctgaggc ggaaagaacc agctataact tcgtatagca tacattatac gaagttatag
     2101 taatcgataa ctcgagaagg atccaaggta ccaagctagc aaaagcggcc gcaggaaccc
     2161 ctagtgatgg agttggccac tccctctctg cgcgctcgct cgctcactga ggccgggcga
     2221 ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc
     2281 agctgcctgc aggggcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca
     2341 caccgcatac gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta agcgcggcgg
     2401 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt
     2461 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
     2521 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
     2581 atttgggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
     2641 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc
     2701 ctatctcggg ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa
     2761 aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgtttacaa
     2821 ttttatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac
     2881 acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca
     2941 gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga
     3001 aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa
     3061 taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
     3121 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
     3181 tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta
     3241 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag
     3301 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca
     3361 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta
     3421 aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc
     3481 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc
     3541 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca
     3601 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc
     3661 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca
     3721 taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac
     3781 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg
     3841 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg
     3901 ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg
     3961 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
     4021 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc
     4081 aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct
     4141 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
     4201 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     4261 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     4321 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     4381 atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     4441 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     4501 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     4561 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     4621 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     4681 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     4741 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     4801 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     4861 tggccttttg ctggcctttt gctcacatgt
//
