LOCUS       sequence-163077-        4863 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  rAAV-based template for genome engineering of protein C-termini 
            containing PQS1 and 3xFLAG tags and a selection cassette.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4863)
  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 4863)
  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-PQS1-3xFLAG
FEATURES             Location/Qualifiers
     source          1..4863
                     /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    192..225
                     /label=FRT3
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision with other FRT3 sites
                     (Bosch et al., 2015)"
     CDS             296..361
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     protein_bind    complement(389..422)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        434..934
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(469..492)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     867..886
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             948..1751
                     /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(1011..1030)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1621..1640
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(1783..1800)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1789..2013
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    complement(2038..2071)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     repeat_region   2126..2266
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2126..2255
                     /label=AAV2 ITR
     rep_origin      2341..2796
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2428..2447)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2638..2659
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2813..2832)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2932..2954
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2992..3010)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3078..3182
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3183..3251,3252..4043)
                     /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(3401..3420)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4214..4802
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4703..4722
                     /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 attcttcaaa tagtatagga acttcactga ccctgatctt
      241 cagagaggcc gtgagcgaaa tcctggagac ctccagattc gcctacctgt acgacgacta
      301 caaagaccat gacggtgatt ataaagatca tgatattgat tacaaggatg acgatgacaa
      361 gggcgagccc gaggcttgaa agcttcggat aacttcgtat agcatacatt atacgaagtt
      421 atcgcgccct accgggtagg ggaggcgctt ttcccaaggc agtctggagc atgcgcttta
      481 gcagccccgc tgggcacttg gcgctacaca agtggcctct ggcctcgcac acattccaca
      541 tccaccggta ggcgccaacc ggctccgttc tttggtggcc ccttcgcgcc accttctact
      601 cctcccctag tcaggaagtt cccccccgcc ccgcagctcg cgtcgtgcag gacgtgacaa
      661 atggaagtag cacgtctcac tagtctcgtg cagatggaca gcaccgctga gcaatggaag
      721 cgggtaggcc tttggggcag cggccaatag cagctttggc tccttcgctt tctgggctca
      781 gaggctggga aggggtgggt ccgggggcgg gctcaggggc gggctcaggg gcggggcggg
      841 cgcccgaagg tcctccggaa gcccggcatt ctgcacgctt caaaagcgca cgtctgccgc
      901 gctgttctcc tcttcctcat ctccgggcct ttcgacctgc agccaatatg ggatcggcca
      961 ttgaacaaga tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct
     1021 atgactgggc acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc
     1081 aggggcgccc ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcagg
     1141 acgaggcagc gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg
     1201 acgttgtcac tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc
     1261 tcctgtcatc tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc
     1321 ggctgcatac gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg
     1381 agcgagcacg tactcggatg gaagccggtc ttgtcaatca ggatgatctg gacgaagagc
     1441 atcaggggct cgcgccagcc gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg
     1501 aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc
     1561 gcttttctgg attcatcgac tgtggccggc tgggtgtggc ggatcgctat caggacatag
     1621 cgttggctac ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg
     1681 tgctttacgg tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg
     1741 agttcttctg aggggatcaa ttctctagag ctcgctgatc agcctcgact gtgccttcta
     1801 gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca
     1861 ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc
     1921 attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata
     1981 gcaggcatgc tggggatgcg gtgggctcta tggcttctga ggcggaaaga accagctata
     2041 acttcgtata gcatacatta tacgaagtta tagtaatcga taactcgaga aggatccaag
     2101 gtaccaagct agcaaaagcg gccgcaggaa cccctagtga tggagttggc cactccctct
     2161 ctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt
     2221 gcccgggcgg cctcagtgag cgagcgagcg cgcagctgcc tgcaggggcg cctgatgcgg
     2281 tattttctcc ttacgcatct gtgcggtatt tcacaccgca tacgtcaaag caaccatagt
     2341 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
     2401 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
     2461 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
     2521 gtgctttacg gcacctcgac cccaaaaaac ttgatttggg tgatggttca cgtagtgggc
     2581 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
     2641 gactcttgtt ccaaactgga acaacactca accctatctc gggctattct tttgatttat
     2701 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
     2761 acgcgaattt taacaaaata ttaacgttta caattttatg gtgcactctc agtacaatct
     2821 gctctgatgc cgcatagtta agccagcccc gacacccgcc aacacccgct gacgcgccct
     2881 gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct
     2941 gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga
     3001 tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca
     3061 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
     3121 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga
     3181 gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc
     3241 ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg
     3301 cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc
     3361 ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat
     3421 cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact
     3481 tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat
     3541 tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga
     3601 tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc
     3661 ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga
     3721 tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag
     3781 cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc
     3841 gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt
     3901 ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct
     3961 acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg
     4021 cctcactgat taagcattgg taactgtcag accaagttta ctcatatata ctttagattg
     4081 atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca
     4141 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     4201 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     4261 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     4321 aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt
     4381 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     4441 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     4501 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     4561 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     4621 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     4681 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     4741 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     4801 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     4861 tgt
//
