LOCUS       sequence-223051-        7400 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  rAAV-based donor template for genome engineering of the TP53 protein
            C-terminus containing a T2A-BirA* module and a selection cassette.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7400)
  AUTHORS   Vandemoortele G, De Sutter D, Moliere A, Pauwels J, Gevaert K, 
            Eyckerman S
  TITLE     A Well-Controlled BioID Design for Endogenous Bait Proteins.
  JOURNAL   J Proteome Res. 2018 Dec 10. doi: 10.1021/acs.jproteome.8b00367.
  PUBMED    30525648
REFERENCE   2  (bases 1 to 7400)
  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: "J Proteome 
            Res. 2018 Dec 10. doi: 10.1021/acs.jproteome.8b00367."
COMMENT     Sequence Label: pAav-TP53-T2A-BirA*
FEATURES             Location/Qualifiers
     source          1..7400
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             14..67
                     /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             join(68..415,416..418,419..1027)
                     /codon_start=1
                     /product="promiscuous R118G mutant of the E. coli biotin 
                     protein ligase (Cronan, 2005)"
                     /label=BirA(R118G)
                     /note="also known as BirA* or BioID"
                     /translation="KDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRDW
                     GVDVFTVPGKGYSLPEPIQLLNAKQILGQLDGGSVAVLPVIDSTNQYLLDRIGELKSGD
                     ACIAEYQQAGRGGRGRKWFSPFGANLYLSMFWRLEQGPAAAIGLSLVIGIVMAEVLRKL
                     GADKVRVKWPNDLYLQDRKLAGILVELTGKTGDAAQIVIGAGINMAMRRVEESVVNQGW
                     ITLQEAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLIIGD
                     KEIFGISRGIDKQGALLLEQDGIIKPWMGGEISLRSAEK"
     protein_bind    1040..1073
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     protein_bind    complement(1077..1110)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        1122..1622
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(1157..1180)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     1555..1574
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             1636..2439
                     /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(1699..1718)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2309..2328
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(2471..2488)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2477..2701
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    complement(2726..2759)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     repeat_region   3639..3779
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3639..3768
                     /label=AAV2 ITR
     rep_origin      3854..4309
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3941..3960)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4151..4172
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4326..4345)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4445..4467
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4505..4523)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4591..4695
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4696..4764,4765..5556)
                     /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(4914..4933)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5727..6315
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6216..6235
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   6377..6506
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
ORIGIN
        1 tatgggcagt ggagagggca gaggaagtct gctaacctgc ggggacgtcg aggagaatcc
       61 tggcccaaag gacaacacgg tgcccctgaa gctgatcgcc ctgctggcca acggcgagtt
      121 ccactctggc gagcagctgg gagagaccct gggaatgagc agagccgcca tcaacaagca
      181 catccagaca ctgagagact ggggagtgga cgtgttcacc gtgcctggca agggctacag
      241 cctgcctgag cctatccagc tgctgaacgc caagcagatc ctgggacagc tggatggcgg
      301 aagcgtggcc gtgctgcctg tgatcgactc caccaatcag tacctgctgg acagaatcgg
      361 agagctgaag tccggcgacg cctgcatcgc cgagtaccag caggctggca gaggaggcag
      421 aggacggaag tggttcagcc cattcggagc caacctgtac ctgtccatgt tctggagact
      481 ggagcaggga cctgctgctg ccatcggact gagtctggtg atcggaatcg tgatggccga
      541 ggtgctgaga aagctgggag ccgacaaggt gagagtgaag tggcctaatg acctgtacct
      601 ccaggaccgc aagctggctg gcatcctggt ggagctgaca ggcaagacag gcgatgccgc
      661 tcagatcgtg atcggagccg gaatcaacat ggccatgaga agagtggagg agagcgtggt
      721 gaaccagggc tggatcaccc tgcaggaggc tggcatcaac ctggaccgga acaccctggc
      781 cgccatgctg atcagagagc tgagagccgc tctggagctg ttcgagcagg agggactggc
      841 tccttacctg agcagatggg agaagctgga caacttcatc aacagacctg tgaagctgat
      901 catcggcgac aaggaaatct tcggcatctc cagaggaatc gacaagcagg gagctctgct
      961 gctggagcag gacggaatca tcaagccctg gatgggcgga gaaatctccc tgagaagcgc
     1021 agagaagtaa tgaaagcttg aagttcctat tctctagaaa gtataggaac ttccggataa
     1081 cttcgtatag catacattat acgaagttat cgcgccctac cgggtagggg aggcgctttt
     1141 cccaaggcag tctggagcat gcgctttagc agccccgctg ggcacttggc gctacacaag
     1201 tggcctctgg cctcgcacac attccacatc caccggtagg cgccaaccgg ctccgttctt
     1261 tggtggcccc ttcgcgccac cttctactcc tcccctagtc aggaagttcc cccccgcccc
     1321 gcagctcgcg tcgtgcagga cgtgacaaat ggaagtagca cgtctcacta gtctcgtgca
     1381 gatggacagc accgctgagc aatggaagcg ggtaggcctt tggggcagcg gccaatagca
     1441 gctttggctc cttcgctttc tgggctcaga ggctgggaag gggtgggtcc gggggcgggc
     1501 tcaggggcgg gctcaggggc ggggcgggcg cccgaaggtc ctccggaagc ccggcattct
     1561 gcacgcttca aaagcgcacg tctgccgcgc tgttctcctc ttcctcatct ccgggccttt
     1621 cgacctgcag ccaatatggg atcggccatt gaacaagatg gattgcacgc aggttctccg
     1681 gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat cggctgctct
     1741 gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac
     1801 ctgtccggtg ccctgaatga actgcaggac gaggcagcgc ggctatcgtg gctggccacg
     1861 acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg
     1921 ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa
     1981 gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca
     2041 ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt
     2101 gtcaatcagg atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc
     2161 aggctcaagg cgcgcatgcc cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc
     2221 ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg
     2281 ggtgtggcgg atcgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt
     2341 ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag
     2401 cgcatcgcct tctatcgcct tcttgacgag ttcttctgag gggatcaatt ctctagagct
     2461 cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc
     2521 gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa
     2581 attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac
     2641 agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg
     2701 gcttctgagg cggaaagaac cagctataac ttcgtatagc atacattata cgaagttata
     2761 gtaatcgata actcgagaag gatccaaggt acccattctc cacttcttgt tccccactga
     2821 cagcctccca cccccatctc tccctcccct gccattttgg gttttgggtc tttgaaccct
     2881 tgcttgcaat aggtgtgcgt cagaagcacc caggacttcc atttgctttg tcccggggct
     2941 ccactgaaca agttggcctg cactggtgtt ttgttgtggg gaggaggatg gggagtagga
     3001 cataccagct tagattttaa ggtttttact gtgagggatg tttgggagat gtaagaaatg
     3061 ttcttgcagt taagggttag tttacaatca gccacattct aggtaggggc ccacttcacc
     3121 gtactaacca gggaagctgt ccctcactgt tgaattttct ctaacttcaa ggcccatatc
     3181 tgtgaaatgc tggcatttgc acctacctca cagagtgcat tgtgagggtt aatgaaataa
     3241 tgtacatctg gccttgaaac caccttttat tacatggggt ctagaacttg acccccttga
     3301 gggtgcttgt tccctctccc tgttggtcgg tgggttggta gtttctacag ttgggcagct
     3361 ggttaggtag agggagttgt caagtctctg ctggcccagc caaaccctgt ctgacaacct
     3421 cttggtgaac cttagtacct aaaaggaaat ctcaccccat cccacaccct ggaggatttc
     3481 atctcttgta tatgatgatc tggatccacc aagacttgtt ttatgctcag ggtcaatttc
     3541 ttttttcttt tttttttttt ctttttcttt gagactgggt ctcgctttgt tgcccaggct
     3601 ggagtggagt ggcgtgatct gctagcaaaa gcggccgcag gaacccctag tgatggagtt
     3661 ggccactccc tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa aggtcgcccg
     3721 acgcccgggc tttgcccggg cggcctcagt gagcgagcga gcgcgcagct gcctgcaggg
     3781 gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcatacgtca
     3841 aagcaaccat agtacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     3901 cgcagcgtga ccgctacact tgccagcgcc ttagcgcccg ctcctttcgc tttcttccct
     3961 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     4021 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgattt gggtgatggt
     4081 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
     4141 ttctttaata gtggactctt gttccaaact ggaacaacac tcaactctat ctcgggctat
     4201 tcttttgatt tataagggat tttgccgatt tcggtctatt ggttaaaaaa tgagctgatt
     4261 taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt ttacaatttt atggtgcact
     4321 ctcagtacaa tctgctctga tgccgcatag ttaagccagc cccgacaccc gccaacaccc
     4381 gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc
     4441 gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgagacga
     4501 aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag
     4561 acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
     4621 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat
     4681 tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg
     4741 gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa
     4801 gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt
     4861 gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt
     4921 ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat
     4981 tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg
     5041 acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta
     5101 cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat
     5161 catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag
     5221 cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa
     5281 ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca
     5341 ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc
     5401 ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt
     5461 atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc
     5521 gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat
     5581 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt
     5641 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     5701 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     5761 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     5821 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta
     5881 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     5941 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     6001 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     6061 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     6121 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     6181 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     6241 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     6301 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     6361 ccttttgctc acatgtcctg caggcagctg cgcgctcgct cgctcactga ggccgcccgg
     6421 gcgtcgggcg acctttggtc gcccggcctc agtgagcgag cgagcgcgca gagagggagt
     6481 ggccaactcc atcactaggg gttcctgcgg ccgcaaacgc gtcagccctg cacagacatt
     6541 tttttagtct tcctccggtt gaatcctata accacattct tgcctcagtg tatccacaga
     6601 acatccaaac ccagggacga gtgtggatac ttctttgcca ttctccgcaa ctcccagccc
     6661 agagctggag ggtctcaagg aggggcctaa taattgtgta atactgaata cagccagagt
     6721 ttcaggtcat atactcagcc ctgccatgca ccggcaggtc ctaggtgacc cccgtcaaac
     6781 tcagtttcct tatatataaa atggggtaag ggggccgggc gcagtggctc acgaatccca
     6841 cactctggga ggccaaggcg agtggatcac ctgaggtcgg gagtttgagc ccagcctgac
     6901 caacatggag aaaccccatc tctactaaaa atacaaaagt agccgggcgt ggtgatgcat
     6961 gcctgtaatc ccagctacct actcgggagg ctgaggcagg agaatcgctt gaacccggga
     7021 ggcagaggtt gcggtgagct gagatctcac cattacactc cagcctgggc aacaagagtg
     7081 aaactccgtc tcaaaaaaga taaataaagt aaaatggggt aagggaagat tacgagacta
     7141 atacacacta atactctgag gtgctcagta aacatatttg catggggtgt ggccaccatc
     7201 ttgatttgaa ttcccgttgt cccagcctta ggcccttcaa agcattggtc agggaaaagg
     7261 ggcacagacc ctctcactca tgtgatgtca tctctcctcc ctgcttctgt ctcctacagc
     7321 cacctgaagt ccaaaaaggg tcagtctacc tcccgccata aaaaactcat gttcaagaca
     7381 gaagggcctg actcagacca
//
