LOCUS       sequence-350134-        6449 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  HDR donor plasmid to introduce the EBFP (T65S/F145Y) cassette to 
            AAVS1 using AAVS1 T2 sgRNA..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6449)
  AUTHORS   Levesque S, Mayorga D, Fiset JP, Goupil C, Duringer A, Loiselle A, 
            Bouchard E, Agudelo D, Doyon Y
  TITLE     Marker-free co-selection for successive rounds of prime editing in 
            human cells.
  JOURNAL   Nat Commun. 2022 Oct 7;13(1):5909. doi: 10.1038/s41467-022-33669-z.
  PUBMED    36207338
REFERENCE   2  (bases 1 to 6449)
  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: "Nat 
            Commun."; date: "2022-10-7"; volume: "13(1):5909. doi"; pages: " 
            10.1038/s41467-022-33669-z"
COMMENT     Sequence Label: AAVS1_Puro_hPGK1_EBFP_Donor
FEATURES             Location/Qualifiers
     source          1..6449
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     misc_feature    320..1123
                     /label=HA-L
                     /note="left homology arm from the adeno-associated virus 
                     integration site (AAVS1) within intron 1 of the human 
                     PPP1R12C gene"
     misc_feature    1130..1155
                     /label=SA
                     /note="splice acceptor site"
     CDS             1179..1232
                     /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             1236..1832
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="TEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIERV
                     TELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLAA
                     QQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETSA
                     PRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     1729..1749
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    1848..1969
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1859..1878)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1913..1932
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        2042..2552
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     2471..2489
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     regulatory      2568..2577
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2574..2576,2577..2579,2580..3293)
                     /codon_start=1
                     /product="enhanced blue variant of GFP"
                     /label=EBFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLSHGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2619..2640)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2880..2899)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3227..3248
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    3302..3526
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     misc_feature    3546..4382
                     /label=HA-R
                     /note="right homology arm from the adeno-associated virus 
                     integration site (AAVS1) within intron 1 of the human 
                     PPP1R12C gene"
     primer_bind     complement(4459..4476)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4630..5218)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4710..4729)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5389..6180,6181..6249))
                     /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     6012..6031
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6250..6354)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6422..6440
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagaa ttcgcccttt gctttctctg accagcattc tctcccctgg gcctgtgccg
      361 ctttctgtct gcagcttgtg gcctgggtca cctctacggc tggcccagat ccttccctgc
      421 cgcctccttc aggttccgtc ttcctccact ccctcttccc cttgctctct gctgtgttgc
      481 tgcccaagga tgctctttcc ggagcacttc cttctcggcg ctgcaccacg tgatgtcctc
      541 tgagcggatc ctccccgtgt ctgggtcctc tccgggcatc tctcctccct cacccaaccc
      601 catgccgtct tcactcgctg ggttcccttt tccttctcct tctggggcct gtgccatctc
      661 tcgtttctta ggatggcctt ctccgacgga tgtctccctt gcgtcccgcc tccccttctt
      721 gtaggcctgc atcatcaccg tttttctgga caaccccaaa gtaccccgtc tccctggctt
      781 tagccacctc tccatcctct tgctttcttt gcctggacac cccgttctcc tgtggattcg
      841 ggtcacctct cactcctttc atttgggcag ctcccctacc ccccttacct ctctagtctg
      901 tgctagctct tccagccccc tgtcatggca tcttccaggg gtccgagagc tcagctagtc
      961 ttcttcctcc aacccgggcc cctatgtcca cttcaggaca gcatgtttgc tgcctccagg
     1021 gatcctgtgt ccccgagctg ggaccacctt atattcccag ggccggttaa tgtggctctg
     1081 gttctgggta cttttatctg tcccctccac cccacagtgg ggcaagcttc tgacctcttc
     1141 tcttcctccc acagggcctc gagagatctg gcggcggaga gggcagagga agtcttctaa
     1201 catgcggtga cgtggaggag aatcccggcc ctagcaccga gtacaagccc acggtgcgcc
     1261 tcgccacccg cgacgacgtc cccagggccg tacgcaccct cgccgccgcg ttcgccgact
     1321 accccgccac gcgccacacc gtcgatccgg accgccacat cgagcgggtc accgagctgc
     1381 aagaactctt cctcacgcgc gtcgggctcg acatcggcaa ggtgtgggtc gcggacgacg
     1441 gcgccgcggt ggcggtctgg accacgccgg agagcgtcga agcgggggcg gtgttcgccg
     1501 agatcggccc gcgcatggcc gagttgagcg gttcccggct ggccgcgcag caacagatgg
     1561 aaggcctcct ggcgccgcac cggcccaagg agcccgcgtg gttcctggcc accgtcggcg
     1621 tctcgcccga ccaccagggc aagggtctgg gcagcgccgt cgtgctcccc ggagtggagg
     1681 cggccgagcg cgccggggtg cccgccttcc tggagacctc cgcgccccgc aacctcccct
     1741 tctacgagcg gctcggcttc accgtcaccg ccgacgtcga ggtgcccgaa ggaccgcgca
     1801 cctggtgcat gacccgcaag cccggtgcct gatctagaca gacatgataa gatacattga
     1861 tgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg
     1921 tgatgctatt gctttatttg taaccattat aagctgcaat aaacaagtta acaacaacaa
     1981 ttgcattcat tttatgtttc aggttcaggg ggaggtgtgg gaggtttttt aatgcatcca
     2041 cggggttggg gttgcgcctt ttccaaggca gccctgggtt tgcgcaggga cgcggctgct
     2101 ctgggcgtgg ttccgggaaa cgcagcggcg ccgaccctgg gtctcgcaca ttcttcacgt
     2161 ccgttcgcag cgtcacccgg atcttcgccg ctacccttgt gggccccccg gcgacgcttc
     2221 ctgctccgcc cctaagtcgg gaaggttcct tgcggttcgc ggcgtgccgg acgtgacaaa
     2281 cggaagccgc acgtctcact agtaccctcg cagacggaca gcgccaggga gcaatggcag
     2341 cgcgccgacc gcgatgggct gtggccaata gcggctgctc agcagggcgc gccgagagca
     2401 gcggccggga aggggcggtg cgggaggcgg ggtgtggggc ggtagtgtgg gccctgttcc
     2461 tgcccgcgcg gtgttccgca ttctgcaagc ctccggagcg cacgtcggca gtcggctccc
     2521 tcgttgaccg aatcaccgac ctctctcccc aggatccgta cgatatcgcc accatggtga
     2581 gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg gacggcgacg
     2641 taaacggcca caagttcagc gtgtccggcg agggcgaggg cgatgccacc tacggcaagc
     2701 tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc accctcgtga
     2761 ccaccctgag ccacggcgtg cagtgcttca gccgctaccc cgaccacatg aagcagcacg
     2821 acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc ttcttcaagg
     2881 acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc
     2941 gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg cacaagctgg
     3001 agtacaacta caacagccac aacgtctata tcatggccga caagcagaag aacggcatca
     3061 aggtgaactt caagatccgc cacaacatcg aggacggcag cgtgcagctc gccgaccact
     3121 accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac cactacctga
     3181 gcacccagtc cgccctgagc aaagacccca acgagaagcg cgatcacatg gtcctgctgg
     3241 agttcgtgac cgccgccggg atcactctcg gcatggacga gctgtacaag tgacctgcag
     3301 gctgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc
     3361 ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt
     3421 ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat
     3481 tgggaagaca atagcaggca tgctggggat gcggtgggct ctatgggtcg acagtactaa
     3541 gctttactag ggacaggatt ggtgacagaa aagccccatc cttaggcctc ctccttccta
     3601 gtctcctgat attgggtcta acccccacct cctgttaggc agattcctta tctggtgaca
     3661 cacccccatt tcctggagcc atctctctcc ttgccagaac ctctaaggtt tgcttacgat
     3721 ggagccagag aggatcctgg gagggagagc ttggcagggg gtgggaggga agggggggat
     3781 gcgtgacctg cccggttctc agtggccacc ctgcgctacc ctctcccaga acctgagctg
     3841 ctctgacgcg gctgtctggt gcgtttcact gatcctggtg ctgcagcttc cttacacttc
     3901 ccaagaggag aagcagtttg gaaaaacaaa atcagaataa gttggtcctg agttctaact
     3961 ttggctcttc acctttctag tccccaattt atattgttcc tccgtgcgtc agttttacct
     4021 gtgagataag gccagtagcc agccccgtcc tggcagggct gtggtgagga ggggggtgtc
     4081 cgtgtggaaa actccctttg tgagaatggt gcgtcctagg tgttcaccag gtcgtggccg
     4141 cctctactcc ctttctcttt ctccatcctt ctttccttaa agagtcccca gtgctatctg
     4201 ggacatattc ctccgcccag agcagggtcc cgcttcccta aggccctgct ctgggcttct
     4261 gggtttgagt ccttggcaag cccaggagag gcgctcaggc ttccctgtcc cccttcctcg
     4321 tccaccatct catgcccctg gctctcctgc cccttcccta caggggttcc tggctctgct
     4381 ctaagggcga attctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     4441 tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg
     4501 agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc
     4561 aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
     4621 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
     4681 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
     4741 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
     4801 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
     4861 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
     4921 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
     4981 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
     5041 gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa
     5101 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
     5161 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
     5221 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg
     5281 gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg
     5341 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt
     5401 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact
     5461 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat
     5521 gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg
     5581 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg
     5641 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat
     5701 tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc
     5761 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt
     5821 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc
     5881 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga
     5941 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc
     6001 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa
     6061 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta
     6121 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg
     6181 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg
     6241 aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat
     6301 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt
     6361 tccccgaaaa gtgccacctg acgtctaaga aaccattatt atcatgacat taacctataa
     6421 aaataggcgt atcacgaggc cctttcgtc
//
