LOCUS       sequence-350132-        6750 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  HDR donor plasmid to introduce the mScarlet-I mTOR signaling 
            indicator (TOSI) cassette to AAVS1 using AAVS1 T2 sgRNA..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6750)
  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 6750)
  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_mScarlet-I-TOSI_Donor
FEATURES             Location/Qualifiers
     source          1..6750
                     /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"
     CDS             2824..3519
                     /codon_start=1
                     /product="bright monomeric red fluorescent protein evolved 
                     from a synthetic template (Bindels et al., 2016)"
                     /label=mScarlet-I
                     /note="matures faster than mScarlet"
                     /translation="MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFSWDILSPQFMYGSRAFIKHPADIPDYYKQSFPEGFKWERVMNFEDGG
                     AVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIK
                     MALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRH
                     STGGMDELYK"
     primer_bind     complement(2957..2975)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3225..3244
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             3520..3567
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     polyA_signal    3603..3827
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     misc_feature    3847..4683
                     /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(4760..4777)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4931..5519)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5011..5030)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5690..6481,6482..6550))
                     /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     6313..6332
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6551..6655)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6723..6741
                     /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 aggggtaccc tcagccttaa ggccacgatg
     2581 gacgtggaaa acgagcagat cctgaacgtg aaccccgctg atcctgacaa cctgagcgac
     2641 agcctgttca gcggcgacga ggaaaatgcc ggcaccgagg aaattaagaa cgagatcaac
     2701 ggcaactgga tcagcgccag cagcatcaat gaggccagaa tcaacgccaa ggccaaaaga
     2761 cggctgcgga agaacagctc tagagattct ggaagaggcg atagcgtgtc cgactccggc
     2821 tccatggtga gcaagggcga ggcagtgatc aaggagttca tgcggttcaa ggtgcacatg
     2881 gagggctcca tgaacggcca cgagttcgag atcgagggcg agggcgaggg ccgcccctac
     2941 gagggcaccc agaccgccaa gctgaaggtg accaagggtg gccccctgcc cttctcctgg
     3001 gacatcctgt cccctcagtt catgtacggc tccagggcct tcatcaagca ccccgccgac
     3061 atccccgact actataagca gtccttcccc gagggcttca agtgggagcg cgtgatgaac
     3121 ttcgaggacg gcggcgccgt gaccgtgacc caggacacct ccctggagga cggcaccctg
     3181 atctacaagg tgaagctccg cggcaccaac ttccctcctg acggccccgt aatgcagaag
     3241 aagacaatgg gctgggaagc atccaccgag cggttgtacc ccgaggacgg cgtgctgaag
     3301 ggcgacatta agatggccct gcgcctgaag gacggcggcc gctacctggc ggacttcaag
     3361 accacctaca aggccaagaa gcccgtgcag atgcccggcg cctacaacgt cgaccgcaag
     3421 ttggacatca cctcccacaa cgaggactac accgtggtgg aacagtacga acgctccgag
     3481 ggccgccact ccaccggcgg catggacgag ctgtacaaga aaaggccggc ggccacgaaa
     3541 aaggccggcc aggcaaaaaa gaaaaagtga taactcgagc ggccgcgttt aaaccctgca
     3601 ggctgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac
     3661 cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg
     3721 tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga
     3781 ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatgggtc gacagtacta
     3841 agctttacta gggacaggat tggtgacaga aaagccccat ccttaggcct cctccttcct
     3901 agtctcctga tattgggtct aacccccacc tcctgttagg cagattcctt atctggtgac
     3961 acacccccat ttcctggagc catctctctc cttgccagaa cctctaaggt ttgcttacga
     4021 tggagccaga gaggatcctg ggagggagag cttggcaggg ggtgggaggg aaggggggga
     4081 tgcgtgacct gcccggttct cagtggccac cctgcgctac cctctcccag aacctgagct
     4141 gctctgacgc ggctgtctgg tgcgtttcac tgatcctggt gctgcagctt ccttacactt
     4201 cccaagagga gaagcagttt ggaaaaacaa aatcagaata agttggtcct gagttctaac
     4261 tttggctctt cacctttcta gtccccaatt tatattgttc ctccgtgcgt cagttttacc
     4321 tgtgagataa ggccagtagc cagccccgtc ctggcagggc tgtggtgagg aggggggtgt
     4381 ccgtgtggaa aactcccttt gtgagaatgg tgcgtcctag gtgttcacca ggtcgtggcc
     4441 gcctctactc cctttctctt tctccatcct tctttcctta aagagtcccc agtgctatct
     4501 gggacatatt cctccgccca gagcagggtc ccgcttccct aaggccctgc tctgggcttc
     4561 tgggtttgag tccttggcaa gcccaggaga ggcgctcagg cttccctgtc ccccttcctc
     4621 gtccaccatc tcatgcccct ggctctcctg ccccttccct acaggggttc ctggctctgc
     4681 tctaagggcg aattctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     4741 ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     4801 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     4861 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     4921 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     4981 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     5041 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     5101 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     5161 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     5221 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     5281 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     5341 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
     5401 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     5461 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     5521 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     5581 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     5641 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     5701 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     5761 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     5821 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     5881 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     5941 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     6001 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     6061 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     6121 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     6181 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     6241 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     6301 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     6361 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     6421 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     6481 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     6541 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     6601 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     6661 ttccccgaaa agtgccacct gacgtctaag aaaccattat tatcatgaca ttaacctata
     6721 aaaataggcg tatcacgagg ccctttcgtc
//
