LOCUS       sequence-476153-        8958 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  All-in-one adenine base editor (ABE8e-nSaCas9) with gRNA targeting 
            NRF1-Ex7 splice site.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8958)
  TITLE     A Scalable Exon Deletion Platform Coupled To Single-Cell 
            Transcriptomics Reveals Exons Shaping Gene Expression Dynamics
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 8958)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pX462-ABE8e-nSaCas9-NRF1-ss-sgRNA
FEATURES             Location/Qualifiers
     source          1..8958
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             16..69
                     /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             70..669
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(70..89)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     566..586
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(697..714)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    703..910
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   919..1059
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   919..1048
                     /label=AAV2 ITR
     rep_origin      1134..1589
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1221..1240)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1431..1452
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(1606..1625)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1725..1747
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1785..1803)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1871..1975
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1976..2044,2045..2836)
                     /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(2194..2213)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3007..3595
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3496..3515
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        3657..3905
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     3657..3677
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     3828..3847
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        3927..4002
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     enhancer        4099..4384
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        4386..4663
                     /label=chicken beta-actin promoter
     intron          4664..4891
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      4903..4912
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4912..4977
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             8752..8778
                     /codon_start=1
                     /product="nuclear localization signal of human c-Myc 
                     proto-oncogene (Dang and Lee, 1988)"
                     /label=c-myc NLS
                     /translation="PAAKRVKLD"
     CDS             8788..8814
                     /codon_start=1
                     /product="nuclear localization signal of human c-Myc 
                     proto-oncogene (Dang and Lee, 1988)"
                     /label=c-myc NLS
                     /translation="PAAKRVKLD"
     CDS             8824..8850
                     /codon_start=1
                     /product="nuclear localization signal of human c-Myc 
                     proto-oncogene (Dang and Lee, 1988)"
                     /label=c-myc NLS
                     /translation="PAAKRVKLD"
     CDS             8860..8886
                     /codon_start=1
                     /product="nuclear localization signal of human c-Myc 
                     proto-oncogene (Dang and Lee, 1988)"
                     /label=c-myc NLS
                     /translation="PAAKRVKLD"
     CDS             8896..8922
                     /codon_start=1
                     /product="nuclear localization signal of human c-Myc 
                     proto-oncogene (Dang and Lee, 1988)"
                     /label=c-myc NLS
                     /translation="PAAKRVKLD"
     CDS             8932..8958
                     /codon_start=1
                     /product="nuclear localization signal of human c-Myc 
                     proto-oncogene (Dang and Lee, 1988)"
                     /label=c-myc NLS
                     /translation="PAAKRVKLD"
ORIGIN
        1 gaattcggca gtggagaggg cagaggaagt ctgctaacat gcggtgacgt cgaggagaat
       61 cctggcccaa tgaccgagta caagcccacg gtgcgcctcg ccacccgcga cgacgtcccc
      121 agggccgtac gcaccctcgc cgccgcgttc gccgactacc ccgccacgcg ccacaccgtc
      181 gatccggacc gccacatcga gcgggtcacc gagctgcaag aactcttcct cacgcgcgtc
      241 gggctcgaca tcggcaaggt gtgggtcgcg gacgacggcg ccgcggtggc ggtctggacc
      301 acgccggaga gcgtcgaagc gggggcggtg ttcgccgaga tcggcccgcg catggccgag
      361 ttgagcggtt cccggctggc cgcgcagcaa cagatggaag gcctcctggc gccgcaccgg
      421 cccaaggagc ccgcgtggtt cctggccacc gtcggagtct cgcccgacca ccagggcaag
      481 ggtctgggca gcgccgtcgt gctccccgga gtggaggcgg ccgagcgcgc cggggtgccc
      541 gccttcctgg agacctccgc gccccgcaac ctccccttct acgagcggct cggcttcacc
      601 gtcaccgccg acgtcgaggt gcccgaagga ccgcgcacct ggtgcatgac ccgcaagccc
      661 ggtgcctgag aattctaact agagctcgct gatcagcctc gactgtgcct tctagttgcc
      721 agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca
      781 ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta
      841 ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagag aatagcaggc
      901 atgctgggga gcggccgcag gaacccctag tgatggagtt ggccactccc tctctgcgcg
      961 ctcgctcgct cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg
     1021 cggcctcagt gagcgagcga gcgcgcagct gcctgcaggg gcgcctgatg cggtattttc
     1081 tccttacgca tctgtgcggt atttcacacc gcatacgtca aagcaaccat agtacgcgcc
     1141 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
     1201 tgccagcgcc ttagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
     1261 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
     1321 acggcacctc gaccccaaaa aacttgattt gggtgatggt tcacgtagtg ggccatcgcc
     1381 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt
     1441 gttccaaact ggaacaacac tcaactctat ctcgggctat tcttttgatt tataagggat
     1501 tttgccgatt tcggtctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa
     1561 ttttaacaaa atattaacgt ttacaatttt atggtgcact ctcagtacaa tctgctctga
     1621 tgccgcatag ttaagccagc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc
     1681 ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg
     1741 tcagaggttt tcaccgtcat caccgaaacg cgcgagacga aagggcctcg tgatacgcct
     1801 atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg
     1861 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     1921 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag
     1981 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt
     2041 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt
     2101 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga
     2161 acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat
     2221 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga
     2281 gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag
     2341 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg
     2401 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg
     2461 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt
     2521 agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg
     2581 gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc
     2641 ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg gaagccgcgg
     2701 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac
     2761 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact
     2821 gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa
     2881 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
     2941 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     3001 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     3061 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     3121 tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca
     3181 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     3241 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     3301 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     3361 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     3421 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     3481 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     3541 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     3601 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgtgagg
     3661 gcctatttcc catgattcct tcatatttgc atatacgata caaggctgtt agagagataa
     3721 ttggaattaa tttgactgta aacacaaaga tattagtaca aaatacgtga cgtagaaagt
     3781 aataatttct tgggtagttt gcagttttaa aattatgttt taaaatggac tatcatatgc
     3841 ttaccgtaac ttgaaagtat ttcgatttct tggctttata tatcttgtgg aaaggacgaa
     3901 acaccgttac tcacctggat aagtgagtta tagtactctg gaaacagaat ctactataac
     3961 aaggcaaaat gccgtgttta tctcgtcaac ttgttggcga gattttttgt tttagagcta
     4021 gaaatagcaa gttaaaataa ggctagtccg tttttagcgc gtgcgccaat tctgcagaca
     4081 aatggctcta gaggtacccg ttacataact tacggtaaat ggcccgcctg gctgaccgcc
     4141 caacgacccc cgcccattga cgtcaatagt aacgccaata gggactttcc attgacgtca
     4201 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
     4261 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt gtgcccagta
     4321 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
     4381 catggtcgag gtgagcccca cgttctgctt cactctcccc atctcccccc cctccccacc
     4441 cccaattttg tatttattta ttttttaatt attttgtgca gcgatggggg cggggggggg
     4501 gggggggcgc gcgccaggcg gggcggggcg gggcgagggg cggggcgggg cgaggcggag
     4561 aggtgcggcg gcagccaatc agagcggcgc gctccgaaag tttcctttta tggcgaggcg
     4621 gcggcggcgg cggccctata aaaagcgaag cgcgcggcgg gcgggagtcg ctgcgcgctg
     4681 ccttcgcccc gtgccccgct ccgccgccgc ctcgcgccgc ccgccccggc tctgactgac
     4741 cgcgttactc ccacaggtga gcgggcggga cggcccttct cctccgggct gtaattagct
     4801 gagcaagagg taagggttta agggatggtt ggttggtggg gtattaatgt ttaattacct
     4861 ggagcacctg cctgaaatca ctttttttca ggttggaccg gtgccaccat ggactataag
     4921 gaccacgacg gagactacaa ggatcatgat attgattaca aagacgatga cgataagatg
     4981 gcctctgagg tggagttttc ccacgagtac tggatgagac atgccctgac cctggccaag
     5041 agggcacggg atgagaggga ggtgcctgtg ggagccgtgc tggtgctgaa caatagagtg
     5101 atcggcgagg gctggaacag agccatcggc ctgcacgacc caacagccca tgccgaaatt
     5161 atggccctga gacagggcgg cctggtcatg cagaactaca gactgattga cgccaccctg
     5221 tacgtgacat tcgagccttg cgtgatgtgc gccggcgcca tgatccactc taggatcggc
     5281 cgcgtggtgt ttggcgtgag gaactcaaaa agaggcgccg caggctccct gatgaacgtg
     5341 ctgaactacc ccggcatgaa tcaccgcgtc gaaattaccg agggaatcct ggcagatgaa
     5401 tgtgccgccc tgctgtgcga tttctatcgg atgcctagac aggtgttcaa tgctcagaag
     5461 aaggcccaga gctccatcaa ctccggagga tctagcggag gatcctctgg cagcgagaca
     5521 ccaggaacaa gcgagtcagc aacaccagag agcagtggcg gcagcagcgg ggggtcagct
     5581 agcgctaagc gaaattacat tctggggctg gccattggca ttacatcagt gggctatggc
     5641 atcattgact acgagacaag ggacgtgatc gacgccggcg tgagactgtt caaggaggcc
     5701 aacgtggaga acaatgaggg ccggagatcc aagaggggag caaggcgcct gaagcggaga
     5761 aggcgccaca gaatccagag agtgaagaag ctgctgttcg attacaacct gctgaccgac
     5821 cactccgagc tgtctggcat caatccttat gaggccagag tgaagggcct gtcccagaag
     5881 ctgtctgagg aggagtttag cgccgccctg ctgcacctgg caaagaggag aggcgtgcac
     5941 aacgtgaatg aggtggagga ggacaccggc aacgagctgt ccacaaagga gcagatcagc
     6001 cgcaattcca aggccctgga ggagaagtat gtggccgagc tgcagctgga gcggctgaag
     6061 aaggatggcg aggtgagggg ctccatcaat cgcttcaaga cctctgacta cgtgaaggag
     6121 gccaagcagc tgctgaaggt gcagaaggcc taccaccagc tggatcagtc ctttatcgat
     6181 acatatatcg acctgctgga gacaaggcgc acatactatg agggaccagg agagggctct
     6241 cccttcggct ggaaggacat caaggagtgg tacgagatgc tgatgggcca ctgcacctat
     6301 tttccagagg agctgagaag cgtgaagtac gcctataacg ccgatctgta caacgccctg
     6361 aatgacctga acaacctggt catcaccagg gatgagaacg agaagctgga gtactatgag
     6421 aagttccaga tcatcgagaa cgtgttcaag cagaagaaga agcctacact gaagcagatc
     6481 gccaaggaga tcctggtgaa cgaggaggac atcaagggct accgcgtgac ctccacaggc
     6541 aagccagagt tcaccaatct gaaggtgtat cacgatatca aggacatcac agcccggaag
     6601 gagatcatcg agaacgccga gctgctggat cagatcgcca agatcctgac catctatcag
     6661 agctccgagg acatccagga ggagctgacc aacctgaata gcgagctgac acaggaggag
     6721 atcgagcaga tcagcaatct gaagggctac accggcacac acaacctgag cctgaaggcc
     6781 atcaatctga tcctggatga gctgtggcac acaaacgaca atcagatcgc catctttaac
     6841 cggctgaagc tggtgccaaa gaaggtggac ctgtcccagc agaaggagat cccaaccaca
     6901 ctggtggacg atttcatcct gtctcccgtg gtgaagcgga gcttcatcca gagcatcaaa
     6961 gtgatcaacg ccatcatcaa gaagtacggc ctgcccaatg atatcatcat cgagctggcc
     7021 agggagaaga actccaagga cgcccagaag atgatcaatg agatgcagaa gaggaaccgc
     7081 cagaccaatg agcggatcga ggagatcatc agaaccacag gcaaggagaa cgccaagtac
     7141 ctgatcgaga agatcaagct gcacgatatg caggagggca agtgtctgta ttctctggag
     7201 gccatccctc tggaggacct gctgaacaat ccattcaact acgaggtgga tcacatcatc
     7261 ccccggagcg tgagcttcga caattctttt aacaataagg tgctggtgaa gcaggaggag
     7321 aacagcaaga agggcaatag gacccctttc cagtacctgt ctagctccga ttctaagatc
     7381 agctacgaga cattcaagaa gcacatcctg aatctggcca agggcaaggg ccgcatcagc
     7441 aagaccaaga aggagtacct gctggaggag cgggacatca acagattctc cgtgcagaag
     7501 gacttcatca accggaatct ggtggacacc agatacgcca cacgcggcct gatgaatctg
     7561 ctgcggtctt atttcagagt gaacaatctg gatgtgaagg tgaagagcat caacggcggc
     7621 ttcacctcct ttctgcggag aaagtggaag tttaagaagg agcgcaacaa gggctataag
     7681 caccacgccg aggatgccct gatcatcgcc aatgccgact tcatctttaa ggagtggaag
     7741 aagctggaca aggccaagaa agtgatggag aaccagatgt tcgaggagaa gcaggccgag
     7801 agcatgcccg agatcgagac agagcaggag tacaaggaga ttttcatcac acctcaccag
     7861 atcaagcaca tcaaggactt caaggactac aagtattctc acagggtgga taagaagccc
     7921 aaccgcaagc tgatcaatga caccctgtat agcacacgga aggacgataa gggcaatacc
     7981 ctgatcgtga acaatctgaa cggcctgtac gacaaggata atgacaagct gaagaagctg
     8041 atcaacaagt ctcccgagaa gctgctgatg taccaccacg atcctcagac atatcagaag
     8101 ctgaagctga tcatggagca gtacggcgac gagaagaacc cactgtataa gtactatgag
     8161 gagacaggca actacctgac aaagtatagc aagaaggata atggccccgt gatcaagaag
     8221 atcaagtact atggcaacaa gctgaatgcc cacctggaca tcaccgacga ttaccctaac
     8281 tctcgcaata aggtggtgaa gctgagcctg aagccatacc ggttcgacgt gtacctggac
     8341 aacggcgtgt ataagtttgt gacagtgaag aatctggatg tgatcaagaa ggagaactac
     8401 tatgaggtga acagcaagtg ctacgaggag gccaagaagc tgaagaagat cagcaaccag
     8461 gccgagttca tcgcctcttt ttacaagaat gacctgatca agatcaatgg cgagctgtat
     8521 agagtgatcg gcgtgaacaa tgatctgctg aacagaatcg aagtgaatat gatcgacatc
     8581 acctacaggg agtatctgga gaacatgaat gataagaggc cccctcatat catcaagacc
     8641 atcgcctcta agacacagag catcaagaag tacagcacag acatcctggg gaacctgtat
     8701 gaagtcaaga gcaagaaaca tcctcagatt atcaagaaag gcagaattgg gcctgctgct
     8761 aaacgtgtta agcttgatgg gggtagcccg gcagccaaga gagtcaaact cgacggcggc
     8821 tccccagcgg caaaaagggt gaaactagac gggggtagcc ccgccgcgaa gcgtgtaaag
     8881 ctggatggag gatcgcctgc ggctaagcga gtcaaattag acggtgggtc accggctgca
     8941 aagcgcgtca aattggat
//
