LOCUS       sequence-440936-        5512 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of 2x-deadEGFP(A111V/L202S) and aptamer-gRNAs 
            to correct both mutations for MOBE3-4.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5512)
  AUTHORS   Cowan QT, Gu S, Gu W, Ranzau BL, Simonson TS, Komor AC
  TITLE     Development of multiplexed orthogonal base editor (MOBE) systems.
  JOURNAL   Nat Biotechnol. 2024 May 21. doi: 10.1038/s41587-024-02240-0.
  PUBMED    38773305
REFERENCE   2  (bases 1 to 5512)
  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 
            Biotechnol. 2024 May 21. doi: 10.1038/s41587-024-02240-0."
COMMENT     Sequence Label: Fluorescent reporter for MOBE3-4
FEATURES             Location/Qualifiers
     source          1..5512
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      659..1247
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1148..1167
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        1385..1633
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        1724..1742
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     enhancer        1949..2328
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2329..2532
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2483..2503
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2574..2593
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2574..2592
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      2600..2609
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2606..2608,2609..2611,2612..3325)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRVEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYSSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2651..2672)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2912..2931)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3259..3280
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3344..3361)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3350..3557
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     3566..3582
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3566..3582
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     misc_RNA        complement(3645..3720)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(3742..3990)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(4060..4076)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4060..4076)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   4083..4223
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4083..4212
                     /label=AAV2 ITR
     rep_origin      4298..4753
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4385..4404)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4595..4616
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4770..4789)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4889..4911
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4949..4967)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5035..5139
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5140..5208,5209..5512,1..488)
                     /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(5358..5377)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg
       61 agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac
      121 cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg
      181 caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat
      241 taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg
      301 ctggctggtt tattgctgat aaatctggag ccggtgagcg tggaagccgc ggtatcattg
      361 cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc
      421 aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc
      481 attggtaact gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt
      541 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt
      601 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
      661 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
      721 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
      781 gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca
      841 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
      901 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
      961 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     1021 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     1081 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     1141 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     1201 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     1261 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgtcg ctagctgtac
     1321 aaaaaagcag gctttaaagg aaccaattca gtcgactgga tccggtacca aggtcgggca
     1381 ggaagagggc ctatttccca tgattccttc atatttgcat atacgataca aggctgttag
     1441 agagataatt agaattaatt tgactgtaaa cacaaagata ttagtacaaa atacgtgacg
     1501 tagaaagtaa taatttcttg ggtagtttgc agttttaaaa ttatgtttta aaatggacta
     1561 tcatatgctt accgtaactt gaaagtattt cgatttcttg gctttatata tcttgtggaa
     1621 aggacgaaac accgctattc aagtacccag tccggtttta gagctagaaa tagcaagtta
     1681 aaataaggct agtccgttat caacttgaaa aagtggcacc ggcacatgag gatcacccat
     1741 gtgccggtgc tttttttaag cttgggccgc tcgaggtacc tctctacata tgacatgtga
     1801 gctgcttgtg tgttggaggt cgctgagtag tgcgcgagca aaatttaagc tacaacaagg
     1861 caaggcttga ccgacaattg catgaagaat ctgcttaggg ttaggcgttt tgcgctgctt
     1921 cgcgatgtac gggccagata tacgcgttga cattgattat tgactagtta ttaatagtaa
     1981 tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac ataacttacg
     2041 gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg
     2101 tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta
     2161 cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt
     2221 gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac cttatgggac
     2281 tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt gatgcggttt
     2341 tggcagtaca tcaatgggcg tggatagcgg tttgactcac ggggatttcc aagtctccac
     2401 cccattgacg tcaatgggag tttgttttgg caccaaaatc aacgggactt tccaaaatgt
     2461 cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat
     2521 ataagcagag ctggtttagt gaaccgtcag atccgctaga gatccgcggc cgctaatacg
     2581 actcactata gggagagccg ccaccatggt gagcaagggc gaggagctgt tcaccggggt
     2641 ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg
     2701 cgagggcgag ggcgatgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg
     2761 caagctgccc gtgccctggc ccaccctcgt gaccaccctg acctatggag tgcagtgctt
     2821 cagccgctac cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg
     2881 ctacgtccag gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgtaga
     2941 ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa
     3001 ggaggacggc aacatcctgg ggcacaagct ggagtacaac tacaacagcc acaacgtcta
     3061 tatcatggcc gacaagcaga agaacggcat caaggtgaac ttcaagatcc gccacaacat
     3121 cgaggacggc agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg
     3181 ccccgtgctg ctgcccgaca accactattc aagtacccag tccgcgctga gcaaagaccc
     3241 caacgagaag cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct
     3301 cggcatggac gagctgtaca agtaactaga gctcgctgat cagcctcgac tgtgccttct
     3361 agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc
     3421 actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt
     3481 cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagagaat
     3541 agcaggcatg ctggggagcg gccgctctag aactagtgga tcccccgggc tgcaggaatt
     3601 cgatacgaat cctgcaggaa aaaaagatgc tcgcaggcat tcaggcaccg actcggtgcc
     3661 actttttcaa gttgataacg gactagcctt attttaactt gctatttcta gctctaaaac
     3721 actacaagac ccgcgtagag cggtgtttcg tcctttccac aagatatata aagccaagaa
     3781 atcgaaatac tttcaagtta cggtaagcat atgatagtcc attttaaaac ataattttaa
     3841 aactgcaaac tacccaagaa attattactt tctacgtcac gtattttgta ctaatatctt
     3901 tgtgtttaca gtcaaattaa ttctaattat ctctctaaca gccttgtatc gtatatgcaa
     3961 atatgaagga atcatgggaa ataggccctc ttcctgcccg accttggtac cggatccagt
     4021 cgactgaatt ggttccttta aagcctgctt ttttgtacag tcatagctgt ttcctgggcc
     4081 gcaggaaccc ctagtgatgg agttggccac tccctctctg cgcgctcgct cgctcactga
     4141 ggccgggcga ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct cagtgagcga
     4201 gcgagcgcgc agctgcctgc aggggcgcct gatgcggtat tttctcctta cgcatctgtg
     4261 cggtatttca caccgcatac gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta
     4321 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccttagcg
     4381 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa
     4441 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc
     4501 aaaaaacttg atttgggtga tggttcacgt agtgggccat cgccctgata gacggttttt
     4561 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca
     4621 acactcaact ctatctcggg ctattctttt gatttataag ggattttgcc gatttcggtc
     4681 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta
     4741 acgtttacaa ttttatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc
     4801 cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca
     4861 tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg
     4921 tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat
     4981 gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga
     5041 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
     5101 ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt
     5161 gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg
     5221 ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg
     5281 gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg
     5341 agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag
     5401 caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca
     5461 gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc ca
//
