LOCUS       sequence-458363-        3359 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Fragmid fragment: (guide cassette) CBE activity-based selection 
            cloning.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3359)
  AUTHORS   Kaplan EG, Steger RJ, Shah ST, Drepanos LM, Griffith AL, Reint G, 
            Doench JG
  TITLE     Activity-based selection for enhanced base editor mutational 
            scanning.
  JOURNAL   Nat Genet. 2025 Nov;57(11):2920-2929. doi: 
            10.1038/s41588-025-02366-0. Epub 2025 Oct 14.
  PUBMED    41087678
REFERENCE   2  (bases 1 to 3359)
  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"; doi: 
            "10.1038/s41588-025-02366-0"; journalName: "Nat Genet"; date: 
            "2025-11"; volume: "57"; issue: "11"; pages: "2920-2929"
COMMENT     Sequence Label: AA568
FEATURES             Location/Qualifiers
     source          1..3359
                     /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"
     primer_bind     364..386
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     promoter        444..692
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     444..464
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     615..634
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     promoter        complement(983..1197)
                     /label=H1 promoter
                     /note="human H1 RNA promoter"
     primer_bind     complement(1043..1062)
                     /label=H1
                     /note="Human H1 promoter, forward primer"
     primer_bind     complement(1269..1285)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1269..1285)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1282..1304)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1293..1309
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(1317..1323,1324..1341,1342..1347))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1362..1383
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1500..1517)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1671..2259)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1751..1770)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2437..3246)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     3160..3179
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3238..3257
                     /label=pENTR-R
                     /note="pENTR vectors, reverse 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 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagagaatt cgagctcggt acctcgcgaa
      421 tacatctaga tgaagacgtc agagagggcc tatttcccat gattccttca tatttgcata
      481 tacgatacaa ggctgttaga gagataatta gaattaattt gactgtaaac acaaagatat
      541 tagtacaaaa tacgtgacgt agaaagtaat aatttcttgg gtagtttgca gttttaaaat
      601 tatgttttaa aatggactat catatgctta ccgtaacttg aaagtatttc gatttcttgg
      661 ctttatatat cttgtggaaa ggacgaaaca ccggagacgc ttttttcgtc tcagtttcag
      721 agctaagcac aagagtgcat agcaagttga aataaggcta gtccgtttac aacttgaaaa
      781 agtggcaccc gagtcgggtg cttttttgaa agctccacag tgcaggggaa agaatagtag
      841 agacccaatg gaaaaaagca ccgactcggt gccactttgc tgtttccagc aaagttgata
      901 acggactagc cttatttaaa cttgctatgc tgtttccagc atagctctta aacccgttca
      961 agttacacgc ctgcgggaaa gagtggtctc atacagaact tataagattc ccaaatccaa
     1021 agacatttca cgtttatggt gatttcccag aacacatagc gacatgcaaa tattgcaggg
     1081 cgccactccc ctgtccctca cagccatctt cctgccaggg cgcacgcgcg ctgggtgttc
     1141 ccgcctagtg acactgggcc cgcgattcct tggagcgggt tgatgacgtc agcgttcgaa
     1201 cacgcgtctt catcggatcc cgggcccgtc gactgcagag gcctgcatgc aagcttggtg
     1261 taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac
     1321 atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca
     1381 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
     1441 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc
     1501 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
     1561 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
     1621 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     1681 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     1741 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     1801 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     1861 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     1921 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     1981 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     2041 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     2101 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     2161 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     2221 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     2281 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
     2341 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaagccca
     2401 atctgaataa tgttacaacc aattaaccaa ttctgattag aaaaactcat cgagcatcaa
     2461 atgaaactgc aatttattca tatcaggatt atcaatacca tatttttgaa aaagccgttt
     2521 ctgtaatgaa ggagaaaact caccgaggca gttccatagg atggcaagat cctggtatcg
     2581 gtctgcgatt ccgactcgtc caacatcaat acaacctatt aatttcccct cgtcaaaaat
     2641 aaggttatca agtgagaaat caccatgagt gacgactgaa tccggtgaga atggcaaaag
     2701 tttatgcatt tctttccaga cttgttcaac aggccagcca ttacgctcgt catcaaaatc
     2761 actcgcatca accaaaccgt tattcattcg tgattgcgcc tgagcgagac gaaatacgcg
     2821 atcgctgtta aaaggacaat tacaaacagg aatcgaatgc aaccggcgca ggaacactgc
     2881 cagcgcatca acaatatttt cacctgaatc aggatattct tctaatacct ggaatgctgt
     2941 ttttccgggg atcgcagtgg tgagtaacca tgcatcatca ggagtacgga taaaatgctt
     3001 gatggtcgga agaggcataa attccgtcag ccagtttagt ctgaccatct catctgtaac
     3061 atcattggca acgctacctt tgccatgttt cagaaacaac tctggcgcat cgggcttccc
     3121 atacaagcga tagattgtcg cacctgattg cccgacatta tcgcgagccc atttataccc
     3181 atataaatca gcatccatgt tggaatttaa tcgcggcctc gacgtttccc gttgaatatg
     3241 gctcataaca ccccttgtat tactgtttat gtaagcagac agttttattg ttcatgatga
     3301 tatattttta tcttgtgcaa tgtaacatca gagattttga gacacgggcc agagctgca
//
