LOCUS       sequence-458371-        3371 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Fragmid fragment: (2A Selection) CBE activity-based selection 
            intron.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3371)
  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 3371)
  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: AA516
FEATURES             Location/Qualifiers
     source          1..3371
                     /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"
     CDS             444..500
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     primer_bind     1091..1111
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(1281..1297)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1281..1297)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1294..1316)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1305..1321
                     /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(1329..1335,1336..1353,1354..1359))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1374..1395
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1512..1529)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1683..2271)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1763..1782)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2449..3258)
                     /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     3172..3191
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3250..3269
                     /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 tgaagacgga agcgcaacaa acttctctct gctgaaacaa gccggagatg
      481 tcgaagagaa tcctggaccg accgagtaca agcccacggt gcgcctcgcc acccgcgacg
      541 acgtccccag ggccgtacgc accctcgccg ccgcgttcgc cgactacccc gccacgcgcc
      601 acaccgtcga tccggaccgc cacatcgagc gggtcaccga gctgcaagaa ctcttcctca
      661 cgcgcgtcgg gctcgacatc ggcaaggtgt gggtcgcgga cgacggcgcc gccgtggcgg
      721 tctggaccac gccggagagc gtcgaagcgg gggcggtgtt cgccgagatc ggcccgcgca
      781 tggccgagtt gagcggttcc cggctggcag gcgtgtaact tgaacggggg ttttaattca
      841 ggtcccggat ccggtggtgg tgcaaatcaa agaactgctc ctcagtggat gttgccttta
      901 cttctagcgc agcaacagat ggaaggcctc ctggcgccgc accggcccaa ggagcccgcg
      961 tggttcctgg ccaccgtcgg agtctcgccc gaccaccagg gcaagggtct gggcagcgcc
     1021 gtcgtgctcc ccggagtgga ggcggccgag cgcgccgggg tgcccgcctt cctggagacc
     1081 tccgcgcccc gcaacctccc cttctacgag cggctcggct tcaccgtcac cgccgacgtc
     1141 gaggtgcccg aaggaccgcg cacctggtgc atgacccgca agcccggtgc cggatcctga
     1201 taaattccag tcttcggaac caaatcggat cccgggcccg tcgactgcag aggcctgcat
     1261 gcaagcttgg tgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca
     1321 attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg
     1381 agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
     1441 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     1501 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
     1561 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     1621 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     1681 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     1741 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     1801 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     1861 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     1921 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     1981 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     2041 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     2101 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
     2161 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     2221 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     2281 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     2341 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     2401 aaatcaagcc caatctgaat aatgttacaa ccaattaacc aattctgatt agaaaaactc
     2461 atcgagcatc aaatgaaact gcaatttatt catatcagga ttatcaatac catatttttg
     2521 aaaaagccgt ttctgtaatg aaggagaaaa ctcaccgagg cagttccata ggatggcaag
     2581 atcctggtat cggtctgcga ttccgactcg tccaacatca atacaaccta ttaatttccc
     2641 ctcgtcaaaa ataaggttat caagtgagaa atcaccatga gtgacgactg aatccggtga
     2701 gaatggcaaa agtttatgca tttctttcca gacttgttca acaggccagc cattacgctc
     2761 gtcatcaaaa tcactcgcat caaccaaacc gttattcatt cgtgattgcg cctgagcgag
     2821 acgaaatacg cgatcgctgt taaaaggaca attacaaaca ggaatcgaat gcaaccggcg
     2881 caggaacact gccagcgcat caacaatatt ttcacctgaa tcaggatatt cttctaatac
     2941 ctggaatgct gtttttccgg ggatcgcagt ggtgagtaac catgcatcat caggagtacg
     3001 gataaaatgc ttgatggtcg gaagaggcat aaattccgtc agccagttta gtctgaccat
     3061 ctcatctgta acatcattgg caacgctacc tttgccatgt ttcagaaaca actctggcgc
     3121 atcgggcttc ccatacaagc gatagattgt cgcacctgat tgcccgacat tatcgcgagc
     3181 ccatttatac ccatataaat cagcatccat gttggaattt aatcgcggcc tcgacgtttc
     3241 ccgttgaata tggctcataa caccccttgt attactgttt atgtaagcag acagttttat
     3301 tgttcatgat gatatatttt tatcttgtgc aatgtaacat cagagatttt gagacacggg
     3361 ccagagctgc a
//
