LOCUS       sequence-496999-        3503 bp    DNA     circular SYN 13-JAN-2026
DEFINITION  Expresses an sgRNA to target the KanR* site on the engineered E. 
            coli MG1655 strain and contains the Repair Template to replace the 
            KanR* on the genome of E. coli MG1655.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3503)
  AUTHORS   Gupta D, Patinios C, Bassett HV, Kibe A, Collins SP, Kamm C, Wang Y,
            Zhao C, Vollen K, Toussaint C, Calvin I, Cullot G, Aird EJ, Polkoff 
            KM, Nguyen-Vo TP, Migur A, Schut F, Al'Abri IS, Achmedov T, Del Re 
            A, Corn JE, Saliba AE, Crook N, Stepanova AN, Alonso JM, Beisel CL
  TITLE     Targeted DNA ADP-ribosylation triggers templated repair in bacteria 
            and base mutagenesis in eukaryotes.
  JOURNAL   Nat Biotechnol. 2025 Sep 4. doi: 10.1038/s41587-025-02802-w.
  PUBMED    40908325
REFERENCE   2  (bases 1 to 3503)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jan 13, 2026 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Biotechnol. 2025 Sep 4. doi: 10.1038/s41587-025-02802-w."
COMMENT     Sequence Label: pCBS-4803
FEATURES             Location/Qualifiers
     source          1..3503
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(472..1284)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="SHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGK
                     PDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKT
                     AFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDAS
                     DFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIA
                     DRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(472..1284)
                     /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="SHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGK
                     PDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKT
                     AFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDAS
                     DFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIA
                     DRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     1195..1214
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        1293..1327
                     /label=J23119 promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119)"
     misc_RNA        1348..1423
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     rep_origin      complement(1821..2409)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1901..1920)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        2512..2616
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2617..2685,2686..3477)
                     /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(2835..2854)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt
       61 tgataatcgc ggccgctgta ctgcagtgca ctagttggac tgttgtgata tgccatatat
      121 gcagtggtgt tcgccggcgc tggttccggc tgtcttgctt agttgtgagt cgccaggcag
      181 tcatcggtat gatctgtaca tgattcgtca gtgtgagcac agaaaagccc gcctttcggc
      241 gggctttgac cactgacgag cagatttctg taagatattc gtgcggaagg gatcactaaa
      301 gaagatctac aacaaaaggc aggtaaaccc gtagaaaccg tgccgcagat ttttgtcgat
      361 cagcaacata tcggcggcta taccgatttt gctgcatggg tgaaagaaaa tctggacgcc
      421 tgatcgtctg acaagcccag ccgcgtgtta caaccaatta accaattctg attagaaaaa
      481 ctcatcgagc atcaaatgaa actgcaattt attcatatca ggattatcaa taccatattt
      541 ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg aggcagttcc ataggatggc
      601 aagatcctgg tatcggtctg cgattccgac tcgtccaaca tcaatacaac ctattaattt
      661 cccctcgtca aaaataaggt tatcaagtga gaaatcacca tgagtgacga ctgaatccgg
      721 tgagaatggc aaaagcttat gcatttcttt ccagacttgt tcaacaggcc agccattacg
      781 ctcgtcatca aaatcactcg catcaaccaa accgttattc attcgtgatt gcgcctgagc
      841 gaggcgaaat acgcgatcgc tgttaaaagg acaattacaa acaggaatcg aatgcaaccg
      901 gcgcaggaac actgccagcg catcaacaat attttcacct gaatcaggat attcttctaa
      961 tacctggaat gctgttttcc cggggatcgc agtggtgagt aaccatgcat catcaggagt
     1021 acggataaaa tgcttgatgg tcggaagagg cataaattcc gtcagccagt ttagtctgac
     1081 catctcatct gtaacatcat tggcaacgct acctttgcca tgtttcagaa acaactctgg
     1141 cgcatcgggc ttcccataca atcgatagat tgtcgcacct gattgcccga cattatcgcg
     1201 agcccattta tacccatata aatcagcatc catgttggaa tttaatcgcg gcctggagca
     1261 agacgtttcc cgttgaatat ggctagggaa cgttgacagc tagctcagtc ctaggtataa
     1321 tgctagctcc tttcctactc aacaggcgtt ttagagctag aaatagcaag ttaaaataag
     1381 gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttt ccggcttatc
     1441 ggtcagtttc acctgattta cgtaaaaacc cgcttcggcg ggtttttgct tttggagggg
     1501 cagaaagatg aatgactgtc cacgacgcta tacccaaaag aaagctgtga gggctcacgt
     1561 cttatttggg caaaggagtc agcggatcac cgtactatgt gtgagtgctc ctatcgcgct
     1621 tgctcctgtg agatacgatg tggccgtgtc tgaccgtggt gcgtcatcta ttcactcact
     1681 tggctgcgat ttattctaga tgcgaattca ggggcggccg cacggttatc cacagaatca
     1741 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     1801 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     1861 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     1921 cctggaagct ccctcgtgcg ctctcctgtt ctgaccctgc cgcttaccgg atacctgtcc
     1981 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     2041 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     2101 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     2161 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     2221 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc
     2281 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     2341 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     2401 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     2461 tcacgttaag ggattttggt catgacaggt ggcacttttc ggggaaatgt gcgcggaacc
     2521 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc
     2581 tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc
     2641 gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg
     2701 gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat
     2761 ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc
     2821 acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa
     2881 ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa
     2941 aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt
     3001 gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct
     3061 tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat
     3121 gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg
     3181 cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg
     3241 atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt
     3301 attgctgata aatctggagc cggtgagcgt gggtcccgcg gtatcattgc agcactgggg
     3361 ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg
     3421 gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg
     3481 tcagaccaag tttactcata tat
//
