LOCUS       sequence-431523-        3444 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Fragmid fragment: (N' terminus) for C>T base editing.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3444)
  AUTHORS   McGee AV, Liu YV, Griffith AL, Szegletes ZM, Wen B, Kraus C, Miller 
            NW, Steger RJ, Escude Velasco B, Bosch JA, Zirin JD, Viswanatha R, 
            Sontheimer EJ, Goodale A, Greene MA, Green TM, Doench JG
  TITLE     Modular vector assembly enables rapid assessment of emerging CRISPR 
            technologies.
  JOURNAL   Cell Genom. 2024 Mar 13;4(3):100519. doi: 
            10.1016/j.xgen.2024.100519.
  PUBMED    38484704
REFERENCE   2  (bases 1 to 3444)
  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: "Cell 
            Genom."; date: "2024-03-13"; volume: "4(3):100519. doi"; pages: " 
            10.1016/j.xgen.2024.100519"
COMMENT     Sequence Label: AA011
FEATURES             Location/Qualifiers
     source          1..3444
                     /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"
     regulatory      444..453
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             489..509
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             510..1193
                     /codon_start=1
                     /product="cytidine deaminase (C to U editing enzyme) from 
                     rat"
                     /label=APOBEC-1
                     /note="can use ssDNA as a substrate (Komor et al., 2016)"
                     /translation="SSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGG
                     RHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLS
                     RYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNE
                     AHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHIL
                     WATGLK"
     primer_bind     complement(1354..1370)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1354..1370)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1367..1389)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1378..1394
                     /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(1402..1408,1409..1426,1427..1432))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1447..1468
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1585..1602)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1756..2344)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1836..1855)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2522..3331)
                     /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     3245..3264
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3323..3342
                     /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 tgaagacgta ggagccacca tggccaaacg gacagccgac ggaagcgagt
      481 tcgagtcacc aaagaagaag cggaaagtct cctcagagac tgggcctgtc gccgtcgatc
      541 caaccctgcg ccgccggatt gaacctcacg agtttgaagt gttctttgac ccccgggagc
      601 tgagaaagga gacatgcctg ctgtacgaga tcaactgggg aggcaggcac tccatctgga
      661 ggcacacctc tcagaacaca aataagcacg tggaggtgaa cttcatcgag aagtttacca
      721 cagagcggta cttctgcccc aataccagat gtagcatcac atggtttctg agctggtccc
      781 cttgcggaga gtgtagcagg gccatcaccg agttcctgtc cagatatcca cacgtgacac
      841 tgtttatcta catcgccagg ctgtatcacc acgcagaccc aaggaatagg cagggcctgc
      901 gcgatctgat cagctccggc gtgaccatcc agatcatgac agagcaggag tccggctact
      961 gctggcggaa cttcgtgaat tattctccta gcaacgaggc ccactggcct aggtacccac
     1021 acctgtgggt gcgcctgtac gtgctggagc tgtattgcat catcctgggc ctgccccctt
     1081 gtctgaatat cctgcggaga aagcagcccc agctgacctt ctttacaatc gccctgcagt
     1141 cttgtcacta tcagaggctg ccaccccaca tcctgtgggc cacaggcctg aagtctggag
     1201 gatctagcgg aggatcctct ggcagcgaga caccaggaac aagcgagtca gcaacaccag
     1261 agagcagtgg cggcagcagc ggcggcagca gtcttcatcg gatcccgggc ccgtcgactg
     1321 cagaggcctg catgcaagct tggtgtaatc atggtcatag ctgtttcctg tgtgaaattg
     1381 ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg
     1441 tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc
     1501 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     1561 gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
     1621 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
     1681 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     1741 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     1801 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     1861 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     1921 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
     1981 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     2041 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     2101 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     2161 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct
     2221 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     2281 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     2341 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     2401 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
     2461 aaaatgaagt tttaaatcaa gcccaatctg aataatgtta caaccaatta accaattctg
     2521 attagaaaaa ctcatcgagc atcaaatgaa actgcaattt attcatatca ggattatcaa
     2581 taccatattt ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg aggcagttcc
     2641 ataggatggc aagatcctgg tatcggtctg cgattccgac tcgtccaaca tcaatacaac
     2701 ctattaattt cccctcgtca aaaataaggt tatcaagtga gaaatcacca tgagtgacga
     2761 ctgaatccgg tgagaatggc aaaagtttat gcatttcttt ccagacttgt tcaacaggcc
     2821 agccattacg ctcgtcatca aaatcactcg catcaaccaa accgttattc attcgtgatt
     2881 gcgcctgagc gagacgaaat acgcgatcgc tgttaaaagg acaattacaa acaggaatcg
     2941 aatgcaaccg gcgcaggaac actgccagcg catcaacaat attttcacct gaatcaggat
     3001 attcttctaa tacctggaat gctgtttttc cggggatcgc agtggtgagt aaccatgcat
     3061 catcaggagt acggataaaa tgcttgatgg tcggaagagg cataaattcc gtcagccagt
     3121 ttagtctgac catctcatct gtaacatcat tggcaacgct acctttgcca tgtttcagaa
     3181 acaactctgg cgcatcgggc ttcccataca agcgatagat tgtcgcacct gattgcccga
     3241 cattatcgcg agcccattta tacccatata aatcagcatc catgttggaa tttaatcgcg
     3301 gcctcgacgt ttcccgttga atatggctca taacacccct tgtattactg tttatgtaag
     3361 cagacagttt tattgttcat gatgatatat ttttatcttg tgcaatgtaa catcagagat
     3421 tttgagacac gggccagagc tgca
//
