LOCUS       sequence-257486-        4349 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  gRNA expression vector for C-to-T base editing reporter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4349)
  AUTHORS   Sakata RC, Ishiguro S, Mori H, Tanaka M, Tatsuno K, Ueda H, Yamamoto
            S, Seki M, Masuyama N, Nishida K, Nishimasu H, Arakawa K, Kondo A, 
            Nureki O, Tomita M, Aburatani H, Yachie N
  TITLE     Base editors for simultaneous introduction of C-to-T and A-to-G 
            mutations.
  JOURNAL   Nat Biotechnol. 2020 Jun 1. pii: 10.1038/s41587-020-0509-0. doi: 
            10.1038/s41587-020-0509-0.
  PUBMED    32483365
REFERENCE   2  (bases 1 to 4349)
  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. 2020 Jun 1. pii: 10.1038/s41587-020-0509-0. doi: 
            10.1038/s41587-020-0509-0."
COMMENT     Sequence Label: pSI-535
FEATURES             Location/Qualifiers
     source          1..4349
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    58..79
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(94..99,100..117,118..124)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    132..148
                     /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)."
     primer_bind     137..159
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     156..172
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     156..172
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        193..211
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        739..987
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     739..759
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     910..929
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        1009..1084
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     polyA_signal    1119..1200
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1156..1175)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(1730..1749)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1731..1749)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1756..1773)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1756..1772)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1765..1787)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      1914..2369
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2001..2020)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2211..2232
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2395..2499
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2500..2568,2569..3360)
                     /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(2718..2737)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3531..4119
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4020..4039
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4273..4290
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa
       61 tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc cggctcgtat
      121 gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg accatgatta
      181 cgccaagctc gaaattaacc ctcactaaag ggaacaaaag ctggagctga tttaaatggg
      241 gacagccccc ccccaaagcc cccagggatg taattacgtc cctcccccgc tagggggcag
      301 cagcgagccg cccggggctc cgctccggtc cggcgctccc cccgcatccc cgagccggca
      361 gcgtgcgggg acagcccggg cacggggaag gtggcacggg atcgctttcc tctgaacgct
      421 tctcgctgct ctttgagcct gcagacacct ggggggatac ggggaaaaag ctttaggctg
      481 agtgcacggg gacagccccc ccccaaagcc cccagggatg taattacgtc cctcccccgc
      541 tagggggcag cagcgagccg cccggggctc cgctccggtc cggcgctccc cccgcatccc
      601 cgagccggca gcgtgcgggg acagcccggg cacggggaag gtggcacggg atcgctttcc
      661 tctgaacgct tctcgctgct ctttgagcct gcagacacct ggggggatac ggggaaaaag
      721 ctttaggctg aagatctaga gggcctattt cccatgattc cttcatattt gcatatacga
      781 tacaaggctg ttagagagat aattagaatt aatttgactg taaacacaaa gatattagta
      841 caaaatacgt gacgtagaaa gtaataattt cttgggtagt ttgcagtttt aaaattatgt
      901 tttaaaatgg actatcatat gcttaccgta acttgaaagt atttcgattt cttggcttta
      961 tatatcttgt ggaaaggacg aaacaccgca cggtcaccct gacacgctgt tttagagcta
     1021 gaaatagcaa gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg
     1081 gtgctttttt gaattcctga cgcacaattg ttgttgttaa cttgtttatt gcagcttata
     1141 atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc
     1201 ctcgaatcac tagtggggac agcccccccc caaagccccc agggatgtaa ttacgtccct
     1261 cccccgctag ggggcagcag cgagccgccc ggggctccgc tccggtccgg cgctcccccc
     1321 gcatccccga gccggcagcg tgcggggaca gcccgggcac ggggaaggtg gcacgggatc
     1381 gctttcctct gaacgcttct cgctgctctt tgagcctgca gacacctggg gggatacggg
     1441 gaaaaagctt taggctgagt gcacggggac agcccccccc caaagccccc agggatgtaa
     1501 ttacgtccct cccccgctag ggggcagcag cgagccgccc ggggctccgc tccggtccgg
     1561 cgctcccccc gcatccccga gccggcagcg tgcggggaca gcccgggcac ggggaaggtg
     1621 gcacgggatc gctttcctct gaacgcttct cgctgctctt tgagcctgca gacacctggg
     1681 gggatacggg gaaaaagctt taggctgagg cgcgcctgta cccaattcgc cctatagtga
     1741 gtcgtattac aattcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt
     1801 tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga
     1861 ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat gggacgcgcc
     1921 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
     1981 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
     2041 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
     2101 acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc
     2161 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt
     2221 gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat
     2281 tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa
     2341 ttttaacaaa atattaacgc ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga
     2401 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
     2461 ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt
     2521 gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg
     2581 ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg
     2641 gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg
     2701 agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag
     2761 caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca
     2821 gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg
     2881 agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc
     2941 gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg
     3001 aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg
     3061 ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac
     3121 tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg
     3181 tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg
     3241 gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact
     3301 atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa
     3361 ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt
     3421 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
     3481 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     3541 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     3601 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     3661 cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct
     3721 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     3781 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     3841 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     3901 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     3961 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     4021 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     4081 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     4141 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct
     4201 gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga
     4261 acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat acgcaaaccg
     4321 cctctccccg cgcgttggcc gattcatta
//
