LOCUS       sequence-257489-        4349 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  gRNA expression vector for A-to-G 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-359
FEATURES             Location/Qualifiers
     source          1..4349
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     9..26
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    143..164
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(179..184,185..202,203..209)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    217..233
                     /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     222..244
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     241..257
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     241..257
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        278..296
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        824..1072
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     824..844
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     995..1014
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        1094..1169
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     polyA_signal    1204..1285
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1241..1260)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(1815..1834)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1816..1834)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1841..1858)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1841..1857)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1850..1872)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      1999..2454
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2086..2105)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2296..2317
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2480..2584
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2585..2653,2654..3445)
                     /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(2803..2822)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3616..4204
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4105..4124
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cccaatacgc aaaccgcctc
       61 tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag
      121 cgggcagtga gcgcaacgca attaatgtga gttagctcac tcattaggca ccccaggctt
      181 tacactttat gcttccggct cgtatgttgt gtggaattgt gagcggataa caatttcaca
      241 caggaaacag ctatgaccat gattacgcca agctcgaaat taaccctcac taaagggaac
      301 aaaagctgga gctgatttaa atggggacag ccccccccca aagcccccag ggatgtaatt
      361 acgtccctcc cccgctaggg ggcagcagcg agccgcccgg ggctccgctc cggtccggcg
      421 ctccccccgc atccccgagc cggcagcgtg cggggacagc ccgggcacgg ggaaggtggc
      481 acgggatcgc tttcctctga acgcttctcg ctgctctttg agcctgcaga cacctggggg
      541 gatacgggga aaaagcttta ggctgagtgc acggggacag ccccccccca aagcccccag
      601 ggatgtaatt acgtccctcc cccgctaggg ggcagcagcg agccgcccgg ggctccgctc
      661 cggtccggcg ctccccccgc atccccgagc cggcagcgtg cggggacagc ccgggcacgg
      721 ggaaggtggc acgggatcgc tttcctctga acgcttctcg ctgctctttg agcctgcaga
      781 cacctggggg gatacgggga aaaagcttta ggctgaagat ctagagggcc tatttcccat
      841 gattccttca tatttgcata tacgatacaa ggctgttaga gagataatta gaattaattt
      901 gactgtaaac acaaagatat tagtacaaaa tacgtgacgt agaaagtaat aatttcttgg
      961 gtagtttgca gttttaaaat tatgttttaa aatggactat catatgctta ccgtaacttg
     1021 aaagtatttc gatttcttgg ctttatatat cttgtggaaa ggacgaaaca ccgccttatg
     1081 accctgacac gctgttttag agctagaaat agcaagttaa aataaggcta gtccgttatc
     1141 aacttgaaaa agtggcaccg agtcggtgct tttttgaatt cctgacgcac aattgttgtt
     1201 gttaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc
     1261 acaaataaag catttttttc actgcctcga atcactagtg gggacagccc ccccccaaag
     1321 cccccaggga tgtaattacg tccctccccc gctagggggc agcagcgagc cgcccggggc
     1381 tccgctccgg tccggcgctc cccccgcatc cccgagccgg cagcgtgcgg ggacagcccg
     1441 ggcacgggga aggtggcacg ggatcgcttt cctctgaacg cttctcgctg ctctttgagc
     1501 ctgcagacac ctggggggat acggggaaaa agctttaggc tgagtgcacg gggacagccc
     1561 ccccccaaag cccccaggga tgtaattacg tccctccccc gctagggggc agcagcgagc
     1621 cgcccggggc tccgctccgg tccggcgctc cccccgcatc cccgagccgg cagcgtgcgg
     1681 ggacagcccg ggcacgggga aggtggcacg ggatcgcttt cctctgaacg cttctcgctg
     1741 ctctttgagc ctgcagacac ctggggggat acggggaaaa agctttaggc tgaggcgcgc
     1801 ctgtacccaa ttcgccctat agtgagtcgt attacaattc actggccgtc gttttacaac
     1861 gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt
     1921 tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca
     1981 gcctgaatgg cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
     2041 ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct
     2101 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
     2161 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg
     2221 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt
     2281 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg
     2341 tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc
     2401 tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttaggtgg
     2461 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
     2521 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa
     2581 gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct
     2641 tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg
     2701 tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg
     2761 ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt
     2821 atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga
     2881 cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga
     2941 attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac
     3001 gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg
     3061 ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac
     3121 gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct
     3181 agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct
     3241 gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg
     3301 gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat
     3361 ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg
     3421 tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat
     3481 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct
     3541 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
     3601 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     3661 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     3721 gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta
     3781 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     3841 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     3901 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     3961 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     4021 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     4081 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     4141 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     4201 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
     4261 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg
     4321 agctgatacc gctcgccgca gccgaacga
//
