LOCUS       sequence-325941-        4883 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  30bp(mismatch17) cas13d gRNA for 1405 editing .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4883)
  AUTHORS   Liu Y, Mao S, Huang S, Li Y, Chen Y, Di M, Huang X, Lv J, Wang X, Ge
            J, Shen S, Zhang X, Liu D, Huang X, Chi T
  TITLE     REPAIRx, a specific yet highly efficient programmable A > I RNA base
            editor.
  JOURNAL   EMBO J. 2020 Nov 16;39(22):e104748. doi: 10.15252/embj.2020104748. 
            Epub 2020 Oct 15.
  PUBMED    33058207
REFERENCE   2  (bases 1 to 4883)
  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: "EMBO J."; 
            date: "2020-11-16"; volume: "39(22):e104748. doi"; pages: " 
            10.15252/embj.2020104748. Epub 2020 Oct 15"
COMMENT     Sequence Label: TC1078-30
FEATURES             Location/Qualifiers
     source          1..4883
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(57..645)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(137..156)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(816..1607,1608..1676))
                     /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     1439..1458
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1677..1781)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      1808..2263
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1895..1914)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2105..2126
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     polyA_signal    2394..2442
                     /note="synthetic polyadenylation signal"
     misc_feature    2456..2547
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     2496..2515
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
     promoter        2626..2874
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2626..2646
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2797..2816
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2876..2911
                     /label=RfxCas13d DR36
                     /note="36-base direct repeat for an RfxCas13d pre-gRNA 
                     (Konermann et al., 2018)"
     misc_RNA        2942..2977
                     /label=RfxCas13d DR36
                     /note="36-base direct repeat for an RfxCas13d pre-gRNA 
                     (Konermann et al., 2018)"
     misc_feature    3107..3224
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        3273..3783
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     3702..3720
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             3805..4404
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(3805..3824)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4301..4321
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    4551..4672
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4562..4581)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4616..4635
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
ORIGIN
        1 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
       61 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
      121 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
      181 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
      241 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
      301 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
      361 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
      421 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
      481 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
      541 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
      601 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
      661 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
      721 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
      781 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
      841 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
      901 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagat
      961 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     1021 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     1081 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
     1141 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     1201 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     1261 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     1321 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     1381 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     1441 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     1501 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     1561 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     1621 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     1681 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     1741 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     1801 ccacctgacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc
     1861 gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt
     1921 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc
     1981 cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt
     2041 agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt
     2101 aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt
     2161 gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa
     2221 aaatttaacg cgaattttaa caaaatatta acgcttacaa tttgccattc gccattcagg
     2281 ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt cgctattacg ccagcccaag
     2341 ctaccatgat aagtaagtaa tattaaggta cgggaggtac ttggagcggc cgcaataaaa
     2401 tatctttatt ttcattacat ctgtgtgttg gttttttgtg tgaatcgata gtactaacat
     2461 acgctctcca tcaaaacaaa acgaaacaaa acaaactagc aaaataggct gtccccagtg
     2521 caagtgcagg tgccagaaca tttctctatc gataggtacc gattagtgaa cggatctcga
     2581 cggtatcgat cacgagacta gcctcgagcg gccgccccct tcaccgaggg cctatttccc
     2641 atgattcctt catatttgca tatacgatac aaggctgtta gagagataat tggaattaat
     2701 ttgactgtaa acacaaagat attagtacaa aatacgtgac gtagaaagta ataatttctt
     2761 gggtagtttg cagttttaaa attatgtttt aaaatggact atcatatgct taccgtaact
     2821 tgaaagtatt tcgatttctt ggctttatat atcttgtgga aaggacgaaa caccgcaagt
     2881 aaacccctac caactggtcg gggtttgaaa ccatgcagac gcggttccac tcgcagccac
     2941 acaagtaaac ccctaccaac tggtcggggt ttgaaacgaa gacttttttt ttcgcttcct
     3001 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     3061 aggcggtaat acggtcctcg agacaaatgg cagtattcat ccacaatttt aaaagaaaag
     3121 gggggattgg ggggtacagt gcaggggaaa gaatagtaga cataatagca acagacatac
     3181 aaactaaaga attacaaaaa caaattacaa aaattcaaaa ttttcgggtt tattacaggg
     3241 acagcagaga tccactttgg ccgcggctcg agggggttgg ggttgcgcct tttccaaggc
     3301 agccctgggt ttgcgcaggg acgcggctgc tctgggcgtg gttccgggaa acgcagcggc
     3361 gccgaccctg ggactcgcac attcttcacg tccgttcgca gcgtcacccg gatcttcgcc
     3421 gctacccttg tgggcccccc ggcgacgctt cctgctccgc ccctaagtcg ggaaggttcc
     3481 ttgcggttcg cggcgtgccg gacgtgacaa acggaagccg cacgtctcac tagtaccctc
     3541 gcagacggac agcgccaggg agcaatggca gcgcgccgac cgcgatgggc tgtggccaat
     3601 agcggctgct cagcagggcg cgccgagagc agcggccggg aaggggcggt gcgggaggcg
     3661 gggtgtgggg cggtagtgtg ggccctgttc ctgcccgcgc ggtgttccgc attctgcaag
     3721 cctccggagc gcacgtcggc agtcggctcc ctcgttgacc gaatcaccga cctctctccc
     3781 cagggggatc caccggagct taccatgacc gagtacaagc ccacggtgcg cctcgccacc
     3841 cgcgacgacg tccccagggc cgtacgcacc ctcgccgccg cgttcgccga ctaccccgcc
     3901 acgcgccaca ccgtcgatcc ggaccgccac atcgagcggg tcaccgagct gcaagaactc
     3961 ttcctcacgc gcgtcgggct cgacatcggc aaggtgtggg tcgcggacga cggcgccgcg
     4021 gtggcggtct ggaccacgcc ggagagcgtc gaagcggggg cggtgttcgc cgagatcggc
     4081 ccgcgcatgg ccgagttgag cggttcccgg ctggccgcgc agcaacagat ggaaggcctc
     4141 ctggcgccgc accggcccaa ggagcccgcg tggttcctgg ccaccgtcgg cgtctcgccc
     4201 gaccaccagg gcaagggtct gggcagcgcc gtcgtgctcc ccggagtgga ggcggccgag
     4261 cgcgccgggg tgcccgcctt cctggaaacc tccgcgcccc gcaacctccc cttctacgag
     4321 cggctcggct tcaccgtcac cgccgacgtc gaggtgcccg aaggaccgcg cacctggtgc
     4381 atgacccgca agcccggtgc ctgacgcccg ccccacgacc cgcagcgccc gaccgaaagg
     4441 agcgcacgac cccatgcatc ggtaccttta agaccaatga cttacaaggc agctgtagat
     4501 cttagccact ttctagagtc ggggcggccg gccgcttcga gcagacatga taagatacat
     4561 tgatgagttt ggacaaacca caactagaat gcagtgaaaa aaatgcttta tttgtgaaat
     4621 ttgtgatgct attgctttat ttgtaaccat tataagctgc aataaacaag ttaacaacaa
     4681 caattgcatt cattttatgt ttcaggttca gggggaggtg tgggaggttt tttaaagcaa
     4741 gtaaaacctc tacaaatgtg gtcgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     4801 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     4861 gggataacgc aggaaagaac atg
//
