LOCUS       sequence-331934-        2957 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  U6-driven expression of control (non-targeting) presgRNA compatible 
            with CasRx. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2957)
  AUTHORS   Chuang YF, Wang PY, Kumar S, Lama S, Lin FL, Liu GS
  TITLE     Methods for in vitro CRISPR/CasRx-Mediated RNA Editing.
  JOURNAL   Front Cell Dev Biol. 2021 Jun 11;9:667879. doi: 
            10.3389/fcell.2021.667879. eCollection 2021.
  PUBMED    34178991
REFERENCE   2  (bases 1 to 2957)
  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: "Front Cell 
            Dev Biol."; date: "2021-06-11"; volume: "9:667879. doi"; pages: " 
            10.3389/fcell.2021.667879. eCollection 2021"
COMMENT     Sequence Label: pM115: CasRx control presgRNA
FEATURES             Location/Qualifiers
     source          1..2957
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     112..131
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        191..226
                     /label=RfxCas13d DR36
                     /note="36-base direct repeat for an RfxCas13d pre-gRNA 
                     (Konermann et al., 2018)"
     misc_RNA        257..292
                     /label=RfxCas13d DR36
                     /note="36-base direct repeat for an RfxCas13d pre-gRNA 
                     (Konermann et al., 2018)"
     rep_origin      374..829
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(461..480)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     671..692
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(846..865)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     965..987
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1025..1043)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1111..1215
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1216..1284,1285..2076)
                     /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(1434..1453)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2247..2835
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2736..2755
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        join(2898..2957,1..189)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2898..2918
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
ORIGIN
        1 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
       61 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
      121 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
      181 cgaaacaccg caagtaaacc cctaccaact ggtcggggtt tgaaactcac cagaagcgta
      241 ccatactcac gaacagcaag taaaccccta ccaactggtc ggggtttgaa actttttttg
      301 aattctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcatacgtca
      361 aagcaaccat agtacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
      421 cgcagcgtga ccgctacact tgccagcgcc ttagcgcccg ctcctttcgc tttcttccct
      481 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
      541 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgattt gggtgatggt
      601 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
      661 ttctttaata gtggactctt gttccaaact ggaacaacac tcaactctat ctcgggctat
      721 tcttttgatt tataagggat tttgccgatt tcggtctatt ggttaaaaaa tgagctgatt
      781 taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt ttacaatttt atggtgcact
      841 ctcagtacaa tctgctctga tgccgcatag ttaagccagc cccgacaccc gccaacaccc
      901 gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc
      961 gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgagacga
     1021 aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag
     1081 acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
     1141 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat
     1201 tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg
     1261 gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa
     1321 gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt
     1381 gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt
     1441 ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat
     1501 tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg
     1561 acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta
     1621 cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat
     1681 catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag
     1741 cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa
     1801 ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca
     1861 ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc
     1921 ggtgagcgtg gaagccgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt
     1981 atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc
     2041 gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat
     2101 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt
     2161 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     2221 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     2281 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     2341 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta
     2401 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     2461 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     2521 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     2581 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     2641 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     2701 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     2761 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     2821 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     2881 ccttttgctc agctagcgag ggcctatttc ccatgattcc ttcatatttg catatacgat
     2941 acaaggctgt tagagag
//
