LOCUS       sequence-305823-        2970 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PspCas13b guide RNA positioned 8bp 5' of Actb 1216 for targeted m6A 
            RNA methylation.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2970)
  AUTHORS   Wilson C, Chen PJ, Miao Z, Liu DR
  TITLE     Programmable m(6)A modification of cellular RNAs with a 
            Cas13-directed methyltransferase.
  JOURNAL   Nat Biotechnol. 2020 Jun 29. pii: 10.1038/s41587-020-0572-6. doi: 
            10.1038/s41587-020-0572-6.
  PUBMED    32601430
REFERENCE   2  (bases 1 to 2970)
  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 29. pii: 10.1038/s41587-020-0572-6. doi: 
            10.1038/s41587-020-0572-6."
COMMENT     Sequence Label: pU6-PspCas13b-gRNA-Actb1216
FEATURES             Location/Qualifiers
     source          1..2970
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    121..142
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(157..162,163..180,181..187)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    195..211
                     /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     219..235
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     219..235
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        249..497
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     249..269
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     420..439
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(589..606)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(589..605)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(598..620)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(814..833)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     933..955
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(993..1011)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1079..1183
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1184..1252,1253..2044)
                     /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(1402..1421)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2215..2803
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2704..2723
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     join(2957..2970,1..4)
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 cgaggaagcg gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca
       61 ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat
      121 taatgtgagt tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg
      181 tatgttgtgt ggaattgtga gcggataaca atttcacgca ggaaacagct atgaccatga
      241 ttacgccaga gggcctattt cccatgattc cttcatattt gcatatacga tacaaggctg
      301 ttagagagat aattggaatt aatttgactg taaacacaaa gatattagta caaaatacgt
      361 gacgtagaaa gtaataattt cttgggtagt ttgcagtttt aaaattatgt tttaaaatgg
      421 actatcatat gcttaccgta acttgaaagt atttcgattt cttggcttta tatatcttgt
      481 ggaaaggacg aaacaccgga agcatttgcg gtggacgatg gaggggccgt tgtggaaggt
      541 ccagttttga ggggctatta caactttttt ggtaccgagc tcgaattcac tggccgtcgt
      601 tttacaacgt cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca
      661 tccccctttc gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca
      721 gttgcgcagc ctgaatggcg aatggcgcct gatgcggtat tttctcctta cgcatctgtg
      781 cggtatttca caccgcatat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt
      841 aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc
      901 ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc
      961 accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat ttttataggt
     1021 taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg
     1081 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca
     1141 ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt
     1201 ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga
     1261 aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga
     1321 actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat
     1381 gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca
     1441 agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt
     1501 cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac
     1561 catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct
     1621 aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga
     1681 gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac
     1741 aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat
     1801 agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg
     1861 ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc
     1921 actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc
     1981 aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg
     2041 gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta
     2101 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg
     2161 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     2221 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     2281 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     2341 agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa
     2401 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     2461 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     2521 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     2581 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     2641 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     2701 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     2761 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     2821 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc
     2881 ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag
     2941 ccgaacgacc gagcgcagcg agtcagtgag
//
