LOCUS       sequence-300742-        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"
     promoter        complement(102..206)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     274..292
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(330..352)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     452..471
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     665..687
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     679..696
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     680..696
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        complement(788..1036)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(846..865)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(1016..1036)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(1050..1066)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1050..1066)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     protein_bind    1074..1090
                     /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)."
     promoter        complement(join(1098..1104,1105..1122,1123..1128))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1143..1164
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1281..1298)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1452..2040)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1532..1551)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2211..2970,1..32,33..101))
                     /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     2834..2853
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
       61 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
      121 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
      181 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa
      241 gaaaccatta ttatcatgac attaacctat aaaaataggc gtatcacgag gccctttcgt
      301 ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc
      361 acagcttgtc tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt
      421 gttggcgggt gtcggggctg gcttaactat gcggcatcag agcagattgt actgagagtg
      481 caccatatgc ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg catcaggcgc
      541 cattcgccat tcaggctgcg caactgttgg gaagggcgat cggtgcgggc ctcttcgcta
      601 ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat taagttgggt aacgccaggg
      661 ttttcccagt cacgacgttg taaaacgacg gccagtgaat tcgagctcgg taccaaaaaa
      721 gttgtaatag cccctcaaaa ctggaccttc cacaacggcc cctccatcgt ccaccgcaaa
      781 tgcttccggt gtttcgtcct ttccacaaga tatataaagc caagaaatcg aaatactttc
      841 aagttacggt aagcatatga tagtccattt taaaacataa ttttaaaact gcaaactacc
      901 caagaaatta ttactttcta cgtcacgtat tttgtactaa tatctttgtg tttacagtca
      961 aattaattcc aattatctct ctaacagcct tgtatcgtat atgcaaatat gaaggaatca
     1021 tgggaaatag gccctctggc gtaatcatgg tcatagctgt ttcctgcgtg aaattgttat
     1081 ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc
     1141 taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     1201 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     1261 attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
     1321 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
     1381 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
     1441 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     1501 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
     1561 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
     1621 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
     1681 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     1741 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     1801 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
     1861 aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg
     1921 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     1981 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
     2041 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
     2101 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
     2161 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
     2221 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
     2281 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
     2341 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
     2401 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
     2461 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
     2521 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
     2581 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
     2641 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
     2701 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
     2761 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
     2821 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
     2881 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
     2941 taacccactc gtgcacccaa ctgatcttca
//
