LOCUS       sequence-231785-        3217 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  gRNA plasmid with hU6 promoter PspCas13b gRNA dual luciferase 
            reporter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3217)
  AUTHORS   Rauch S, He C, Dickinson BC
  TITLE     Targeted m(6)A Reader Proteins To Study Epitranscriptomic Regulation
            of Single RNAs.
  JOURNAL   J Am Chem Soc. 2018 Sep 26;140(38):11974-11981. doi: 
            10.1021/jacs.8b05012. Epub 2018 Sep 18.
  PUBMED    30183280
REFERENCE   2  (bases 1 to 3217)
  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"; doi: 
            "10.1021/jacs.8b05012"; journalName: "J Am Chem Soc"; date: 
            "2018-09-26- 26"; volume: "140"; issue: "38"; pages: "11974-11981"
COMMENT     Sequence Label: hU6 promoter PspCas13b gRNA dual luciferase re...
FEATURES             Location/Qualifiers
     source          1..3217
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     53..75
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     67..84
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     68..84
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     94..113
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        94..112
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        127..375
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     127..147
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(497..517)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(497..515)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(536..552)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(536..552)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(549..571)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    560..576
                     /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(584..590,591..608,609..614))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    629..650
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(767..784)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(938..1526)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(1697..2488,2489..2557))
                     /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     2320..2339
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2558..2662)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2688..3143)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2825..2846)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3037..3056
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca
       61 cgacgttgta aaacgacggc cagtgagcgc gcgtaatacg actcactata gggcgaattg
      121 ggtaccgagg gcctatttcc catgattcct tcatatttgc atatacgata caaggctgtt
      181 agagagataa ttggaattaa tttgactgta aacacaaaga tattagtaca aaatacgtga
      241 cgtagaaagt aataatttct tgggtagttt gcagttttaa aattatgttt taaaatggac
      301 tatcatatgc ttatcgtaac ttgaaagtat ttcgatttct tggctttata tatcttgtgg
      361 aaaggacgaa acaccggcct gcaggtcgac tctagactcg aggctagcga gctcgtttaa
      421 acaactagtt gtggaaggtc cagttttgag gggctattac aacttttttt ccgcggtgga
      481 gctccagctt ttgttccctt tagtgagggt taattgcgcg cttggcgtaa tcatggtcat
      541 agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa
      601 gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc
      661 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
      721 aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact
      781 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
      841 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
      901 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
      961 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     1021 gatactaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     1081 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     1141 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     1201 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     1261 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     1321 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa
     1381 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     1441 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     1501 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     1561 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     1621 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     1681 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     1741 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     1801 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
     1861 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     1921 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     1981 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
     2041 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     2101 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     2161 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     2221 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     2281 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
     2341 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     2401 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     2461 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     2521 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
     2581 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     2641 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctaaa ttgtaagcgt
     2701 taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata
     2761 ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtgt
     2821 tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg
     2881 aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcaccctaat caagtttttt
     2941 ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc gatttagagc
     3001 ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga aaggagcggg
     3061 cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac ccgccgcgct
     3121 taatgcgccg ctacagggcg cgtcccattc gccattcagg ctgcgcaact gttgggaagg
     3181 gcgatcggtg cgggcctctt cgctattacg ccagctg
//
