LOCUS       sequence-227047-        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"
     rep_origin      complement(30..458)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(140..161)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     352..371
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     585..607
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     599..616
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     600..616
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     626..645
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        626..644
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        659..907
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     659..679
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(1029..1049)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(1029..1047)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1068..1084)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1068..1084)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1081..1103)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1092..1108
                     /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(1116..1122,1123..1140,1141..1146))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1161..1182
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1299..1316)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1470..2058)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1550..1569)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2229..3020,3021..3089))
                     /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     2852..2871
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3090..3194)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
       61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
      121 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
      181 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
      241 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
      301 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
      361 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
      421 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg
      481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
      541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
      601 taaaacgacg gccagtgagc gcgcgtaata cgactcacta tagggcgaat tgggtaccga
      661 gggcctattt cccatgattc cttcatattt gcatatacga tacaaggctg ttagagagat
      721 aattggaatt aatttgactg taaacacaaa gatattagta caaaatacgt gacgtagaaa
      781 gtaataattt cttgggtagt ttgcagtttt aaaattatgt tttaaaatgg actatcatat
      841 gcttatcgta acttgaaagt atttcgattt cttggcttta tatatcttgt ggaaaggacg
      901 aaacaccggc ctgcaggtcg actctagact cgaggctagc gagctcgttt aaacaactag
      961 ttgtggaagg tccagttttg aggggctatt acaacttttt ttccgcggtg gagctccagc
     1021 ttttgttccc tttagtgagg gttaattgcg cgcttggcgt aatcatggtc atagctgttt
     1081 cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag
     1141 tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg
     1201 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
     1261 gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc
     1321 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     1381 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     1441 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     1501 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     1561 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     1621 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     1681 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     1741 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     1801 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     1861 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag gacagtattt
     1921 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     1981 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     2041 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     2101 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     2161 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     2221 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     2281 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
     2341 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca
     2401 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     2461 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     2521 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
     2581 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
     2641 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     2701 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     2761 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
     2821 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
     2881 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     2941 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
     3001 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
     3061 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
     3121 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     3181 ataggggttc cgcgcacatt tccccgaaaa gtgccac
//
