LOCUS       sequence-240585-        3096 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression, Genome editing, gRNA scaffold.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3096)
  AUTHORS   Teng F, Li J, Cui T, Xu K, Guo L, Gao Q, Feng G, Chen C, Han D, Zhou
            Q, Li W
  TITLE     Enhanced mammalian genome editing by new Cas12a orthologs with 
            optimized crRNA scaffolds.
  JOURNAL   Genome Biol. 2019 Feb 5;20(1):15. doi: 10.1186/s13059-019-1620-8.
  PUBMED    30717767
REFERENCE   2  (bases 1 to 3096)
  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: "Genome 
            Biol."; date: "2019-02-5"; volume: "20(1):15. doi"; pages: " 
            10.1186/s13059-019-1620-8"
COMMENT     Sequence Label: pUC19-U6-crRNA4n96
FEATURES             Location/Qualifiers
     source          1..3096
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     55..77
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     69..86
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     70..86
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        93..341
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     93..113
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     264..283
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(566..582)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(566..582)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(579..601)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    590..606
                     /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(614..620,621..638,639..644))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    659..680
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(797..814)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(968..1556)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1048..1067)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1727..2518,2519..2587))
                     /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     2350..2369
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2588..2692)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2760..2778
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(2816..2838)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2938..2957
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 tggcgaaagg gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt
       61 cacgacgttg taaaacgacg gccagtgaat tcgagggcct atttcccatg attccttcat
      121 atttgcatat acgatacaag gctgttagag agataattgg aattaatttg actgtaaaca
      181 caaagatatt agtacaaaat acgtgacgta gaaagtaata atttcttggg tagtttgcag
      241 ttttaaaatt atgttttaaa atggactatc atatgcttac cgtaacttga aagtatttcg
      301 atttcttggc tttatatatc ttgtggaaag gacgaaacac cggaacaaag caccagtggt
      361 ctagtggtgg aatagtaccc tgccacggta cagacccggg ttcgattccc ggctggtgca
      421 atttctacta tggtagatag agaccgagag agggtctcaa acaaagcacc agtggtctag
      481 tggtggaata gtaccctgcc acggtacaga cccgggttcg attcccggct ggtgcatttt
      541 tttttttaag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc
      601 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat
      661 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
      721 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
      781 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
      841 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
      901 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
      961 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     1021 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     1081 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     1141 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     1201 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     1261 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     1321 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     1381 ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc
     1441 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     1501 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     1561 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     1621 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     1681 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     1741 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     1801 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     1861 taccgcgaga gccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     1921 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     1981 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     2041 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     2101 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     2161 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     2221 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     2281 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     2341 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     2401 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     2461 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     2521 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     2581 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
     2641 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
     2701 cccgaaaagt gccacctgac gtctaagaaa ccattattat catgacatta acctataaaa
     2761 ataggcgtat cacgaggccc tttcgtctcg cgcgtttcgg tgatgacggt gaaaacctct
     2821 gacacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc gggagcagac
     2881 aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggctggctt aactatgcgg
     2941 catcagagca gattgtactg agagtgcacc atatgcggtg tgaaataccg cacagatgcg
     3001 taaggagaaa ataccgcatc aggcgccatt cgccattcag gctgcgcaac tgttgggaag
     3061 ggcgatcggt gcgggcctct tcgctattac gccagc
//
