LOCUS       sequence-235332-        3058 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 3058)
  AUTHORS   Teng F, Cui T, Feng G, Guo L, Xu K, Gao Q, Li T, Li J, Zhou Q, Li W
  TITLE     Repurposing CRISPR-Cas12b for mammalian genome engineering.
  JOURNAL   Cell Discov. 2018 Nov 27;4:63. doi: 10.1038/s41421-018-0069-3. 
            eCollection 2018.
  PUBMED    30510770
REFERENCE   2  (bases 1 to 3058)
  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: "Cell 
            Discov."; date: "2018-11-27"; volume: "4:63. doi"; pages: " 
            10.1038/s41421-018-0069-3. eCollection 2018"
COMMENT     Sequence Label: pUC19-U6-AksgRNA
FEATURES             Location/Qualifiers
     source          1..3058
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     13..35
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     27..44
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     28..44
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        51..299
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     51..71
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     222..241
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(486..502)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(486..502)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(499..521)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    510..526
                     /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(534..540,541..558,559..564))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    579..600
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(717..734)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(888..1476)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(968..987)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1647..2438,2439..2507))
                     /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     2270..2289
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2508..2612)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2680..2698
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(2736..2758)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2858..2877
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 cgccagggtt ttcccagtca cgacgttgta aaacgacggc cagtgaattc gagggcctat
       61 ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag ataattggaa
      121 ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga aagtaataat
      181 ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat atgcttaccg
      241 taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga cgaaacaccg
      301 gtcgtctata ggacggcgag gacaacggga agtgccaatg tgctctttcc aagagcaaac
      361 accccgttgg cttcaagatg accgctcgct cagcgatctg acaacggatc gctgagcgag
      421 cggtctgaga agtggcacag agaccgagag agggtctcat tttttttaag cttggcgtaa
      481 tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata
      541 cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta
      601 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
      661 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg
      721 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
      781 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
      841 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
      901 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
      961 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     1021 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     1081 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     1141 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     1201 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     1261 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     1321 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     1381 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     1441 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     1501 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
     1561 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     1621 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
     1681 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     1741 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga gccacgctca
     1801 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     1861 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     1921 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     1981 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     2041 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     2101 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     2161 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     2221 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     2281 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     2341 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     2401 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     2461 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     2521 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     2581 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac
     2641 gtctaagaaa ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc
     2701 tttcgtctcg cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggag
     2761 acggtcacag cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca
     2821 gcgggtgttg gcgggtgtcg gggctggctt aactatgcgg catcagagca gattgtactg
     2881 agagtgcacc atatgcggtg tgaaataccg cacagatgcg taaggagaaa ataccgcatc
     2941 aggcgccatt cgccattcag gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct
     3001 tcgctattac gccagctggc gaaaggggga tgtgctgcaa ggcgattaag ttgggtaa
//
