LOCUS       sequence-235351-        3066 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 3066)
  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 3066)
  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-AmsgRNA
FEATURES             Location/Qualifiers
     source          1..3066
                     /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(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"
     primer_bind     complement(1018..1037)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     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
     primer_bind     2730..2748
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(2786..2808)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2908..2927
                     /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 cggggaattg ccgatctata
      361 ggacggcaga ttcaacggga tgtgccaatg cactctttcc aggagtgaac accccgttgg
      421 cttcaacatg atcgcccgct caacggtccg atgtcggatc gttgagcggg cgatctgaga
      481 agtggcacag agaccgagag agggtctcat tttttttaag 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 gataccaggc 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 gccacgctca 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 gccacctgac gtctaagaaa
     2701 ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc tttcgtctcg
     2761 cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggag acggtcacag
     2821 cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca gcgggtgttg
     2881 gcgggtgtcg gggctggctt aactatgcgg catcagagca gattgtactg agagtgcacc
     2941 atatgcggtg tgaaataccg cacagatgcg taaggagaaa ataccgcatc aggcgccatt
     3001 cgccattcag gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct tcgctattac
     3061 gccagc
//
