LOCUS       sequence-235330-        3135 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 3135)
  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 3135)
  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-AagRNA
FEATURES             Location/Qualifiers
     source          1..3135
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     62..84
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     76..93
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     77..93
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        100..348
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     100..120
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     271..290
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(612..628)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(612..628)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(625..647)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    636..652
                     /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(660..666,667..684,685..690))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    705..726
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(843..860)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1014..1602)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1094..1113)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1773..2564,2565..2633))
                     /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     2396..2415
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2634..2738)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2806..2824
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(2862..2884)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2984..3003
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 cgccagctgg cgaaaggggg atgtgctgca aggcgattaa gttgggtaac gccagggttt
       61 tcccagtcac gacgttgtaa aacgacggcc agtgaattcg agggcctatt tcccatgatt
      121 ccttcatatt tgcatatacg atacaaggct gttagagaga taattggaat taatttgact
      181 gtaaacacaa agatattagt acaaaatacg tgacgtagaa agtaataatt tcttgggtag
      241 tttgcagttt taaaattatg ttttaaaatg gactatcata tgcttaccgt aacttgaaag
      301 tatttcgatt tcttggcttt atatatcttg tggaaaggac gaaacaccgg gtctaaagga
      361 cagaattttt caacgggtgt gccaatggcc actttccagg tggcaaagcc cgttgaactt
      421 ctcaaaaaga acgctcgctc agtgttctga caacaaagca ccagtggtct agtggtggaa
      481 tagtaccctg ccacggtaca gacccgggtt cgattcccgg ctggtgcagt cggatcactg
      541 agcgagcgat ctgagaagtg gcacagagac cgagagaggg tctcattttt tttaagcttg
      601 gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac
      661 aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc
      721 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
      781 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct
      841 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
      901 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
      961 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     1021 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     1081 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     1141 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     1201 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     1261 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     1321 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     1381 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     1441 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     1501 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     1561 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     1621 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     1681 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     1741 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     1801 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     1861 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagagcca
     1921 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     1981 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     2041 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
     2101 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     2161 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     2221 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     2281 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     2341 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
     2401 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     2461 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     2521 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     2581 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     2641 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     2701 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
     2761 cctgacgtct aagaaaccat tattatcatg acattaacct ataaaaatag gcgtatcacg
     2821 aggccctttc gtctcgcgcg tttcggtgat gacggtgaaa acctctgaca catgcagctc
     2881 ccggagacgg tcacagcttg tctgtaagcg gatgccggga gcagacaagc ccgtcagggc
     2941 gcgtcagcgg gtgttggcgg gtgtcggggc tggcttaact atgcggcatc agagcagatt
     3001 gtactgagag tgcaccatat gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac
     3061 cgcatcaggc gccattcgcc attcaggctg cgcaactgtt gggaagggcg atcggtgcgg
     3121 gcctcttcgc tatta
//
