LOCUS       sequence-235352-        3053 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 3053)
  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 3053)
  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-BssgRNA
FEATURES             Location/Qualifiers
     source          1..3053
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     36..58
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     50..67
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     51..67
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        74..322
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     74..94
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     245..264
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(504..520)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(504..520)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(517..539)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    528..544
                     /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(552..558,559..576,577..582))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    597..618
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(735..752)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(906..1494)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(986..1005)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1665..2456,2457..2525))
                     /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     2288..2307
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2526..2630)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2698..2716
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(2754..2776)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2876..2895
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 tgcaaggcga ttaagttggg taacgccagg gttttcccag tcacgacgtt gtaaaacgac
       61 ggccagtgaa ttcgagggcc tatttcccat gattccttca tatttgcata tacgatacaa
      121 ggctgttaga gagataattg gaattaattt gactgtaaac acaaagatat tagtacaaaa
      181 tacgtgacgt agaaagtaat aatttcttgg gtagtttgca gttttaaaat tatgttttaa
      241 aatggactat catatgctta ccgtaacttg aaagtatttc gatttcttgg ctttatatat
      301 cttgtggaaa ggacgaaaca ccggccataa gtcgacttac atatccgtgc gtgtgcatta
      361 tgggcccatc cacaggtcta ttcccacgga taatcacgac tttccactaa gctttcgaat
      421 gttcgaaagc ttagtggaaa gcttcgtggt tagcacagag accgagagag ggtctcattt
      481 tttttggatc cggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc
      541 acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga
      601 gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
      661 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
      721 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
      781 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
      841 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
      901 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
      961 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     1021 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     1081 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     1141 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     1201 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     1261 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     1321 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca
     1381 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     1441 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     1501 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     1561 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     1621 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     1681 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     1741 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     1801 ccgcgagagc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     1861 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     1921 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     1981 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     2041 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     2101 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     2161 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     2221 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     2281 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     2341 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     2401 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     2461 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     2521 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     2581 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     2641 cgaaaagtgc cacctgacgt ctaagaaacc attattatca tgacattaac ctataaaaat
     2701 aggcgtatca cgaggccctt tcgtctcgcg cgtttcggtg atgacggtga aaacctctga
     2761 cacatgcagc tcccggagac ggtcacagct tgtctgtaag cggatgccgg gagcagacaa
     2821 gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg gctggcttaa ctatgcggca
     2881 tcagagcaga ttgtactgag agtgcaccat atgcggtgtg aaataccgca cagatgcgta
     2941 aggagaaaat accgcatcag gcgccattcg ccattcaggc tgcgcaactg ttgggaaggg
     3001 cgatcggtgc gggcctcttc gctattacgc cagctggcga aagggggatg tgc
//
