LOCUS       sequence-235353-        3057 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 3057)
  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 3057)
  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-AasgRNA
FEATURES             Location/Qualifiers
     source          1..3057
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     52..74
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     66..83
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     67..83
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        90..338
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     90..110
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     261..280
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(524..540)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(524..540)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(537..559)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    548..564
                     /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(572..578,579..596,597..602))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    617..638
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(755..772)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(926..1514)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1006..1025)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1685..2476,2477..2545))
                     /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     2308..2327
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2546..2650)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2718..2736
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(2774..2796)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2896..2915
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 cgaaaggggg atgtgctgca aggcgattaa gttgggtaac gccagggttt tcccagtcac
       61 gacgttgtaa aacgacggcc agtgaattcg agggcctatt tcccatgatt ccttcatatt
      121 tgcatatacg atacaaggct gttagagaga taattggaat taatttgact gtaaacacaa
      181 agatattagt acaaaatacg tgacgtagaa agtaataatt tcttgggtag tttgcagttt
      241 taaaattatg ttttaaaatg gactatcata tgcttaccgt aacttgaaag tatttcgatt
      301 tcttggcttt atatatcttg tggaaaggac gaaacaccgg tctaaaggac agaatttttc
      361 aacgggtgtg ccaatggcca ctttccaggt ggcaaagccc gttgaacttc tcaaaaagaa
      421 cgctcgctca gtgttctgac gtcggatcac tgagcgagcg atctgagaag tggcacagag
      481 accgagagag ggtctcattt tttttaagct tggcgtaatc atggtcatag ctgtttcctg
      541 tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta
      601 aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg
      661 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
      721 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
      781 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
      841 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
      901 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
      961 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     1021 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     1081 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     1141 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     1201 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     1261 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     1321 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta
     1381 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     1441 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     1501 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     1561 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     1621 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     1681 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     1741 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     1801 gccccagtgc tgcaatgata ccgcgagagc cacgctcacc ggctccagat ttatcagcaa
     1861 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     1921 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     1981 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     2041 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     2101 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     2161 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     2221 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     2281 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     2341 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     2401 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     2461 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     2521 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     2581 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     2641 gggttccgcg cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc attattatca
     2701 tgacattaac ctataaaaat aggcgtatca cgaggccctt tcgtctcgcg cgtttcggtg
     2761 atgacggtga aaacctctga cacatgcagc tcccggagac ggtcacagct tgtctgtaag
     2821 cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg
     2881 gctggcttaa ctatgcggca tcagagcaga ttgtactgag agtgcaccat atgcggtgtg
     2941 aaataccgca cagatgcgta aggagaaaat accgcatcag gcgccattcg ccattcaggc
     3001 tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc gctattacgc cagctgg
//
