LOCUS       sequence-232181-        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     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     364..386
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     378..395
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     379..395
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        402..650
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     402..422
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     573..592
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(837..853)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(837..853)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(850..872)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    861..877
                     /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(885..891,892..909,910..915))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    930..951
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1068..1085)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1239..1827)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1319..1338)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1998..2789,2790..2858))
                     /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     2621..2640
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2859..2963)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3031..3049
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgagggccta tttcccatga
      421 ttccttcata tttgcatata cgatacaagg ctgttagaga gataattgga attaatttga
      481 ctgtaaacac aaagatatta gtacaaaata cgtgacgtag aaagtaataa tttcttgggt
      541 agtttgcagt tttaaaatta tgttttaaaa tggactatca tatgcttacc gtaacttgaa
      601 agtatttcga tttcttggct ttatatatct tgtggaaagg acgaaacacc ggtcgtctat
      661 aggacggcga ggacaacggg aagtgccaat gtgctctttc caagagcaaa caccccgttg
      721 gcttcaagat gaccgctcgc tcagcgatct gacaacggat cgctgagcga gcggtctgag
      781 aagtggcaca gagaccgaga gagggtctca ttttttttaa gcttggcgta atcatggtca
      841 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga
      901 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg
      961 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     1021 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
     1081 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     1141 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     1201 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     1261 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     1321 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     1381 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     1441 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     1501 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     1561 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     1621 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga
     1681 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     1741 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     1801 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     1861 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     1921 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     1981 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     2041 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     2101 ggcttaccat ctggccccag tgctgcaatg ataccgcgtg agccacgctc accggctcca
     2161 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     2221 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     2281 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     2341 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     2401 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     2461 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     2521 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     2581 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     2641 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     2701 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     2761 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     2821 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     2881 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     2941 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtctaagaa
     3001 accattatta tcatgacatt aacctataaa aataggcgta tcacgaggcc ctttcgtc
//
