LOCUS       sequence-235334-        3005 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 3005)
  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 3005)
  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-AasgRNA3.8
FEATURES             Location/Qualifiers
     source          1..3005
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     58..80
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     72..89
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     73..89
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        96..344
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     96..116
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     267..286
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(478..494)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(478..494)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(491..513)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    502..518
                     /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(526..532,533..550,551..556))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    571..592
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(709..726)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(880..1468)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(960..979)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1639..2430,2431..2499))
                     /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     2262..2281
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2500..2604)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2672..2690
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(2728..2750)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2850..2869
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 agctggcgaa agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc
       61 agtcacgacg ttgtaaaacg acggccagtg aattcgaggg cctatttccc atgattcctt
      121 catatttgca tatacgatac aaggctgtta gagagataat tggaattaat ttgactgtaa
      181 acacaaagat attagtacaa aatacgtgac gtagaaagta ataatttctt gggtagtttg
      241 cagttttaaa attatgtttt aaaatggact atcatatgct taccgtaact tgaaagtatt
      301 tcgatttctt ggctttatat atcttgtgga aaggacgaaa caccgggtct aaaggacaga
      361 atttttcaac gggtgtgcca atggccactt tccaggtggc aaagcccgtt gaacttcaag
      421 cgaagtggca cagagaccga gagagggtct catttttttt agcttggcgt aatcatggtc
      481 atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg
      541 aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt
      601 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
      661 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga
      721 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat
      781 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca
      841 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
      901 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
      961 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
     1021 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
     1081 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
     1141 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
     1201 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
     1261 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
     1321 aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
     1381 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
     1441 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
     1501 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat
     1561 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
     1621 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
     1681 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga
     1741 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc
     1801 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac
     1861 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc
     1921 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc
     1981 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc
     2041 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt
     2101 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc
     2161 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg
     2221 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag
     2281 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat
     2341 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc
     2401 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa
     2461 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta
     2521 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa
     2581 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga
     2641 aaccattatt atcatgacat taacctataa aaataggcgt atcacgaggc cctttcgtct
     2701 cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg agacggtcac
     2761 agcttgtctg taagcggatg ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt
     2821 tggcgggtgt cggggctggc ttaactatgc ggcatcagag cagattgtac tgagagtgca
     2881 ccatatgcgg tgtgaaatac cgcacagatg cgtaaggaga aaataccgca tcaggcgcca
     2941 ttcgccattc aggctgcgca actgttggga agggcgatcg gtgcgggcct cttcgctatt
     3001 acgcc
//
