LOCUS       sequence-235530-        8640 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  Mammalian expression, Transcription regulation, deactivated 
            AaCas12b.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 8640)
  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 8640)
  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: pCAG-AaCas12b(R911A)-2AeGFP
FEATURES             Location/Qualifiers
     source          1..8640
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     intron          102..1118
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1042..1064
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1126..1145
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     promoter        1179..1197
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1209..1229
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4629..4649
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4656..4745
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     CDS             4767..4820
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             join(4821..4823,4824..4826,4827..5540)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(4821..4823,4824..4826,4827..5537)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(4866..4887)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     5474..5495
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    5583..5704
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5620..5639)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5674..5693
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5747..5763)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5747..5763)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5760..5782)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5771..5787
                     /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(5795..5801,5802..5819,5820..5825))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5840..5861
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5978..5995)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6149..6737)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6229..6248)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6908..7699,7700..7768))
                     /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     7531..7550
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7769..7873)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7952..7971)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        8143..8522
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt tccttttatg gcgaggcggc
       61 ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc gggagtcgct gcgcgctgcc
      121 ttcgccccgt gccccgctcc gccgccgcct cgcgccgccc gccccggctc tgactgaccg
      181 cgttactccc acaggtgagc gggcgggacg gcccttctcc tccgggctgt aattagcgct
      241 tggtttaatg acggcttgtt tcttttctgt ggctgcgtga aagccttgag gggctccggg
      301 agggcccttt gtgcgggggg agcggctcgg ggggtgcgtg cgtgtgtgtg tgcgtgggga
      361 gcgccgcgtg cggctccgcg ctgcccggcg gctgtgagcg ctgcgggcgc ggcgcggggc
      421 tttgtgcgct ccgcagtgtg cgcgagggga gcgcggccgg gggcggtgcc ccgcggtgcg
      481 gggggggctg cgaggggaac aaaggctgcg tgcggggtgt gtgcgtgggg gggtgagcag
      541 ggggtgtggg cgcgtcggtc gggctgcaac cccccctgca cccccctccc cgagttgctg
      601 agcacggccc ggcttcgggt gcggggctcc gtacggggcg tggcgcgggg ctcgccgtgc
      661 cgggcggggg gtggcggcag gtgggggtgc cgggcggggc ggggccgcct cgggccgggg
      721 agggctcggg ggaggggcgc ggcggccccc ggagcgccgg cggctgtcga ggcgcggcga
      781 gccgcagcca ttgcctttta tggtaatcgt gcgagagggc gcagggactt cctttgtccc
      841 aaatctgtgc ggagccgaaa tctgggaggc gccgccgcac cccctctagc gggcgcgggg
      901 cgaagcggtg cggcgccggc aggaaggaaa tgggcgggga gggccttcgt gcgtcgccgc
      961 gccgccgtcc ccttctccct ctccagcctc ggggctgtcc gcggggggac ggctgccttc
     1021 gggggggacg gggcagggcg gggttcggct tctggcgtgt gaccggcggc tctagcgcct
     1081 ctgctaacca tgttcatgcc ttcttctttt tcctacagct cctgggcaac gtgctggtta
     1141 ttgtgctgtc tcatcatttt ggcaaagcta gtgaattcta atacgactca ctataggccg
     1201 ccaccatgcc caagaagaag aggaaggttc ccgggatggc cgtgaagagc atgaaggtga
     1261 agctgcgcct ggacaacatg cccgagatcc gcgccggcct gtggaagctg cacaccgagg
     1321 tgaacgccgg cgtgcgctac tacaccgagt ggctgagcct gctgcgccag gagaacctgt
     1381 accgccgcag ccccaacggc gacggcgagc aggagtgcta caagaccgcc gaggagtgca
     1441 aggccgagct gctggagcgc ctgcgcgccc gccaggtgga gaacggccac tgcggccccg
     1501 ccggcagcga cgacgagctg ctgcagctgg cccgccagct gtacgagctg ctggtgcccc
     1561 aggccatcgg cgccaagggc gacgcccagc agatcgcccg caagttcctg agccccctgg
     1621 ccgacaagga cgccgtgggc ggcctgggca tcgccaaggc cggcaacaag ccccgctggg
     1681 tgcgcatgcg cgaggccggc gagcccggct gggaggagga gaaggccaag gccgaggccc
     1741 gcaagagcac cgaccgcacc gccgacgtgc tgcgcgccct ggccgacttc ggcctgaagc
     1801 ccctgatgcg cgtgtacacc gacagcgaca tgagcagcgt gcagtggaag cccctgcgca
     1861 agggccaggc cgtgcgcacc tgggaccgcg acatgttcca gcaggccatc gagcgcatga
     1921 tgagctggga gagctggaac cagcgcgtgg gcgaggccta cgccaagctg gtggagcaga
     1981 agagccgctt cgagcagaag aacttcgtgg gccaggagca cctggtgcag ctggtgaacc
     2041 agctgcagca ggacatgaag gaggccagcc acggcctgga gagcaaggag cagaccgccc
     2101 actacctgac cggccgcgcc ctgcgcggca gcgacaaggt gttcgagaag tgggagaagc
     2161 tggaccccga cgcccccttc gacctgtacg acaccgagat caagaacgtg cagcgccgca
     2221 acacccgccg cttcggcagc cacgacctgt tcgccaagct ggccgagccc aagtaccagg
     2281 ccctgtggcg cgaggacgcc agcttcctga cccgctacgc cgtgtacaac agcatcgtgc
     2341 gcaagctgaa ccacgccaag atgttcgcca ccttcaccct gcccgacgcc accgcccacc
     2401 ccatctggac ccgcttcgac aagctgggcg gcaacctgca ccagtacacc ttcctgttca
     2461 acgagttcgg cgagggccgc cacgccatcc gcttccagaa gctgctgacc gtggaggacg
     2521 gcgtggccaa ggaggtggac gacgtgaccg tgcccatcag catgagcgcc cagctggacg
     2581 acctgctgcc ccgcgacccc cacgagctgg tggccctgta cttccaggac tacggcgccg
     2641 agcagcacct ggccggcgag ttcggcggcg ccaagatcca gtaccgccgc gaccagctga
     2701 accacctgca cgcccgccgc ggcgcccgcg acgtgtacct gaacctgagc gtgcgcgtgc
     2761 agagccagag cgaggcccgc ggcgagcgcc gcccccccta cgccgccgtg ttccgcctgg
     2821 tgggcgacaa ccaccgcgcc ttcgtgcact tcgacaagct gagcgactac ctggccgagc
     2881 accccgacga cggcaagctg ggcagcgagg gcctgctgag cggcctgcgc gtgatgagcg
     2941 tggacctggg cctgcgcacc agcgccagca tcagcgtgtt ccgcgtggcc cgcaaggacg
     3001 agctgaagcc caacagcgag ggccgcgtgc ccttctgctt ccccatcgag ggcaacgaga
     3061 acctggtggc cgtgcacgag cgcagccagc tgctgaagct gcccggcgag accgagagca
     3121 aggacctgcg cgccatccgc gaggagcgcc agcgcaccct gcgccagctg cgcacccagc
     3181 tggcctacct gcgcctgctg gtgcgctgcg gcagcgagga cgtgggccgc cgcgagcgca
     3241 gctgggccaa gctgatcgag cagcccatgg acgccaacca gatgaccccc gactggcgcg
     3301 aggccttcga ggacgagctg cagaagctga agagcctgta cggcatctgc ggcgaccgcg
     3361 agtggaccga ggccgtgtac gagagcgtgc gccgcgtgtg gcgccacatg ggcaagcagg
     3421 tgcgcgactg gcgcaaggac gtgcgcagcg gcgagcgccc caagatccgc ggctaccaga
     3481 aggacgtggt gggcggcaac agcatcgagc agatcgagta cctggagcgc cagtacaagt
     3541 tcctgaagag ctggagcttc ttcggcaagg tgagcggcca ggtgatccgc gccgagaagg
     3601 gcagccgctt cgccatcacc ctgcgcgagc acatcgacca cgccaaggag gaccgcctga
     3661 agaagctggc cgaccgcatc atcatggagg ccctgggcta cgtgtacgcc ctggacgacg
     3721 agcgcggcaa gggcaagtgg gtggccaagt accccccctg ccagctgatc ctgctggagg
     3781 agctgagcga gtaccagttc aacaacgacc gcccccccag cgagaacaac cagctgatgc
     3841 agtggagcca ccgcggcgtg ttccaggagc tgctgaacca ggcccaggtg cacgacctgc
     3901 tggtgggcac catgtacgcc gccttcagca gccgcttcga cgcccgcacc ggcgcccccg
     3961 gcatcgcctg ccgccgcgtg cccgcccgct gcgcccgcga gcagaacccc gagcccttcc
     4021 cctggtggct gaacaagttc gtggccgagc acaagctgga cggctgcccc ctgcgcgccg
     4081 acgacctgat ccccaccggc gagggcgagt tcttcgtgag ccccttcagc gccgaggagg
     4141 gcgacttcca ccagatccac gccgacctga acgccgccca gaacctgcag cgccgcctgt
     4201 ggagcgactt cgacatcagc cagatccgcc tgcgctgcga ctggggcgag gtggacggcg
     4261 agcccgtgct gatcccccgc accaccggca agcgcaccgc cgacagctac ggcaacaagg
     4321 tgttctacac caagaccggc gtgacctact acgagcgcga gcgcggcaag aagcgccgca
     4381 aggtgttcgc ccaggaggag ctgagcgagg aggaggccga gctgctggtg gaggccgacg
     4441 aggcccgcga gaagagcgtg gtgctgatgc gcgaccccag cggcatcatc aaccgcggcg
     4501 actggacccg ccagaaggag ttctggagca tggtgaacca gcgcatcgag ggctacctgg
     4561 tgaagcagat ccgcagccgc gtgcgcctgc aggagagcgc ctgcgagaac accggcgaca
     4621 tcgctagccc aaagaagaag aggaaagtct ctagataccc ttatgatgtt ccagattatg
     4681 ccggataccc atacgatgtc cctgactatg caggctccta cccttatgac gtcccagact
     4741 acgccggatc caggtccggc ggcggagagg gcagaggaag tcttctaaca tgcggtgacg
     4801 tggaggagaa tcccggccca atggtgagca agggcgagga gctgttcacc ggggtggtgc
     4861 ccatcctggt cgagctggac ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg
     4921 gcgagggcga tgccacctac ggcaagctga ccctgaagtt catctgcacc accggcaagc
     4981 tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc
     5041 gctaccccga ccacatgaag cagcacgact tcttcaagtc cgccatgccc gaaggctacg
     5101 tccaggagcg caccatcttc ttcaaggacg acggcaacta caagacccgc gccgaggtga
     5161 agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg
     5221 acggcaacat cctggggcac aagctggagt acaactacaa cagccacaac gtctatatca
     5281 tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa gatccgccac aacatcgagg
     5341 acggcagcgt gcagctcgcc gaccactacc agcagaacac ccccatcggc gacggccccg
     5401 tgctgctgcc cgacaaccac tacctgagca cccagtccgc cctgagcaaa gaccccaacg
     5461 agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc actctcggca
     5521 tggacgagct gtacaagtaa ctgcagcgcg gggatctcat gctggagttc ttcgcccacc
     5581 ccaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca
     5641 caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat
     5701 cttattaatt aacgtcgacc tctagctaga gcttggcgta atcatggtca tagctgtttc
     5761 ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt
     5821 gtaaagccta gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc
     5881 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     5941 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct
     6001 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     6061 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
     6121 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     6181 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     6241 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     6301 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     6361 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     6421 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     6481 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     6541 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg
     6601 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     6661 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     6721 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     6781 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     6841 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     6901 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     6961 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     7021 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
     7081 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     7141 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     7201 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
     7261 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     7321 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     7381 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     7441 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     7501 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa
     7561 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     7621 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     7681 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     7741 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
     7801 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     7861 taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtcgacgga tcgggagatc
     7921 gatctcccga tcccctaggg tcgactctca gtacaatctg ctctgatgcc gcatagttaa
     7981 gccagtatct gctccctgct tgtgtgttgg aggtcgctga gtagtgcgcg agcaaaattt
     8041 aagctacaac aaggcaaggc ttgaccgaca attgcatgaa gaatctgctt agggttaggc
     8101 gttttgcgct gcttcgcgat gtacgggcca gatatacgcg ttgacattga ttattgacta
     8161 gttattaata gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg
     8221 ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga
     8281 cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat
     8341 gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa
     8401 gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca
     8461 tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc gctattacca
     8521 tggtcgaggt gagccccacg ttctgcttca ctctccccat ctcccccccc tccccacccc
     8581 caattttgta tttatttatt ttttaattat tttgtgcagc gatgggggcg gggggggggg
//
