LOCUS       sequence-232189-        8695 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression, Transcription regulation, deactivated 
            AaCas12b.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8695)
  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 8695)
  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(D977A)-2AeGFP
FEATURES             Location/Qualifiers
     source          1..8695
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        8..387
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        390..665
                     /label=chicken beta-actin promoter
     intron          666..1678
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1602..1624
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1686..1705
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     promoter        1739..1757
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1769..1789
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5189..5209
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5216..5305
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     CDS             5327..5380
                     /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(5381..5383,5384..5386,5387..6100)
                     /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(5381..5383,5384..5386,5387..6097)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(5426..5447)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     6034..6055
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    6143..6264
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6180..6199)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6234..6253
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6307..6323)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6307..6323)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6320..6342)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6331..6347
                     /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(6355..6361,6362..6379,6380..6385))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6400..6421
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6538..6555)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6709..7297)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6789..6808)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7468..8259,8260..8328))
                     /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     8091..8110
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8329..8433)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(8512..8531)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 acgcgttgac attgattatt gactagttat taatagtaat caattacggg gtcattagtt
       61 catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga
      121 ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca
      181 atagggactt tccattgacg tcaatgggtg gactatttac ggtaaactgc ccacttggca
      241 gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg
      301 cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc
      361 tacgtattag tcatcgctat taccatgggt cgaggtgagc cccacgttct gcttcactct
      421 ccccatctcc cccccctccc cacccccaat tttgtattta tttatttttt aattattttg
      481 tgcagcgatg ggggcggggg gggggggggc gcgcgccagg cggggcgggg cggggcgagg
      541 ggcggggcgg ggcgaggcgg agaggtgcgg cggcagccaa tcagagcggc gcgctccgaa
      601 agtttccttt tatggcgagg cggcggcggc ggcggcccta taaaaagcga agcgcgcggc
      661 gggcgggagt cgctgcgttg ccttcgcccc gtgccccgct ccgcgccgcc tcgcgccgcc
      721 cgccccggct ctgactgacc gcgttactcc cacaggtgag cgggcgggac ggcccttctc
      781 ctccgggctg taattagcgc ttggtttaat gacggctcgt ttcttttctg tggctgcgtg
      841 aaagccttaa agggctccgg gagggccctt tgtgcggggg ggagcggctc ggggggtgcg
      901 tgcgtgtgtg tgtgcgtggg gagcgccgcg tgcggcccgc gctgcccggc ggctgtgagc
      961 gctgcgggcg cggcgcgggg ctttgtgcgc tccgcgtgtg cgcgagggga gcgcggccgg
     1021 gggcggtgcc ccgcggtgcg ggggggctgc gaggggaaca aaggctgcgt gcggggtgtg
     1081 tgcgtggggg ggtgagcagg gggtgtgggc gcggcggtcg ggctgtaacc cccccctgca
     1141 cccccctccc cgagttgctg agcacggccc ggcttcgggt gcggggctcc gtacggggcg
     1201 tggcgcgggg ctcgccgtgc cgggcggggg gtggcggcag gtgggggtgc cgggcggggc
     1261 ggggccgcct cgggccgggg agggctcggg ggaggggcgc ggcggccccc ggagcgccgg
     1321 cggctgtcga ggcgcggcga gccgcagcca ttgcctttta tggtaatcgt gcgagagggc
     1381 gcagggactt cctttgtccc aaatctgtgc ggagccgaaa tctgggaggc gccgccgcac
     1441 cccctctagc gggcgcgggg cgaagcggtg cggcgccggc aggaaggaaa tgggcgggga
     1501 gggccttcgt gcgtcgccgc gccgccgtcc ccttctccat ctccagcctc ggggctgtcc
     1561 gcagggggac ggctgccttc gggggggacg gggcagggcg gggttcggct tctggcgtgt
     1621 gaccggcggc tctagcgcct ctgctaacca tgttcatgcc ttcttctttt tcctacagct
     1681 cctgggcaac gtgctggtta ttgtgctgtc tcatcatttt ggcaaagcta gtgaattcta
     1741 atacgactca ctataggccg ccaccatgcc caagaagaag aggaaggttc ccgggatggc
     1801 cgtgaagagc atgaaggtga agctgcgcct ggacaacatg cccgagatcc gcgccggcct
     1861 gtggaagctg cacaccgagg tgaacgccgg cgtgcgctac tacaccgagt ggctgagcct
     1921 gctgcgccag gagaacctgt accgccgcag ccccaacggc gacggcgagc aggagtgcta
     1981 caagaccgcc gaggagtgca aggccgagct gctggagcgc ctgcgcgccc gccaggtgga
     2041 gaacggccac tgcggccccg ccggcagcga cgacgagctg ctgcagctgg cccgccagct
     2101 gtacgagctg ctggtgcccc aggccatcgg cgccaagggc gacgcccagc agatcgcccg
     2161 caagttcctg agccccctgg ccgacaagga cgccgtgggc ggcctgggca tcgccaaggc
     2221 cggcaacaag ccccgctggg tgcgcatgcg cgaggccggc gagcccggct gggaggagga
     2281 gaaggccaag gccgaggccc gcaagagcac cgaccgcacc gccgacgtgc tgcgcgccct
     2341 ggccgacttc ggcctgaagc ccctgatgcg cgtgtacacc gacagcgaca tgagcagcgt
     2401 gcagtggaag cccctgcgca agggccaggc cgtgcgcacc tgggaccgcg acatgttcca
     2461 gcaggccatc gagcgcatga tgagctggga gagctggaac cagcgcgtgg gcgaggccta
     2521 cgccaagctg gtggagcaga agagccgctt cgagcagaag aacttcgtgg gccaggagca
     2581 cctggtgcag ctggtgaacc agctgcagca ggacatgaag gaggccagcc acggcctgga
     2641 gagcaaggag cagaccgccc actacctgac cggccgcgcc ctgcgcggca gcgacaaggt
     2701 gttcgagaag tgggagaagc tggaccccga cgcccccttc gacctgtacg acaccgagat
     2761 caagaacgtg cagcgccgca acacccgccg cttcggcagc cacgacctgt tcgccaagct
     2821 ggccgagccc aagtaccagg ccctgtggcg cgaggacgcc agcttcctga cccgctacgc
     2881 cgtgtacaac agcatcgtgc gcaagctgaa ccacgccaag atgttcgcca ccttcaccct
     2941 gcccgacgcc accgcccacc ccatctggac ccgcttcgac aagctgggcg gcaacctgca
     3001 ccagtacacc ttcctgttca acgagttcgg cgagggccgc cacgccatcc gcttccagaa
     3061 gctgctgacc gtggaggacg gcgtggccaa ggaggtggac gacgtgaccg tgcccatcag
     3121 catgagcgcc cagctggacg acctgctgcc ccgcgacccc cacgagctgg tggccctgta
     3181 cttccaggac tacggcgccg agcagcacct ggccggcgag ttcggcggcg ccaagatcca
     3241 gtaccgccgc gaccagctga accacctgca cgcccgccgc ggcgcccgcg acgtgtacct
     3301 gaacctgagc gtgcgcgtgc agagccagag cgaggcccgc ggcgagcgcc gcccccccta
     3361 cgccgccgtg ttccgcctgg tgggcgacaa ccaccgcgcc ttcgtgcact tcgacaagct
     3421 gagcgactac ctggccgagc accccgacga cggcaagctg ggcagcgagg gcctgctgag
     3481 cggcctgcgc gtgatgagcg tggacctggg cctgcgcacc agcgccagca tcagcgtgtt
     3541 ccgcgtggcc cgcaaggacg agctgaagcc caacagcgag ggccgcgtgc ccttctgctt
     3601 ccccatcgag ggcaacgaga acctggtggc cgtgcacgag cgcagccagc tgctgaagct
     3661 gcccggcgag accgagagca aggacctgcg cgccatccgc gaggagcgcc agcgcaccct
     3721 gcgccagctg cgcacccagc tggcctacct gcgcctgctg gtgcgctgcg gcagcgagga
     3781 cgtgggccgc cgcgagcgca gctgggccaa gctgatcgag cagcccatgg acgccaacca
     3841 gatgaccccc gactggcgcg aggccttcga ggacgagctg cagaagctga agagcctgta
     3901 cggcatctgc ggcgaccgcg agtggaccga ggccgtgtac gagagcgtgc gccgcgtgtg
     3961 gcgccacatg ggcaagcagg tgcgcgactg gcgcaaggac gtgcgcagcg gcgagcgccc
     4021 caagatccgc ggctaccaga aggacgtggt gggcggcaac agcatcgagc agatcgagta
     4081 cctggagcgc cagtacaagt tcctgaagag ctggagcttc ttcggcaagg tgagcggcca
     4141 ggtgatccgc gccgagaagg gcagccgctt cgccatcacc ctgcgcgagc acatcgacca
     4201 cgccaaggag gaccgcctga agaagctggc cgaccgcatc atcatggagg ccctgggcta
     4261 cgtgtacgcc ctggacgacg agcgcggcaa gggcaagtgg gtggccaagt accccccctg
     4321 ccagctgatc ctgctggagg agctgagcga gtaccagttc aacaacgacc gcccccccag
     4381 cgagaacaac cagctgatgc agtggagcca ccgcggcgtg ttccaggagc tgctgaacca
     4441 ggcccaggtg cacgacctgc tggtgggcac catgtacgcc gccttcagca gccgcttcga
     4501 cgcccgcacc ggcgcccccg gcatccgctg ccgccgcgtg cccgcccgct gcgcccgcga
     4561 gcagaacccc gagcccttcc cctggtggct gaacaagttc gtggccgagc acaagctgga
     4621 cggctgcccc ctgcgcgccg acgacctgat ccccaccggc gagggcgagt tcttcgtgag
     4681 ccccttcagc gccgaggagg gcgacttcca ccagatccac gccgccctga acgccgccca
     4741 gaacctgcag cgccgcctgt ggagcgactt cgacatcagc cagatccgcc tgcgctgcga
     4801 ctggggcgag gtggacggcg agcccgtgct gatcccccgc accaccggca agcgcaccgc
     4861 cgacagctac ggcaacaagg tgttctacac caagaccggc gtgacctact acgagcgcga
     4921 gcgcggcaag aagcgccgca aggtgttcgc ccaggaggag ctgagcgagg aggaggccga
     4981 gctgctggtg gaggccgacg aggcccgcga gaagagcgtg gtgctgatgc gcgaccccag
     5041 cggcatcatc aaccgcggcg actggacccg ccagaaggag ttctggagca tggtgaacca
     5101 gcgcatcgag ggctacctgg tgaagcagat ccgcagccgc gtgcgcctgc aggagagcgc
     5161 ctgcgagaac accggcgaca tcgctagccc aaagaagaag aggaaagtct ctagataccc
     5221 ttatgatgtt ccagattatg ccggataccc atacgatgtc cctgactatg caggctccta
     5281 cccttatgac gtcccagact acgccggatc caggtccggc ggcggagagg gcagaggaag
     5341 tcttctaaca tgcggtgacg tggaggagaa tcccggccca atggtgagca agggcgagga
     5401 gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa
     5461 gttcagcgtg tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagtt
     5521 catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta
     5581 cggcgtgcag tgcttcagcc gctaccccga ccacatgaag cagcacgact tcttcaagtc
     5641 cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta
     5701 caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa
     5761 gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacaa
     5821 cagccacaac gtctatatca tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa
     5881 gatccgccac aacatcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac
     5941 ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagca cccagtccgc
     6001 cctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc
     6061 cgccgggatc actctcggca tggacgagct gtacaagtaa ctgcagcgcg gggatctcat
     6121 gctggagttc ttcgcccacc ccaacttgtt tattgcagct tataatggtt acaaataaag
     6181 caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt
     6241 gtccaaactc atcaatgtat cttattaatt aacgtcgacc tctagctaga gcttggcgta
     6301 atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat
     6361 acgagccgga agcataaagt gtaaagccta gggtgcctaa tgagtgagct aactcacatt
     6421 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
     6481 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc
     6541 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     6601 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     6661 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     6721 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     6781 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     6841 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     6901 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     6961 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     7021 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     7081 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     7141 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     7201 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     7261 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     7321 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     7381 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     7441 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     7501 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     7561 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
     7621 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     7681 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     7741 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
     7801 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     7861 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     7921 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     7981 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     8041 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
     8101 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     8161 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     8221 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     8281 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     8341 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     8401 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga
     8461 cgtcgacgga tcgggagatc gatctcccga tcccctaggg tcgactctca gtacaatctg
     8521 ctctgatgcc gcatagttaa gccagtatct gctccctgct tgtgtgttgg aggtcgctga
     8581 gtagtgcgcg agcaaaattt aagctacaac aaggcaaggc ttgaccgaca attgcatgaa
     8641 gaatctgctt agggttaggc gttttgcgct gcttcgcgat gtacgggcca gatat
//
