LOCUS       sequence-235358-        8622 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 8622)
  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 8622)
  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..8622
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        160..539
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     intron          741..1757
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1681..1703
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1765..1784
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     promoter        1818..1836
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1848..1868
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5268..5288
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5295..5384
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     CDS             5406..5459
                     /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(5460..5462,5463..5465,5466..6179)
                     /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(5460..5462,5463..5465,5466..6176)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(5505..5526)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     6113..6134
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    6222..6343
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6259..6278)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6313..6332
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6386..6402)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6386..6402)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6399..6421)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6410..6426
                     /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(6434..6440,6441..6458,6459..6464))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6479..6500
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6617..6634)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6788..7376)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6868..6887)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7547..8338,8339..8407))
                     /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     8170..8189
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8408..8512)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(8591..8610)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 agtatctgct ccctgcttgt gtgttggagg tcgctgagta gtgcgcgagc aaaatttaag
       61 ctacaacaag gcaaggcttg accgacaatt gcatgaagaa tctgcttagg gttaggcgtt
      121 ttgcgctgct tcgcgatgta cgggccagat atacgcgttg acattgatta ttgactagtt
      181 attaatagta atcaattacg gggtcattag ttcatagccc atatatggag ttccgcgtta
      241 cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt
      301 caataatgac gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg
      361 tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagta
      421 cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga
      481 ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgg
      541 tcgaggtgag ccccacgttc tgcttcactc tccccatctc ccccccctcc ccacccccaa
      601 ttttgtattt atttattttt taattatttt gtgcagcgat gggggcgggg ggggggggga
      661 gcggcgcgct ccgaaagttt ccttttatgg cgaggcggcg gcggcggcgg ccctataaaa
      721 agcgaagcgc gcggcgggcg ggagtcgctg cgcgctgcct tcgccccgtg ccccgctccg
      781 ccgccgcctc gcgccgcccg ccccggctct gactgaccgc gttactccca caggtgagcg
      841 ggcgggacgg cccttctcct ccgggctgta attagcgctt ggtttaatga cggcttgttt
      901 cttttctgtg gctgcgtgaa agccttgagg ggctccggga gggccctttg tgcgggggga
      961 gcggctcggg gggtgcgtgc gtgtgtgtgt gcgtggggag cgccgcgtgc ggctccgcgc
     1021 tgcccggcgg ctgtgagcgc tgcgggcgcg gcgcggggct ttgtgcgctc cgcagtgtgc
     1081 gcgaggggag cgcggccggg ggcggtgccc cgcggtgcgg ggggggctgc gaggggaaca
     1141 aaggctgcgt gcggggtgtg tgcgtggggg ggtgagcagg gggtgtgggc gcgtcggtcg
     1201 ggctgcaacc ccccctgcac ccccctcccc gagttgctga gcacggcccg gcttcgggtg
     1261 cggggctccg tacggggcgt ggcgcggggc tcgccgtgcc gggcgggggg tggcggcagg
     1321 tgggggtgcc gggcggggcg gggccgcctc gggccgggga gggctcgggg gaggggcgcg
     1381 gcggcccccg gagcgccggc ggctgtcgag gcgcggcgag ccgcagccat tgccttttat
     1441 ggtaatcgtg cgagagggcg cagggacttc ctttgtccca aatctgtgcg gagccgaaat
     1501 ctgggaggcg ccgccgcacc ccctctagcg ggcgcggggc gaagcggtgc ggcgccggca
     1561 ggaaggaaat gggcggggag ggccttcgtg cgtcgccgcg ccgccgtccc cttctccctc
     1621 tccagcctcg gggctgtccg cggggggacg gctgccttcg ggggggacgg ggcagggcgg
     1681 ggttcggctt ctggcgtgtg accggcggct ctagcgcctc tgctaaccat gttcatgcct
     1741 tcttcttttt cctacagctc ctgggcaacg tgctggttat tgtgctgtct catcattttg
     1801 gcaaagctag tgaattctaa tacgactcac tataggccgc caccatgccc aagaagaaga
     1861 ggaaggttcc cgggatggcc gtgaagagca tgaaggtgaa gctgcgcctg gacaacatgc
     1921 ccgagatccg cgccggcctg tggaagctgc acaccgaggt gaacgccggc gtgcgctact
     1981 acaccgagtg gctgagcctg ctgcgccagg agaacctgta ccgccgcagc cccaacggcg
     2041 acggcgagca ggagtgctac aagaccgccg aggagtgcaa ggccgagctg ctggagcgcc
     2101 tgcgcgcccg ccaggtggag aacggccact gcggccccgc cggcagcgac gacgagctgc
     2161 tgcagctggc ccgccagctg tacgagctgc tggtgcccca ggccatcggc gccaagggcg
     2221 acgcccagca gatcgcccgc aagttcctga gccccctggc cgacaaggac gccgtgggcg
     2281 gcctgggcat cgccaaggcc ggcaacaagc cccgctgggt gcgcatgcgc gaggccggcg
     2341 agcccggctg ggaggaggag aaggccaagg ccgaggcccg caagagcacc gaccgcaccg
     2401 ccgacgtgct gcgcgccctg gccgacttcg gcctgaagcc cctgatgcgc gtgtacaccg
     2461 acagcgacat gagcagcgtg cagtggaagc ccctgcgcaa gggccaggcc gtgcgcacct
     2521 gggaccgcga catgttccag caggccatcg agcgcatgat gagctgggag agctggaacc
     2581 agcgcgtggg cgaggcctac gccaagctgg tggagcagaa gagccgcttc gagcagaaga
     2641 acttcgtggg ccaggagcac ctggtgcagc tggtgaacca gctgcagcag gacatgaagg
     2701 aggccagcca cggcctggag agcaaggagc agaccgccca ctacctgacc ggccgcgccc
     2761 tgcgcggcag cgacaaggtg ttcgagaagt gggagaagct ggaccccgac gcccccttcg
     2821 acctgtacga caccgagatc aagaacgtgc agcgccgcaa cacccgccgc ttcggcagcc
     2881 acgacctgtt cgccaagctg gccgagccca agtaccaggc cctgtggcgc gaggacgcca
     2941 gcttcctgac ccgctacgcc gtgtacaaca gcatcgtgcg caagctgaac cacgccaaga
     3001 tgttcgccac cttcaccctg cccgacgcca ccgcccaccc catctggacc cgcttcgaca
     3061 agctgggcgg caacctgcac cagtacacct tcctgttcaa cgagttcggc gagggccgcc
     3121 acgccatccg cttccagaag ctgctgaccg tggaggacgg cgtggccaag gaggtggacg
     3181 acgtgaccgt gcccatcagc atgagcgccc agctggacga cctgctgccc cgcgaccccc
     3241 acgagctggt ggccctgtac ttccaggact acggcgccga gcagcacctg gccggcgagt
     3301 tcggcggcgc caagatccag taccgccgcg accagctgaa ccacctgcac gcccgccgcg
     3361 gcgcccgcga cgtgtacctg aacctgagcg tgcgcgtgca gagccagagc gaggcccgcg
     3421 gcgagcgccg ccccccctac gccgccgtgt tccgcctggt gggcgacaac caccgcgcct
     3481 tcgtgcactt cgacaagctg agcgactacc tggccgagca ccccgacgac ggcaagctgg
     3541 gcagcgaggg cctgctgagc ggcctgcgcg tgatgagcgt ggacctgggc ctgcgcacca
     3601 gcgccagcat cagcgtgttc cgcgtggccc gcaaggacga gctgaagccc aacagcgagg
     3661 gccgcgtgcc cttctgcttc cccatcgagg gcaacgagaa cctggtggcc gtgcacgagc
     3721 gcagccagct gctgaagctg cccggcgaga ccgagagcaa ggacctgcgc gccatccgcg
     3781 aggagcgcca gcgcaccctg cgccagctgc gcacccagct ggcctacctg cgcctgctgg
     3841 tgcgctgcgg cagcgaggac gtgggccgcc gcgagcgcag ctgggccaag ctgatcgagc
     3901 agcccatgga cgccaaccag atgacccccg actggcgcga ggccttcgag gacgagctgc
     3961 agaagctgaa gagcctgtac ggcatctgcg gcgaccgcga gtggaccgag gccgtgtacg
     4021 agagcgtgcg ccgcgtgtgg cgccacatgg gcaagcaggt gcgcgactgg cgcaaggacg
     4081 tgcgcagcgg cgagcgcccc aagatccgcg gctaccagaa ggacgtggtg ggcggcaaca
     4141 gcatcgagca gatcgagtac ctggagcgcc agtacaagtt cctgaagagc tggagcttct
     4201 tcggcaaggt gagcggccag gtgatccgcg ccgagaaggg cagccgcttc gccatcaccc
     4261 tgcgcgagca catcgaccac gccaaggagg accgcctgaa gaagctggcc gaccgcatca
     4321 tcatggaggc cctgggctac gtgtacgccc tggacgacga gcgcggcaag ggcaagtggg
     4381 tggccaagta ccccccctgc cagctgatcc tgctggagga gctgagcgag taccagttca
     4441 acaacgaccg cccccccagc gagaacaacc agctgatgca gtggagccac cgcggcgtgt
     4501 tccaggagct gctgaaccag gcccaggtgc acgacctgct ggtgggcacc atgtacgccg
     4561 ccttcagcag ccgcttcgac gcccgcaccg gcgcccccgg catccgctgc cgccgcgtgc
     4621 ccgcccgctg cgcccgcgag cagaaccccg agcccttccc ctggtggctg aacaagttcg
     4681 tggccgagca caagctggac ggctgccccc tgcgcgccga cgacctgatc cccaccggcg
     4741 agggcgagtt cttcgtgagc cccttcagcg ccgaggaggg cgacttccac cagatccacg
     4801 ccgccctgaa cgccgcccag aacctgcagc gccgcctgtg gagcgacttc gacatcagcc
     4861 agatccgcct gcgctgcgac tggggcgagg tggacggcga gcccgtgctg atcccccgca
     4921 ccaccggcaa gcgcaccgcc gacagctacg gcaacaaggt gttctacacc aagaccggcg
     4981 tgacctacta cgagcgcgag cgcggcaaga agcgccgcaa ggtgttcgcc caggaggagc
     5041 tgagcgagga ggaggccgag ctgctggtgg aggccgacga ggcccgcgag aagagcgtgg
     5101 tgctgatgcg cgaccccagc ggcatcatca accgcggcga ctggacccgc cagaaggagt
     5161 tctggagcat ggtgaaccag cgcatcgagg gctacctggt gaagcagatc cgcagccgcg
     5221 tgcgcctgca ggagagcgcc tgcgagaaca ccggcgacat cgctagccca aagaagaaga
     5281 ggaaagtctc tagataccct tatgatgttc cagattatgc cggataccca tacgatgtcc
     5341 ctgactatgc aggctcctac ccttatgacg tcccagacta cgccggatcc aggtccggcg
     5401 gcggagaggg cagaggaagt cttctaacat gcggtgacgt ggaggagaat cccggcccaa
     5461 tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg
     5521 gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg
     5581 gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc
     5641 tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc
     5701 agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct
     5761 tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg
     5821 tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca
     5881 agctggagta caactacaac agccacaacg tctatatcat ggccgacaag cagaagaacg
     5941 gcatcaaggt gaacttcaag atccgccaca acatcgagga cggcagcgtg cagctcgccg
     6001 accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact
     6061 acctgagcac ccagtccgcc ctgagcaaag accccaacga gaagcgcgat cacatggtcc
     6121 tgctggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg tacaagtaac
     6181 tgcagcgcgg ggatctcatg ctggagttct tcgcccaccc caacttgttt attgcagctt
     6241 ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac
     6301 tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttattaatta acgtcgacct
     6361 ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc
     6421 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctag ggtgcctaat
     6481 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     6541 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     6601 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
     6661 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     6721 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     6781 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     6841 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     6901 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     6961 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     7021 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     7081 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     7141 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     7201 ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc
     7261 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     7321 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     7381 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     7441 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     7501 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     7561 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     7621 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     7681 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     7741 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     7801 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     7861 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     7921 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     7981 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     8041 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     8101 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     8161 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     8221 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     8281 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     8341 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     8401 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
     8461 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
     8521 cccgaaaagt gccacctgac gtcgacggat cgggagatcg atctcccgat cccctagggt
     8581 cgactctcag tacaatctgc tctgatgccg catagttaag cc
//
