LOCUS       sequence-235529-        8637 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  Mammalian expression, Genome editing.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 8637)
  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 8637)
  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-AmCas12b-2AeGFP
FEATURES             Location/Qualifiers
     source          1..8637
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     enhancer        71..450
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     intron          616..1632
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1556..1578
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1640..1659
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     promoter        1693..1711
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1723..1743
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5194..5214
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5221..5310
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     CDS             5332..5385
                     /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(5386..5388,5389..5391,5392..6105)
                     /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(5386..5388,5389..5391,5392..6102)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(5431..5452)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(5692..5711)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     6039..6060
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    6148..6269
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6185..6204)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6239..6258
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6312..6328)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6312..6328)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6325..6347)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6336..6352
                     /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(6360..6366,6367..6384,6385..6390))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6405..6426
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6543..6560)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6714..7302)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6794..6813)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7473..8264,8265..8333))
                     /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     8096..8115
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8334..8438)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(8517..8536)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 tgcatgaaga atctgcttag ggttaggcgt tttgcgctgc ttcgcgatgt acgggccaga
       61 tatacgcgtt gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta
      121 gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc
      181 tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg
      241 ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg
      301 gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa
      361 tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac
      421 atctacgtat tagtcatcgc tattaccatg gtcgaggtga gccccacgtt ctgcttcact
      481 ctccccatct cccccccctc cccaccccca attttgtatt tatttatttt ttaattattt
      541 tgtgcagcga tgggggcggg gggggggggg cggcggcggc ggcggcccta taaaaagcga
      601 agcgcgcggc gggcgggagt cgctgcgcgc tgccttcgcc ccgtgccccg ctccgccgcc
      661 gcctcgcgcc gcccgccccg gctctgactg accgcgttac tcccacaggt gagcgggcgg
      721 gacggccctt ctcctccggg ctgtaattag cgcttggttt aatgacggct tgtttctttt
      781 ctgtggctgc gtgaaagcct tgaggggctc cgggagggcc ctttgtgcgg ggggagcggc
      841 tcggggggtg cgtgcgtgtg tgtgtgcgtg gggagcgccg cgtgcggctc cgcgctgccc
      901 ggcggctgtg agcgctgcgg gcgcggcgcg gggctttgtg cgctccgcag tgtgcgcgag
      961 gggagcgcgg ccgggggcgg tgccccgcgg tgcggggggg gctgcgaggg gaacaaaggc
     1021 tgcgtgcggg gtgtgtgcgt gggggggtga gcagggggtg tgggcgcgtc ggtcgggctg
     1081 caaccccccc tgcacccccc tccccgagtt gctgagcacg gcccggcttc gggtgcgggg
     1141 ctccgtacgg ggcgtggcgc ggggctcgcc gtgccgggcg gggggtggcg gcaggtgggg
     1201 gtgccgggcg gggcggggcc gcctcgggcc ggggagggct cgggggaggg gcgcggcggc
     1261 ccccggagcg ccggcggctg tcgaggcgcg gcgagccgca gccattgcct tttatggtaa
     1321 tcgtgcgaga gggcgcaggg acttcctttg tcccaaatct gtgcggagcc gaaatctggg
     1381 aggcgccgcc gcaccccctc tagcgggcgc ggggcgaagc ggtgcggcgc cggcaggaag
     1441 gaaatgggcg gggagggcct tcgtgcgtcg ccgcgccgcc gtccccttct ccctctccag
     1501 cctcggggct gtccgcgggg ggacggctgc cttcgggggg gacggggcag ggcggggttc
     1561 ggcttctggc gtgtgaccgg cggctctagc gcctctgcta accatgttca tgccttcttc
     1621 tttttcctac agctcctggg caacgtgctg gttattgtgc tgtctcatca ttttggcaaa
     1681 gctagtgaat tctaatacga ctcactatag gccgccacca tgcccaagaa gaagaggaag
     1741 gttcccggga tgaacgtggc cgtgaagagc atcaaggtga agctgatgct gggccacctg
     1801 cccgagatcc gcgagggcct gtggcacctg cacgaggccg tgaacctggg cgtgcgctac
     1861 tacaccgagt ggctggccct gctgcgccag ggcaacctgt accgccgcgg caaggacggc
     1921 gcccaggagt gctacatgac cgccgagcag tgccgccagg agctgctggt gcgcctgcgc
     1981 gaccgccaga agcgcaacgg ccacaccggc gaccccggca ccgacgagga gctgctgggc
     2041 gtggcccgcc gcctgtacga gctgctggtg ccccagagcg tgggcaagaa gggccaggcc
     2101 cagatgctgg ccagcggctt cctgagcccc ctggccgacc ccaagagcga gggcggcaag
     2161 ggcaccagca agagcggccg caagcccgcc tggatgggca tgaaggaggc cggcgacagc
     2221 cgctgggtgg aggccaaggc ccgctacgag gccaacaagg ccaaggaccc caccaagcag
     2281 gtgatcgcca gcctggagat gtacggcctg cgccccctgt tcgacgtgtt caccgagacc
     2341 tacaagacca tccgctggat gcccctgggc aagcaccagg gcgtgcgcgc ctgggaccgc
     2401 gacatgttcc agcagagcct ggagcgcctg atgagctggg agagctggaa cgagcgcgtg
     2461 ggcgccgagt tcgcccgcct ggtggaccgc cgcgaccgct tccgcgagaa gcacttcacc
     2521 ggccaggagc acctggtggc cctggcccag cgcctggagc aggagatgaa ggaggccagc
     2581 cccggcttcg agagcaagag cagccaggcc caccgcatca ccaagcgcgc cctgcgcggc
     2641 gccgacggca tcatcgacga ctggctgaag ctgagcgagg gcgagcccgt ggaccgcttc
     2701 gacgagatcc tgcgcaagcg ccaggcccag aacccccgcc gcttcggcag ccacgacctg
     2761 ttcctgaagc tggccgagcc cgtgttccag cccctgtggc gcgaggaccc cagcttcctg
     2821 agccgctggg ccagctacaa cgaggtgctg aacaagctgg aggacgccaa gcagttcgcc
     2881 accttcaccc tgcccagccc ctgcagcaac cccgtgtggg cccgcttcga gaacgccgag
     2941 ggcaccaaca tcttcaagta cgacttcctg ttcgaccact tcggcaaggg ccgccacggc
     3001 gtgcgcttcc agcgcatgat cgtgatgcgc gacggcgtgc ccaccgaggt ggagggcatc
     3061 gtggtgccca tcgcccccag ccgccagctg gacgccctgg cccccaacga cgccgccagc
     3121 cccatcgacg tgttcgtggg cgaccccgcc gcccccggcg ccttccgcgg ccagttcggc
     3181 ggcgccaaga tccagtaccg ccgcagcgcc ctggtgcgca agggccgccg cgaggagaag
     3241 gcctacctgt gcggcttccg cctgcccagc cagcgccgca ccggcacccc cgccgacgac
     3301 gccggcgagg tgttcctgaa cctgagcctg cgcgtggaga gccagagcga gcaggccggc
     3361 cgccgcaacc ccccctacgc cgccgtgttc cacatcagcg accagacccg ccgcgtgatc
     3421 gtgcgctacg gcgagatcga gcgctacctg gccgagcacc ccgacaccgg catccccggc
     3481 agccgcggcc tgaccagcgg cctgcgcgtg atgagcgtgg acctgggcct gcgcaccagc
     3541 gccgccatca gcgtgttccg cgtggcccac cgcgacgagc tgacccccga cgcccacggc
     3601 cgccagccct tcttcttccc catccacggc atggaccacc tggtggccct gcacgagcgc
     3661 agccacctga tccgcctgcc cggcgagacc gagagcaaga aggtgcgcag catccgcgag
     3721 cagcgcctgg accgcctgaa ccgcctgcgc agccagatgg ccagcctgcg cctgctggtg
     3781 cgcaccggcg tgctggacga gcagaagcgc gaccgcaact gggagcgcct gcagagcagc
     3841 atggagcgcg gcggcgagcg catgcccagc gactggtggg acctgttcca ggcccaggtg
     3901 cgctacctgg cccagcaccg cgacgccagc ggcgaggcct ggggccgcat ggtgcaggcc
     3961 gccgtgcgca ccctgtggcg ccagctggcc aagcaggtgc gcgactggcg caaggaggtg
     4021 cgccgcaacg ccgacaaggt gaagatccgc ggcatcgccc gcgacgtgcc cggcggccac
     4081 agcctggccc agctggacta cctggagcgc cagtaccgct tcctgcgcag ctggagcgcc
     4141 ttcagcgtgc aggccggcca ggtggtgcgc gccgagcgcg acagccgctt cgccgtggcc
     4201 ctgcgcgagc acatcgacaa cggcaagaag gaccgcctga agaagctggc cgaccgcatc
     4261 ctgatggagg ccctgggcta cgtgtacgtg accgacggcc gccgcgccgg ccagtggcag
     4321 gccgtgtacc ccccctgcca gctggtgctg ctggaggagc tgagcgagta ccgcttcagc
     4381 aacgaccgcc cccccagcga gaacagccag ctgatggtgt ggagccaccg cggcgtgctg
     4441 gaggagctga tccaccaggc ccaggtgcac gacgtgctgg tgggcaccat ccccgccgcc
     4501 ttcagcagcc gcttcgacgc ccgcaccggc gcccccggca tccgctgccg ccgcgtgccc
     4561 agcatccccc tgaaggacgc ccccagcatc cccatctggc tgagccacta cctgaagcag
     4621 accgagcgcg acgccgccgc cctgcgcccc ggcgagctga tccccaccgg cgacggcgag
     4681 ttcctggtga cccccgccgg ccgcggcgcc agcggcgtgc gcgtggtgca cgccgacatc
     4741 aacgccgccc acaacctgca gcgccgcctg tgggagaact tcgacctgag cgacatccgc
     4801 gtgcgctgcg accgccgcga gggcaaggac ggcaccgtgg tgctgatccc ccgcctgacc
     4861 aaccagcgcg tgaaggagcg ctacagcggc gtgatcttca ccagcgagga cggcgtgagc
     4921 ttcaccgtgg gcgacgccaa gacccgccgc cgcagcagcg ccagccaggg cgagggcgac
     4981 gacctgagcg acgaggagca ggagctgctg gccgaggccg acgacgcccg cgagcgcagc
     5041 gtggtgctgt tccgcgaccc cagcggcttc gtgaacggcg gccgctggac cgcccagcgc
     5101 gccttctggg gcatggtgca caaccgcatc gagaccctgc tggccgagcg cttcagcgtg
     5161 agcggcgccg ccgagaaggt gcgcggcgct agcccaaaga agaagaggaa agtctctaga
     5221 tacccttatg atgttccaga ttatgccgga tacccatacg atgtccctga ctatgcaggc
     5281 tcctaccctt atgacgtccc agactacgcc ggatccaggt ccggcggcgg agagggcaga
     5341 ggaagtcttc taacatgcgg tgacgtggag gagaatcccg gcccaatggt gagcaagggc
     5401 gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc
     5461 cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa gctgaccctg
     5521 aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccctg
     5581 acctacggcg tgcagtgctt cagccgctac cccgaccaca tgaagcagca cgacttcttc
     5641 aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa ggacgacggc
     5701 aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag
     5761 ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac
     5821 tacaacagcc acaacgtcta tatcatggcc gacaagcaga agaacggcat caaggtgaac
     5881 ttcaagatcc gccacaacat cgaggacggc agcgtgcagc tcgccgacca ctaccagcag
     5941 aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct gagcacccag
     6001 tccgccctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct ggagttcgtg
     6061 accgccgccg ggatcactct cggcatggac gagctgtaca agtaactgca gcgcggggat
     6121 ctcatgctgg agttcttcgc ccaccccaac ttgtttattg cagcttataa tggttacaaa
     6181 taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt
     6241 ggtttgtcca aactcatcaa tgtatcttat taattaacgt cgacctctag ctagagcttg
     6301 gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac
     6361 aacatacgag ccggaagcat aaagtgtaaa gcctagggtg cctaatgagt gagctaactc
     6421 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     6481 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct
     6541 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
     6601 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     6661 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     6721 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     6781 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     6841 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     6901 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     6961 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     7021 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     7081 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     7141 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     7201 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     7261 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     7321 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     7381 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     7441 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     7501 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     7561 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
     7621 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     7681 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     7741 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
     7801 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     7861 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     7921 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     7981 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     8041 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
     8101 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     8161 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     8221 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     8281 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     8341 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     8401 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
     8461 cctgacgtcg acggatcggg agatcgatct cccgatcccc tagggtcgac tctcagtaca
     8521 atctgctctg atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc
     8581 gctgagtagt gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaat
//
