LOCUS       sequence-241099-        7014 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 7014)
  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 7014)
  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-2AeGFP
FEATURES             Location/Qualifiers
     source          1..7014
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     primer_bind     11..30
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     promoter        64..82
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             94..114
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3514..3534
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3541..3630
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     CDS             3652..3705
                     /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(3706..3708,3709..3711,3712..4425)
                     /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(3706..3708,3709..3711,3712..4422)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3751..3772)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     4359..4380
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    4468..4589
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4505..4524)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4559..4578
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4632..4648)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4632..4648)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4645..4667)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4656..4672
                     /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(4680..4686,4687..4704,4705..4710))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4725..4746
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4863..4880)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5034..5622)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5114..5133)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5793..6584,6585..6653))
                     /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     6416..6435
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6654..6758)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6837..6856)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 cagctcctgg gcaacgtgct ggttattgtg ctgtctcatc attttggcaa agctagtgaa
       61 ttctaatacg actcactata ggccgccacc atgcccaaga agaagaggaa ggttcccggg
      121 atggccgtga agagcatgaa ggtgaagctg cgcctggaca acatgcccga gatccgcgcc
      181 ggcctgtgga agctgcacac cgaggtgaac gccggcgtgc gctactacac cgagtggctg
      241 agcctgctgc gccaggagaa cctgtaccgc cgcagcccca acggcgacgg cgagcaggag
      301 tgctacaaga ccgccgagga gtgcaaggcc gagctgctgg agcgcctgcg cgcccgccag
      361 gtggagaacg gccactgcgg ccccgccggc agcgacgacg agctgctgca gctggcccgc
      421 cagctgtacg agctgctggt gccccaggcc atcggcgcca agggcgacgc ccagcagatc
      481 gcccgcaagt tcctgagccc cctggccgac aaggacgccg tgggcggcct gggcatcgcc
      541 aaggccggca acaagccccg ctgggtgcgc atgcgcgagg ccggcgagcc cggctgggag
      601 gaggagaagg ccaaggccga ggcccgcaag agcaccgacc gcaccgccga cgtgctgcgc
      661 gccctggccg acttcggcct gaagcccctg atgcgcgtgt acaccgacag cgacatgagc
      721 agcgtgcagt ggaagcccct gcgcaagggc caggccgtgc gcacctggga ccgcgacatg
      781 ttccagcagg ccatcgagcg catgatgagc tgggagagct ggaaccagcg cgtgggcgag
      841 gcctacgcca agctggtgga gcagaagagc cgcttcgagc agaagaactt cgtgggccag
      901 gagcacctgg tgcagctggt gaaccagctg cagcaggaca tgaaggaggc cagccacggc
      961 ctggagagca aggagcagac cgcccactac ctgaccggcc gcgccctgcg cggcagcgac
     1021 aaggtgttcg agaagtggga gaagctggac cccgacgccc ccttcgacct gtacgacacc
     1081 gagatcaaga acgtgcagcg ccgcaacacc cgccgcttcg gcagccacga cctgttcgcc
     1141 aagctggccg agcccaagta ccaggccctg tggcgcgagg acgccagctt cctgacccgc
     1201 tacgccgtgt acaacagcat cgtgcgcaag ctgaaccacg ccaagatgtt cgccaccttc
     1261 accctgcccg acgccaccgc ccaccccatc tggacccgct tcgacaagct gggcggcaac
     1321 ctgcaccagt acaccttcct gttcaacgag ttcggcgagg gccgccacgc catccgcttc
     1381 cagaagctgc tgaccgtgga ggacggcgtg gccaaggagg tggacgacgt gaccgtgccc
     1441 atcagcatga gcgcccagct ggacgacctg ctgccccgcg acccccacga gctggtggcc
     1501 ctgtacttcc aggactacgg cgccgagcag cacctggccg gcgagttcgg cggcgccaag
     1561 atccagtacc gccgcgacca gctgaaccac ctgcacgccc gccgcggcgc ccgcgacgtg
     1621 tacctgaacc tgagcgtgcg cgtgcagagc cagagcgagg cccgcggcga gcgccgcccc
     1681 ccctacgccg ccgtgttccg cctggtgggc gacaaccacc gcgccttcgt gcacttcgac
     1741 aagctgagcg actacctggc cgagcacccc gacgacggca agctgggcag cgagggcctg
     1801 ctgagcggcc tgcgcgtgat gagcgtggac ctgggcctgc gcaccagcgc cagcatcagc
     1861 gtgttccgcg tggcccgcaa ggacgagctg aagcccaaca gcgagggccg cgtgcccttc
     1921 tgcttcccca tcgagggcaa cgagaacctg gtggccgtgc acgagcgcag ccagctgctg
     1981 aagctgcccg gcgagaccga gagcaaggac ctgcgcgcca tccgcgagga gcgccagcgc
     2041 accctgcgcc agctgcgcac ccagctggcc tacctgcgcc tgctggtgcg ctgcggcagc
     2101 gaggacgtgg gccgccgcga gcgcagctgg gccaagctga tcgagcagcc catggacgcc
     2161 aaccagatga cccccgactg gcgcgaggcc ttcgaggacg agctgcagaa gctgaagagc
     2221 ctgtacggca tctgcggcga ccgcgagtgg accgaggccg tgtacgagag cgtgcgccgc
     2281 gtgtggcgcc acatgggcaa gcaggtgcgc gactggcgca aggacgtgcg cagcggcgag
     2341 cgccccaaga tccgcggcta ccagaaggac gtggtgggcg gcaacagcat cgagcagatc
     2401 gagtacctgg agcgccagta caagttcctg aagagctgga gcttcttcgg caaggtgagc
     2461 ggccaggtga tccgcgccga gaagggcagc cgcttcgcca tcaccctgcg cgagcacatc
     2521 gaccacgcca aggaggaccg cctgaagaag ctggccgacc gcatcatcat ggaggccctg
     2581 ggctacgtgt acgccctgga cgacgagcgc ggcaagggca agtgggtggc caagtacccc
     2641 ccctgccagc tgatcctgct ggaggagctg agcgagtacc agttcaacaa cgaccgcccc
     2701 cccagcgaga acaaccagct gatgcagtgg agccaccgcg gcgtgttcca ggagctgctg
     2761 aaccaggccc aggtgcacga cctgctggtg ggcaccatgt acgccgcctt cagcagccgc
     2821 ttcgacgccc gcaccggcgc ccccggcatc cgctgccgcc gcgtgcccgc ccgctgcgcc
     2881 cgcgagcaga accccgagcc cttcccctgg tggctgaaca agttcgtggc cgagcacaag
     2941 ctggacggct gccccctgcg cgccgacgac ctgatcccca ccggcgaggg cgagttcttc
     3001 gtgagcccct tcagcgccga ggagggcgac ttccaccaga tccacgccga cctgaacgcc
     3061 gcccagaacc tgcagcgccg cctgtggagc gacttcgaca tcagccagat ccgcctgcgc
     3121 tgcgactggg gcgaggtgga cggcgagccc gtgctgatcc cccgcaccac cggcaagcgc
     3181 accgccgaca gctacggcaa caaggtgttc tacaccaaga ccggcgtgac ctactacgag
     3241 cgcgagcgcg gcaagaagcg ccgcaaggtg ttcgcccagg aggagctgag cgaggaggag
     3301 gccgagctgc tggtggaggc cgacgaggcc cgcgagaaga gcgtggtgct gatgcgcgac
     3361 cccagcggca tcatcaaccg cggcgactgg acccgccaga aggagttctg gagcatggtg
     3421 aaccagcgca tcgagggcta cctggtgaag cagatccgca gccgcgtgcg cctgcaggag
     3481 agcgcctgcg agaacaccgg cgacatcgct agcccaaaga agaagaggaa agtctctaga
     3541 tacccttatg atgttccaga ttatgccgga tacccatacg atgtccctga ctatgcaggc
     3601 tcctaccctt atgacgtccc agactacgcc ggatccaggt ccggcggcgg agagggcaga
     3661 ggaagtcttc taacatgcgg tgacgtggag gagaatcccg gcccaatggt gagcaagggc
     3721 gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc
     3781 cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa gctgaccctg
     3841 aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccctg
     3901 acctacggcg tgcagtgctt cagccgctac cccgaccaca tgaagcagca cgacttcttc
     3961 aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa ggacgacggc
     4021 aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag
     4081 ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac
     4141 tacaacagcc acaacgtcta tatcatggcc gacaagcaga agaacggcat caaggtgaac
     4201 ttcaagatcc gccacaacat cgaggacggc agcgtgcagc tcgccgacca ctaccagcag
     4261 aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct gagcacccag
     4321 tccgccctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct ggagttcgtg
     4381 accgccgccg ggatcactct cggcatggac gagctgtaca agtaactgca gcgcggggat
     4441 ctcatgctgg agttcttcgc ccaccccaac ttgtttattg cagcttataa tggttacaaa
     4501 taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt
     4561 ggtttgtcca aactcatcaa tgtatcttat taattaacgt cgacctctag ctagagcttg
     4621 gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac
     4681 aacatacgag ccggaagcat aaagtgtaaa gcctagggtg cctaatgagt gagctaactc
     4741 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     4801 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct
     4861 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
     4921 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     4981 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     5041 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     5101 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     5161 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     5221 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     5281 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     5341 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     5401 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     5461 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     5521 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     5581 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     5641 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     5701 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     5761 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     5821 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     5881 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
     5941 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     6001 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     6061 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
     6121 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     6181 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     6241 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     6301 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     6361 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
     6421 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     6481 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     6541 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     6601 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     6661 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     6721 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
     6781 cctgacgtcg acggatcggg agatcgatct cccgatcccc tagggtcgac tctcagtaca
     6841 atctgctctg atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc
     6901 gctgagtagt gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc
     6961 atgaagaatc tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggcc
//
