LOCUS       sequence-235433-        8636 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 8636)
  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 8636)
  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-BsCas12b-2AeGFP
FEATURES             Location/Qualifiers
     source          1..8636
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     enhancer        8..387
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        389..665
                     /label=chicken beta-actin promoter
     intron          666..1682
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1606..1628
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1690..1709
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     promoter        1743..1761
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1773..1862
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     CDS             1869..1889
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5226..5246
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5268..5321
                     /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(5322..5324,5325..5327,5328..6041)
                     /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(5322..5324,5325..5327,5328..6038)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(5367..5388)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(5628..5647)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5975..5996
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    6084..6205
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6121..6140)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6175..6194
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6248..6264)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6248..6264)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6261..6283)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6272..6288
                     /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(6296..6302,6303..6320,6321..6326))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6341..6362
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6479..6496)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6650..7238)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6730..6749)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7409..8200,8201..8269))
                     /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     8032..8051
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8270..8374)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(8453..8472)
                     /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 gagtatttac ggtaaactgc ccacttggca
      241 gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg
      301 cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc
      361 tacgtattag tcatcgctat taccatggtc gaggtgagcc ccacgttctg cttcactctc
      421 cccatctccc ccccctcccc acccccaatt ttgtatttat ttatttttta attattttgt
      481 gcagcgatgg gggcgggggg ggggggggnc gcgcgccrgg cggggcgggg sggggcgrgg
      541 ggsggggsgg ggcgaggcgg agaggtgcgg cggcagccaa tcagagcggc gcgctccgaa
      601 agtttccttt tatggcgagg cggcggcggc ggcggcccta taaaaagcga agcgcgcggc
      661 gggcgggagt cgctgcgcgc tgccttcgcc ccgtgccccg ctccgccgcc gcctcgcgcc
      721 gcccgccccg gctctgactg accgcgttac tcccacaggt gagcgggcgg gacggccctt
      781 ctcctccggg ctgtaattag cgcttggttt aatgacggct tgtttctttt ctgtggctgc
      841 gtgaaagcct tgaggggctc cgggagggcc ctttgtgcgg ggggagcggc tcggggggtg
      901 cgtgcgtgtg tgtgtgcgtg gggagcgccg cgtgcggctc cgcgctgccc ggcggctgtg
      961 agcgctgcgg gcgcggcgcg gggctttgtg cgctccgcag tgtgcgcgag gggagcgcgg
     1021 ccgggggcgg tgccccgcgg tgcggggggg gctgcgaggg gaacaaaggc tgcgtgcggg
     1081 gtgtgtgcgt gggggggtga gcagggggtg tgggcgcgtc ggtcgggctg caaccccccc
     1141 tgcacccccc tccccgagtt gctgagcacg gcccggcttc gggtgcgggg ctccgtacgg
     1201 ggcgtggcgc ggggctcgcc gtgccgggcg gggggtggcg gcaggtgggg gtgccgggcg
     1261 gggcggggcc gcctcgggcc ggggagggct cgggggaggg gcgcggcggc ccccggagcg
     1321 ccggcggctg tcgaggcgcg gcgagccgca gccattgcct tttatggtaa tcgtgcgaga
     1381 gggcgcaggg acttcctttg tcccaaatct gtgcggagcc gaaatctggg aggcgccgcc
     1441 gcaccccctc tagcgggcgc ggggcgaagc ggtgcggcgc cggcaggaag gaaatgggcg
     1501 gggagggcct tcgtgcgtcg ccgcgccgcc gtccccttct ccctctccag cctcggggct
     1561 gtccgcgggg ggacggctgc cttcgggggg gacggggcag ggcggggttc ggcttctggc
     1621 gtgtgaccgg cggctctagc gcctctgcta accatgttca tgccttcttc tttttcctac
     1681 agctcctggg caacgtgctg gttattgtgc tgtctcatca ttttggcaaa gctagtgaat
     1741 tctaatacga ctcactatag gccgccacca tgtaccctta tgatgttcca gattatgccg
     1801 gatacccata cgatgtccct gactatgcag gctcctaccc ttatgacgtc ccagactacg
     1861 ccaccggtcc caagaagaag aggaaggttc ccgggatggc catccgcagc atcaagctga
     1921 agctgaagac ccacaccggc cccgaggccc agaacctgcg caagggcatc tggcgcaccc
     1981 accgcctgct gaacgagggc gtggcctact acatgaagat gctgctgctg ttccgccagg
     2041 agagcaccgg cgagcgcccc aaggaggagc tgcaggagga gctgatctgc cacatccgcg
     2101 agcagcagca gcgcaaccag gccgacaaga acacccaggc cctgcccctg gacaaggccc
     2161 tggaggccct gcgccagctg tacgagctgc tggtgcccag cagcgtgggc cagagcggcg
     2221 acgcccagat catcagccgc aagttcctga gccccctggt ggaccccaac agcgagggcg
     2281 gcaagggcac cagcaaggcc ggcgccaagc ccacctggca gaagaagaag gaggccaacg
     2341 accccacctg ggagcaggac tacgagaagt ggaagaagcg ccgcgaggag gaccccaccg
     2401 ccagcgtgat caccaccctg gaggagtacg gcatccgccc catcttcccc ctgtacacca
     2461 acaccgtgac cgacatcgcc tggctgcccc tgcagagcaa ccagttcgtg cgcacctggg
     2521 accgcgacat gctgcagcag gccatcgagc gcctgctgag ctgggagagc tggaacaagc
     2581 gcgtgcagga ggagtacgcc aagctgaagg agaagatggc ccagctgaac gagcagctgg
     2641 agggcggcca ggagtggatc agcctgctgg agcagtacga ggagaaccgc gagcgcgagc
     2701 tgcgcgagaa catgaccgcc gccaacgaca agtaccgcat caccaagcgc cagatgaagg
     2761 gctggaacga gctgtacgag ctgtggagca ccttccccgc cagcgccagc cacgagcagt
     2821 acaaggaggc cctgaagcgc gtgcagcagc gcctgcgcgg ccgcttcggc gacgcccact
     2881 tcttccagta cctgatggag gagaagaacc gcctgatctg gaagggcaac ccccagcgca
     2941 tccactactt cgtggcccgc aacgagctga ccaagcgcct ggaggaggcc aagcagagcg
     3001 ccaccatgac cctgcccaac gcccgcaagc accccctgtg ggtgcgcttc gacgcccgcg
     3061 gcggcaacct gcaggactac tacctgaccg ccgaggccga caagccccgc agccgccgct
     3121 tcgtgacctt cagccagctg atctggccca gcgagagcgg ctggatggag aagaaggacg
     3181 tggaggtgga gctggccctg agccgccagt tctaccagca ggtgaagctg ctgaagaacg
     3241 acaagggcaa gcagaagatc gagttcaagg acaagggcag cggcagcacc ttcaacggcc
     3301 acctgggcgg cgccaagctg cagctggagc gcggcgacct ggagaaggag gagaagaact
     3361 tcgaggacgg cgagatcggc agcgtgtacc tgaacgtggt gatcgacttc gagcccctgc
     3421 aggaggtgaa gaacggccgc gtgcaggccc cctacggcca ggtgctgcag ctgatccgcc
     3481 gccccaacga gttccccaag gtgaccacct acaagagcga gcagctggtg gagtggatca
     3541 aggccagccc ccagcacagc gccggcgtgg agagcctggc cagcggcttc cgcgtgatga
     3601 gcatcgacct gggcctgcgc gccgccgccg ccaccagcat cttcagcgtg gaggagagca
     3661 gcgacaagaa cgccgccgac ttcagctact ggatcgaggg cacccccctg gtggccgtgc
     3721 accagcgcag ctacatgctg cgcctgcccg gcgagcaggt ggagaagcag gtgatggaga
     3781 agcgcgacga gcgcttccag ctgcaccagc gcgtgaagtt ccagatccgc gtgctggccc
     3841 agatcatgcg catggccaac aagcagtacg gcgaccgctg ggacgagctg gacagcctga
     3901 agcaggccgt ggagcagaag aagagccccc tggaccagac cgaccgcacc ttctgggagg
     3961 gcatcgtgtg cgacctgacc aaggtgctgc cccgcaacga ggccgactgg gagcaggccg
     4021 tggtgcagat ccaccgcaag gccgaggagt acgtgggcaa ggccgtgcag gcctggcgca
     4081 agcgcttcgc cgccgacgag cgcaagggca tcgccggcct gagcatgtgg aacatcgagg
     4141 agctggaggg cctgcgcaag ctgctgatca gctggagccg ccgcacccgc aacccccagg
     4201 aggtgaaccg cttcgagcgc ggccacacca gccaccagcg cctgctgacc cacatccaga
     4261 acgtgaagga ggaccgcctg aagcagctga gccacgccat cgtgatgacc gccctgggct
     4321 acgtgtacga cgagcgcaag caggagtggt gcgccgagta ccccgcctgc caggtgatcc
     4381 tgttcgagaa cctgagccag taccgcagca acctggaccg cagcaccaag gagaacagca
     4441 ccctgatgaa gtgggcccac cgcagcatcc ccaagtacgt gcacatgcag gccgagccct
     4501 acggcatcca gatcggcgac gtgcgcgccg agtacagcag ccgcttctac gccaagaccg
     4561 gcacccccgg catccgctgc aagaaggtgc gcggccagga cctgcagggc cgccgcttcg
     4621 agaacctgca gaagcgcctg gtgaacgagc agttcctgac cgaggagcag gtgaagcagc
     4681 tgcgccccgg cgacatcgtg cccgacgaca gcggcgagct gttcatgacc ctgaccgacg
     4741 gcagcggcag caaggaggtg gtgttcctgc aggccgacat caacgccgcc cacaacctgc
     4801 agaagcgctt ctggcagcgc tacaacgagc tgttcaaggt gagctgccgc gtgatcgtgc
     4861 gcgacgagga ggagtacctg gtgcccaaga ccaagagcgt gcaggccaag ctgggcaagg
     4921 gcctgttcgt gaagaagagc gacaccgcct ggaaggacgt gtacgtgtgg gacagccagg
     4981 ccaagctgaa gggcaagacc accttcaccg aggagagcga gagccccgag cagctggagg
     5041 acttccagga gatcatcgag gaggccgagg aggccaaggg cacctaccgc accctgttcc
     5101 gcgaccccag cggcgtgttc ttccccgaga gcgtgtggta cccccagaag gacttctggg
     5161 gcgaggtgaa gcgcaagctg tacggcaagc tgcgcgagcg cttcctgacc aaggcccgcg
     5221 ctagcccaaa gaagaagagg aaagtctcta gaaggtccgg cggcggagag ggcagaggaa
     5281 gtcttctaac atgcggtgac gtggaggaga atcccggccc aatggtgagc aagggcgagg
     5341 agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca
     5401 agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt
     5461 tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accctgacct
     5521 acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac ttcttcaagt
     5581 ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact
     5641 acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga
     5701 agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca
     5761 acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag gtgaacttca
     5821 agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca
     5881 cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc acccagtccg
     5941 ccctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg
     6001 ccgccgggat cactctcggc atggacgagc tgtacaagta actgcagcgc ggggatctca
     6061 tgctggagtt cttcgcccac cccaacttgt ttattgcagc ttataatggt tacaaataaa
     6121 gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt
     6181 tgtccaaact catcaatgta tcttattaat taacgtcgac ctctagctag agcttggcgt
     6241 aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca
     6301 tacgagccgg aagcataaag tgtaaagcct agggtgccta atgagtgagc taactcacat
     6361 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
     6421 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct
     6481 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     6541 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     6601 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     6661 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     6721 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     6781 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     6841 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     6901 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     6961 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     7021 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     7081 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     7141 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     7201 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     7261 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
     7321 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     7381 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
     7441 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     7501 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct
     7561 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
     7621 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     7681 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
     7741 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     7801 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
     7861 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
     7921 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
     7981 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
     8041 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
     8101 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
     8161 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
     8221 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
     8281 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
     8341 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg
     8401 acgtcgacgg atcgggagat cgatctcccg atcccctagg gtcgactctc agtacaatct
     8461 gctctgatgc cgcatagtta agccagtatc tgctccctgc ttgtgtgttg gaggtcgctg
     8521 agtagtgcgc gagcaaaatt taagctacaa caaggcaagg cttgaccgac aattgcatga
     8581 agaatctgct tagggttagg cgttttgcgc tgcttcgcga tgtacgggcc agatat
//
