LOCUS       sequence-232176-        8632 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression, Genome editing.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8632)
  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 8632)
  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..8632
                     /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..1858
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     CDS             1865..1885
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5222..5242
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5264..5317
                     /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(5318..5320,5321..5323,5324..6037)
                     /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(5318..5320,5321..5323,5324..6034)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(5363..5384)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(5624..5643)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5971..5992
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    6080..6201
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6117..6136)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6171..6190
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6244..6260)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6244..6260)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6257..6279)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6268..6284
                     /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(6292..6298,6299..6316,6317..6322))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6337..6358
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6475..6492)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6646..7234)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6726..6745)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7405..8196,8197..8265))
                     /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     8028..8047
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8266..8370)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(8449..8468)
                     /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 ccaccatgta cccttatgat gttccagatt atgccggata
     1801 cccatacgat gtccctgact atgcaggctc ctacccttat gacgtcccag actacgccac
     1861 cggtcccaag aagaagagga aggttcccgg gatggccatc cgcagcatca agctgaagct
     1921 gaagacccac accggccccg aggcccagaa cctgcgcaag ggcatctggc gcacccaccg
     1981 cctgctgaac gagggcgtgg cctactacat gaagatgctg ctgctgttcc gccaggagag
     2041 caccggcgag cgccccaagg aggagctgca ggaggagctg atctgccaca tccgcgagca
     2101 gcagcagcgc aaccaggccg acaagaacac ccaggccctg cccctggaca aggccctgga
     2161 ggccctgcgc cagctgtacg agctgctggt gcccagcagc gtgggccaga gcggcgacgc
     2221 ccagatcatc agccgcaagt tcctgagccc cctggtggac cccaacagcg agggcggcaa
     2281 gggcaccagc aaggccggcg ccaagcccac ctggcagaag aagaaggagg ccaacgaccc
     2341 cacctgggag caggactacg agaagtggaa gaagcgccgc gaggaggacc ccaccgccag
     2401 cgtgatcacc accctggagg agtacggcat ccgccccatc ttccccctgt acaccaacac
     2461 cgtgaccgac atcgcctggc tgcccctgca gagcaaccag ttcgtgcgca cctgggaccg
     2521 cgacatgctg cagcaggcca tcgagcgcct gctgagctgg gagagctgga acaagcgcgt
     2581 gcaggaggag tacgccaagc tgaaggagaa gatggcccag ctgaacgagc agctggaggg
     2641 cggccaggag tggatcagcc tgctggagca gtacgaggag aaccgcgagc gcgagctgcg
     2701 cgagaacatg accgccgcca acgacaagta ccgcatcacc aagcgccaga tgaagggctg
     2761 gaacgagctg tacgagctgt ggagcacctt ccccgccagc gccagccacg agcagtacaa
     2821 ggaggccctg aagcgcgtgc agcagcgcct gcgcggccgc ttcggcgacg cccacttctt
     2881 ccagtacctg atggaggaga agaaccgcct gatctggaag ggcaaccccc agcgcatcca
     2941 ctacttcgtg gcccgcaacg agctgaccaa gcgcctggag gaggccaagc agagcgccac
     3001 catgaccctg cccaacgccc gcaagcaccc cctgtgggtg cgcttcgacg cccgcggcgg
     3061 caacctgcag gactactacc tgaccgccga ggccgacaag ccccgcagcc gccgcttcgt
     3121 gaccttcagc cagctgatct ggcccagcga gagcggctgg atggagaaga aggacgtgga
     3181 ggtggagctg gccctgagcc gccagttcta ccagcaggtg aagctgctga agaacgacaa
     3241 gggcaagcag aagatcgagt tcaaggacaa gggcagcggc agcaccttca acggccacct
     3301 gggcggcgcc aagctgcagc tggagcgcgg cgacctggag aaggaggaga agaacttcga
     3361 ggacggcgag atcggcagcg tgtacctgaa cgtggtgatc gacttcgagc ccctgcagga
     3421 ggtgaagaac ggccgcgtgc aggcccccta cggccaggtg ctgcagctga tccgccgccc
     3481 caacgagttc cccaaggtga ccacctacaa gagcgagcag ctggtggagt ggatcaaggc
     3541 cagcccccag cacagcgccg gcgtggagag cctggccagc ggcttccgcg tgatgagcat
     3601 cgacctgggc ctgcgcgccg ccgccgccac cagcatcttc agcgtggagg agagcagcga
     3661 caagaacgcc gccgacttca gctactggat cgagggcacc cccctggtgg ccgtgcacca
     3721 gcgcagctac atgctgcgcc tgcccggcga gcaggtggag aagcaggtga tggagaagcg
     3781 cgacgagcgc ttccagctgc accagcgcgt gaagttccag atccgcgtgc tggcccagat
     3841 catgcgcatg gccaacaagc agtacggcga ccgctgggac gagctggaca gcctgaagca
     3901 ggccgtggag cagaagaaga gccccctgga ccagaccgac cgcaccttct gggagggcat
     3961 cgtgtgcgac ctgaccaagg tgctgccccg caacgaggcc gactgggagc aggccgtggt
     4021 gcagatccac cgcaaggccg aggagtacgt gggcaaggcc gtgcaggcct ggcgcaagcg
     4081 cttcgccgcc gacgagcgca agggcatcgc cggcctgagc atgtggaaca tcgaggagct
     4141 ggagggcctg cgcaagctgc tgatcagctg gagccgccgc acccgcaacc cccaggaggt
     4201 gaaccgcttc gagcgcggcc acaccagcca ccagcgcctg ctgacccaca tccagaacgt
     4261 gaaggaggac cgcctgaagc agctgagcca cgccatcgtg atgaccgccc tgggctacgt
     4321 gtacgacgag cgcaagcagg agtggtgcgc cgagtacccc gcctgccagg tgatcctgtt
     4381 cgagaacctg agccagtacc gcagcaacct ggaccgcagc accaaggaga acagcaccct
     4441 gatgaagtgg gcccaccgca gcatccccaa gtacgtgcac atgcaggccg agccctacgg
     4501 catccagatc ggcgacgtgc gcgccgagta cagcagccgc ttctacgcca agaccggcac
     4561 ccccggcatc cgctgcaaga aggtgcgcgg ccaggacctg cagggccgcc gcttcgagaa
     4621 cctgcagaag cgcctggtga acgagcagtt cctgaccgag gagcaggtga agcagctgcg
     4681 ccccggcgac atcgtgcccg acgacagcgg cgagctgttc atgaccctga ccgacggcag
     4741 cggcagcaag gaggtggtgt tcctgcaggc cgacatcaac gccgcccaca acctgcagaa
     4801 gcgcttctgg cagcgctaca acgagctgtt caaggtgagc tgccgcgtga tcgtgcgcga
     4861 cgaggaggag tacctggtgc ccaagaccaa gagcgtgcag gccaagctgg gcaagggcct
     4921 gttcgtgaag aagagcgaca ccgcctggaa ggacgtgtac gtgtgggaca gccaggccaa
     4981 gctgaagggc aagaccacct tcaccgagga gagcgagagc cccgagcagc tggaggactt
     5041 ccaggagatc atcgaggagg ccgaggaggc caagggcacc taccgcaccc tgttccgcga
     5101 ccccagcggc gtgttcttcc ccgagagcgt gtggtacccc cagaaggact tctggggcga
     5161 ggtgaagcgc aagctgtacg gcaagctgcg cgagcgcttc ctgaccaagg cccgcgctag
     5221 cccaaagaag aagaggaaag tctctagaag gtccggcggc ggagagggca gaggaagtct
     5281 tctaacatgc ggtgacgtgg aggagaatcc cggcccaatg gtgagcaagg gcgaggagct
     5341 gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt
     5401 cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc aagctgaccc tgaagttcat
     5461 ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg
     5521 cgtgcagtgc ttcagccgct accccgacca catgaagcag cacgacttct tcaagtccgc
     5581 catgcccgaa ggctacgtcc aggagcgcac catcttcttc aaggacgacg gcaactacaa
     5641 gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg
     5701 catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca actacaacag
     5761 ccacaacgtc tatatcatgg ccgacaagca gaagaacggc atcaaggtga acttcaagat
     5821 ccgccacaac atcgaggacg gcagcgtgca gctcgccgac cactaccagc agaacacccc
     5881 catcggcgac ggccccgtgc tgctgcccga caaccactac ctgagcaccc agtccgccct
     5941 gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg tgaccgccgc
     6001 cgggatcact ctcggcatgg acgagctgta caagtaactg cagcgcgggg atctcatgct
     6061 ggagttcttc gcccacccca acttgtttat tgcagcttat aatggttaca aataaagcaa
     6121 tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc
     6181 caaactcatc aatgtatctt attaattaac gtcgacctct agctagagct tggcgtaatc
     6241 atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg
     6301 agccggaagc ataaagtgta aagcctaggg tgcctaatga gtgagctaac tcacattaat
     6361 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     6421 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct
     6481 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
     6541 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
     6601 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     6661 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     6721 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     6781 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     6841 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     6901 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     6961 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     7021 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     7081 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     7141 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     7201 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
     7261 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
     7321 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     7381 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
     7441 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     7501 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc
     7561 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     7621 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     7681 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg
     7741 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     7801 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     7861 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     7921 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     7981 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
     8041 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     8101 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     8161 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     8221 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca
     8281 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
     8341 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt
     8401 cgacggatcg ggagatcgat ctcccgatcc cctagggtcg actctcagta caatctgctc
     8461 tgatgccgca tagttaagcc agtatctgct ccctgcttgt gtgttggagg tcgctgagta
     8521 gtgcgcgagc aaaatttaag ctacaacaag gcaaggcttg accgacaatt gcatgaagaa
     8581 tctgcttagg gttaggcgtt ttgcgctgct tcgcgatgta cgggccagat at
//
