LOCUS       sequence-232177-        8746 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 8746)
  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 8746)
  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..8746
                     /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..1789
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5240..5260
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5267..5356
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     CDS             5378..5431
                     /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(5432..5434,5435..5437,5438..6151)
                     /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(5432..5434,5435..5437,5438..6148)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(5477..5498)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(5738..5757)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     6085..6106
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    6194..6315
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6231..6250)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6285..6304
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6358..6374)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6358..6374)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6371..6393)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6382..6398
                     /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(6406..6412,6413..6430,6431..6436))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6451..6472
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6589..6606)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6760..7348)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6840..6859)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7519..8310,8311..8379))
                     /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     8142..8161
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8380..8484)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(8563..8582)
                     /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 ccaccatgcc caagaagaag aggaaggttc ccgggatgaa
     1801 cgtggccgtg aagagcatca aggtgaagct gatgctgggc cacctgcccg agatccgcga
     1861 gggcctgtgg cacctgcacg aggccgtgaa cctgggcgtg cgctactaca ccgagtggct
     1921 ggccctgctg cgccagggca acctgtaccg ccgcggcaag gacggcgccc aggagtgcta
     1981 catgaccgcc gagcagtgcc gccaggagct gctggtgcgc ctgcgcgacc gccagaagcg
     2041 caacggccac accggcgacc ccggcaccga cgaggagctg ctgggcgtgg cccgccgcct
     2101 gtacgagctg ctggtgcccc agagcgtggg caagaagggc caggcccaga tgctggccag
     2161 cggcttcctg agccccctgg ccgaccccaa gagcgagggc ggcaagggca ccagcaagag
     2221 cggccgcaag cccgcctgga tgggcatgaa ggaggccggc gacagccgct gggtggaggc
     2281 caaggcccgc tacgaggcca acaaggccaa ggaccccacc aagcaggtga tcgccagcct
     2341 ggagatgtac ggcctgcgcc ccctgttcga cgtgttcacc gagacctaca agaccatccg
     2401 ctggatgccc ctgggcaagc accagggcgt gcgcgcctgg gaccgcgaca tgttccagca
     2461 gagcctggag cgcctgatga gctgggagag ctggaacgag cgcgtgggcg ccgagttcgc
     2521 ccgcctggtg gaccgccgcg accgcttccg cgagaagcac ttcaccggcc aggagcacct
     2581 ggtggccctg gcccagcgcc tggagcagga gatgaaggag gccagccccg gcttcgagag
     2641 caagagcagc caggcccacc gcatcaccaa gcgcgccctg cgcggcgccg acggcatcat
     2701 cgacgactgg ctgaagctga gcgagggcga gcccgtggac cgcttcgacg agatcctgcg
     2761 caagcgccag gcccagaacc cccgccgctt cggcagccac gacctgttcc tgaagctggc
     2821 cgagcccgtg ttccagcccc tgtggcgcga ggaccccagc ttcctgagcc gctgggccag
     2881 ctacaacgag gtgctgaaca agctggagga cgccaagcag ttcgccacct tcaccctgcc
     2941 cagcccctgc agcaaccccg tgtgggcccg cttcgagaac gccgagggca ccaacatctt
     3001 caagtacgac ttcctgttcg accacttcgg caagggccgc cacggcgtgc gcttccagcg
     3061 catgatcgtg atgcgcgacg gcgtgcccac cgaggtggag ggcatcgtgg tgcccatcgc
     3121 ccccagccgc cagctggacg ccctggcccc caacgacgcc gccagcccca tcgacgtgtt
     3181 cgtgggcgac cccgccgccc ccggcgcctt ccgcggccag ttcggcggcg ccaagatcca
     3241 gtaccgccgc agcgccctgg tgcgcaaggg ccgccgcgag gagaaggcct acctgtgcgg
     3301 cttccgcctg cccagccagc gccgcaccgg cacccccgcc gacgacgccg gcgaggtgtt
     3361 cctgaacctg agcctgcgcg tggagagcca gagcgagcag gccggccgcc gcaacccccc
     3421 ctacgccgcc gtgttccaca tcagcgacca gacccgccgc gtgatcgtgc gctacggcga
     3481 gatcgagcgc tacctggccg agcaccccga caccggcatc cccggcagcc gcggcctgac
     3541 cagcggcctg cgcgtgatga gcgtggacct gggcctgcgc accagcgccg ccatcagcgt
     3601 gttccgcgtg gcccaccgcg acgagctgac ccccgacgcc cacggccgcc agcccttctt
     3661 cttccccatc cacggcatgg accacctggt ggccctgcac gagcgcagcc acctgatccg
     3721 cctgcccggc gagaccgaga gcaagaaggt gcgcagcatc cgcgagcagc gcctggaccg
     3781 cctgaaccgc ctgcgcagcc agatggccag cctgcgcctg ctggtgcgca ccggcgtgct
     3841 ggacgagcag aagcgcgacc gcaactggga gcgcctgcag agcagcatgg agcgcggcgg
     3901 cgagcgcatg cccagcgact ggtgggacct gttccaggcc caggtgcgct acctggccca
     3961 gcaccgcgac gccagcggcg aggcctgggg ccgcatggtg caggccgccg tgcgcaccct
     4021 gtggcgccag ctggccaagc aggtgcgcga ctggcgcaag gaggtgcgcc gcaacgccga
     4081 caaggtgaag atccgcggca tcgcccgcga cgtgcccggc ggccacagcc tggcccagct
     4141 ggactacctg gagcgccagt accgcttcct gcgcagctgg agcgccttca gcgtgcaggc
     4201 cggccaggtg gtgcgcgccg agcgcgacag ccgcttcgcc gtggccctgc gcgagcacat
     4261 cgacaacggc aagaaggacc gcctgaagaa gctggccgac cgcatcctga tggaggccct
     4321 gggctacgtg tacgtgaccg acggccgccg cgccggccag tggcaggccg tgtacccccc
     4381 ctgccagctg gtgctgctgg aggagctgag cgagtaccgc ttcagcaacg accgcccccc
     4441 cagcgagaac agccagctga tggtgtggag ccaccgcggc gtgctggagg agctgatcca
     4501 ccaggcccag gtgcacgacg tgctggtggg caccatcccc gccgccttca gcagccgctt
     4561 cgacgcccgc accggcgccc ccggcatccg ctgccgccgc gtgcccagca tccccctgaa
     4621 ggacgccccc agcatcccca tctggctgag ccactacctg aagcagaccg agcgcgacgc
     4681 cgccgccctg cgccccggcg agctgatccc caccggcgac ggcgagttcc tggtgacccc
     4741 cgccggccgc ggcgccagcg gcgtgcgcgt ggtgcacgcc gacatcaacg ccgcccacaa
     4801 cctgcagcgc cgcctgtggg agaacttcga cctgagcgac atccgcgtgc gctgcgaccg
     4861 ccgcgagggc aaggacggca ccgtggtgct gatcccccgc ctgaccaacc agcgcgtgaa
     4921 ggagcgctac agcggcgtga tcttcaccag cgaggacggc gtgagcttca ccgtgggcga
     4981 cgccaagacc cgccgccgca gcagcgccag ccagggcgag ggcgacgacc tgagcgacga
     5041 ggagcaggag ctgctggccg aggccgacga cgcccgcgag cgcagcgtgg tgctgttccg
     5101 cgaccccagc ggcttcgtga acggcggccg ctggaccgcc cagcgcgcct tctggggcat
     5161 ggtgcacaac cgcatcgaga ccctgctggc cgagcgcttc agcgtgagcg gcgccgccga
     5221 gaaggtgcgc ggcgctagcc caaagaagaa gaggaaagtc tctagatacc cttatgatgt
     5281 tccagattat gccggatacc catacgatgt ccctgactat gcaggctcct acccttatga
     5341 cgtcccagac tacgccggat ccaggtccgg cggcggagag ggcagaggaa gtcttctaac
     5401 atgcggtgac gtggaggaga atcccggccc aatggtgagc aagggcgagg agctgttcac
     5461 cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt
     5521 gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac
     5581 caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accctgacct acggcgtgca
     5641 gtgcttcagc cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc
     5701 cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg
     5761 cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga
     5821 cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca acagccacaa
     5881 cgtctatatc atggccgaca agcagaagaa cggcatcaag gtgaacttca agatccgcca
     5941 caacatcgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg
     6001 cgacggcccc gtgctgctgc ccgacaacca ctacctgagc acccagtccg ccctgagcaa
     6061 agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat
     6121 cactctcggc atggacgagc tgtacaagta actgcagcgc ggggatctca tgctggagtt
     6181 cttcgcccac cccaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat
     6241 cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact
     6301 catcaatgta tcttattaat taacgtcgac ctctagctag agcttggcgt aatcatggtc
     6361 atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg
     6421 aagcataaag tgtaaagcct agggtgccta atgagtgagc taactcacat taattgcgtt
     6481 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
     6541 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga
     6601 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat
     6661 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca
     6721 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
     6781 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
     6841 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
     6901 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
     6961 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
     7021 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
     7081 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
     7141 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
     7201 aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
     7261 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
     7321 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
     7381 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat
     7441 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
     7501 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
     7561 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga
     7621 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc
     7681 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac
     7741 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc
     7801 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc
     7861 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc
     7921 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt
     7981 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc
     8041 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg
     8101 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag
     8161 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat
     8221 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc
     8281 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa
     8341 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta
     8401 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa
     8461 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtcgacgg
     8521 atcgggagat cgatctcccg atcccctagg gtcgactctc agtacaatct gctctgatgc
     8581 cgcatagtta agccagtatc tgctccctgc ttgtgtgttg gaggtcgctg agtagtgcgc
     8641 gagcaaaatt taagctacaa caaggcaagg cttgaccgac aattgcatga agaatctgct
     8701 tagggttagg cgttttgcgc tgcttcgcga tgtacgggcc agatat
//
