LOCUS       sequence-232178-        8749 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 8749)
  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 8749)
  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-AkCas12b-2AeGFP
FEATURES             Location/Qualifiers
     source          1..8749
                     /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             5339..5359
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5381..5434
                     /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(5435..5437,5438..5440,5441..6154)
                     /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(5435..5437,5438..5440,5441..6151)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(5480..5501)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(5741..5760)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     6088..6109
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    6197..6318
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6234..6253)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6288..6307
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6361..6377)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6361..6377)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6374..6396)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6385..6401
                     /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(6409..6415,6416..6433,6434..6439))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6454..6475
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6592..6609)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6763..7351)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6843..6862)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7522..8313,8314..8382))
                     /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     8145..8164
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8383..8487)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(8566..8585)
                     /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 gatggccgtg aagagcatca aggtgaagct
     1921 gcgcctgagc gagtgccccg acatcctggc cggcatgtgg cagctgcacc gcgccaccaa
     1981 cgccggcgtg cgctactaca ccgagtgggt gagcctgatg cgccaggaga tcctgtacag
     2041 ccgcggcccc gacggcggcc agcagtgcta catgaccgcc gaggactgcc agcgcgagct
     2101 gctgcgccgc ctgcgcaacc gccagctgca caacggccgc caggaccagc ccggcaccga
     2161 cgccgacctg ctggccatca gccgccgcct gtacgagatc ctggtgctgc agagcatcgg
     2221 caagcgcggc gacgcccagc agatcgccag cagcttcctg agccccctgg tggaccccaa
     2281 cagcaagggc ggccgcggcg aggccaagag cggccgcaag cccgcctggc agaagatgcg
     2341 cgaccagggc gacccccgct gggtggccgc ccgcgagaag tacgagcagc gcaaggccgt
     2401 ggaccccagc aaggagatcc tgaacagcct ggacgccctg ggcctgcgcc ccctgttcgc
     2461 cgtgttcacc gagacctacc gcagcggcgt ggactggaag cccctgggca agagccaggg
     2521 cgtgcgcacc tgggaccgcg acatgttcca gcaggccctg gagcgcctga tgagctggga
     2581 gagctggaac cgccgcgtgg gcgaggagta cgcccgcctg ttccagcaga agatgaagtt
     2641 cgagcaggag cacttcgccg agcagagcca cctggtgaag ctggcccgcg ccctggaggc
     2701 cgacatgcgc gccgccagcc agggcttcga ggccaagcgc ggcaccgccc accagatcac
     2761 ccgccgcgcc ctgcgcggcg ccgaccgcgt gttcgagatc tggaagagca tccccgagga
     2821 ggccctgttc agccagtacg acgaggtgat ccgccaggtg caggccgaga agcgccgcga
     2881 cttcggcagc cacgacctgt tcgccaagct ggccgagccc aagtaccagc ccctgtggcg
     2941 cgccgacgag accttcctga cccgctacgc cctgtacaac ggcgtgctgc gcgacctgga
     3001 gaaggcccgc cagttcgcca ccttcaccct gcccgacgcc tgcgtgaacc ccatctggac
     3061 ccgcttcgag agcagccagg gcagcaacct gcacaagtac gagttcctgt tcgaccacct
     3121 gggccccggc cgccacgccg tgcgcttcca gcgcctgctg gtggtggaga gcgagggcgc
     3181 caaggagcgc gacagcgtgg tggtgcccgt ggcccccagc ggccagctgg acaagctggt
     3241 gctgcgcgag gaggagaaga gcagcgtggc cctgcacctg cacgacaccg cccgccccga
     3301 cggcttcatg gccgagtggg ccggcgccaa gctgcagtac gagcgcagca ccctggcccg
     3361 caaggcccgc cgcgacaagc agggcatgcg cagctggcgc cgccagccca gcatgctgat
     3421 gagcgccgcc cagatgctgg aggacgccaa gcaggccggc gacgtgtacc tgaacatcag
     3481 cgtgcgcgtg aagagcccca gcgaggtgcg cggccagcgc cgccccccct acgccgccct
     3541 gttccgcatc gacgacaagc agcgccgcgt gaccgtgaac tacaacaagc tgagcgccta
     3601 cctggaggag caccccgaca agcagatccc cggcgccccc ggcctgctga gcggcctgcg
     3661 cgtgatgagc gtggacctgg gcctgcgcac cagcgccagc atcagcgtgt tccgcgtggc
     3721 caagaaggag gaggtggagg ccctgggcga cggccgcccc ccccactact accccatcca
     3781 cggcaccgac gacctggtgg ccgtgcacga gcgcagccac ctgatccaga tgcccggcga
     3841 gaccgagacc aagcagctgc gcaagctgcg cgaggagcgc caggccgtgc tgcgccccct
     3901 gttcgcccag ctggccctgc tgcgcctgct ggtgcgctgc ggcgccgccg acgagcgcat
     3961 ccgcacccgc agctggcagc gcctgaccaa gcagggccgc gagttcacca agcgcctgac
     4021 ccccagctgg cgcgaggccc tggagctgga gctgacccgc ctggaggcct actgcggccg
     4081 cgtgcccgac gacgagtgga gccgcatcgt ggaccgcacc gtgatcgccc tgtggcgccg
     4141 catgggcaag caggtgcgcg actggcgcaa gcaggtgaag agcggcgcca aggtgaaggt
     4201 gaagggctac cagctggacg tggtgggcgg caacagcctg gcccagatcg actacctgga
     4261 gcagcagtac aagttcctgc gccgctggag cttcttcgcc cgcgccagcg gcctggtggt
     4321 gcgcgccgac cgcgagagcc acttcgccgt ggccctgcgc cagcacatcg agaacgccaa
     4381 gcgcgaccgc ctgaagaagc tggccgaccg catcctgatg gaggccctgg gctacgtgta
     4441 cgaggccagc ggcccccgcg agggccagtg gaccgcccag caccccccct gccagctgat
     4501 catcctggag gagctgagcg cctaccgctt cagcgacgac cgccccccca gcgagaacag
     4561 caagctgatg gcctggggcc accgcggcat cctggaggag ctggtgaacc aggcccaggt
     4621 gcacgacgtg ctggtgggca ccgtgtacgc cgccttcagc agccgcttcg acgcccgcac
     4681 cggcgccccc ggcgtgcgct gccgccgcgt gcccgcccgc ttcgtgggcg ccaccgtgga
     4741 cgacagcctg cccctgtggc tgaccgagtt cctggacaag caccgcctgg acaagaacct
     4801 gctgcgcccc gacgacgtga tccccaccgg cgagggcgag ttcctggtga gcccctgcgg
     4861 cgaggaggcc gcccgcgtgc gccaggtgca cgccgacatc aacgccgccc agaacctgca
     4921 gcgccgcctg tggcagaact tcgacatcac cgagctgcgc ctgcgctgcg acgtgaagat
     4981 gggcggcgag ggcaccgtgc tggtgccccg cgtgaacaac gcccgcgcca agcagctgtt
     5041 cggcaagaag gtgctggtga gccaggacgg cgtgaccttc ttcgagcgca gccagaccgg
     5101 cggcaagccc cacagcgaga agcagaccga cctgaccgac aaggagctgg agctgatcgc
     5161 cgaggccgac gaggcccgcg ccaagagcgt ggtgctgttc cgcgacccca gcggccacat
     5221 cggcaagggc cactggatcc gccagcgcga gttctggagc ctggtgaagc agcgcatcga
     5281 gagccacacc gccgagcgca tccgcgtgcg cggcgtgggc agcagcctgg acgctagccc
     5341 aaagaagaag aggaaagtct ctagaaggtc cggcggcgga gagggcagag gaagtcttct
     5401 aacatgcggt gacgtggagg agaatcccgg cccaatggtg agcaagggcg aggagctgtt
     5461 caccggggtg gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag
     5521 cgtgtccggc gagggcgagg gcgatgccac ctacggcaag ctgaccctga agttcatctg
     5581 caccaccggc aagctgcccg tgccctggcc caccctcgtg accaccctga cctacggcgt
     5641 gcagtgcttc agccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat
     5701 gcccgaaggc tacgtccagg agcgcaccat cttcttcaag gacgacggca actacaagac
     5761 ccgcgccgag gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat
     5821 cgacttcaag gaggacggca acatcctggg gcacaagctg gagtacaact acaacagcca
     5881 caacgtctat atcatggccg acaagcagaa gaacggcatc aaggtgaact tcaagatccg
     5941 ccacaacatc gaggacggca gcgtgcagct cgccgaccac taccagcaga acacccccat
     6001 cggcgacggc cccgtgctgc tgcccgacaa ccactacctg agcacccagt ccgccctgag
     6061 caaagacccc aacgagaagc gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg
     6121 gatcactctc ggcatggacg agctgtacaa gtaactgcag cgcggggatc tcatgctgga
     6181 gttcttcgcc caccccaact tgtttattgc agcttataat ggttacaaat aaagcaatag
     6241 catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa
     6301 actcatcaat gtatcttatt aattaacgtc gacctctagc tagagcttgg cgtaatcatg
     6361 gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc
     6421 cggaagcata aagtgtaaag cctagggtgc ctaatgagtg agctaactca cattaattgc
     6481 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
     6541 cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac
     6601 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     6661 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     6721 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     6781 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     6841 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     6901 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     6961 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     7021 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     7081 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     7141 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     7201 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     7261 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     7321 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     7381 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     7441 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     7501 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     7561 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     7621 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     7681 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     7741 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     7801 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     7861 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     7921 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     7981 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     8041 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     8101 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     8161 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     8221 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     8281 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     8341 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     8401 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     8461 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcga
     8521 cggatcggga gatcgatctc ccgatcccct agggtcgact ctcagtacaa tctgctctga
     8581 tgccgcatag ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg
     8641 cgcgagcaaa atttaagcta caacaaggca aggcttgacc gacaattgca tgaagaatct
     8701 gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg gccagatat
//
