LOCUS       sequence-373798-        8698 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 8698)
  AUTHORS   Chen Y, Hu Y, Wang X, Luo S, Yang N, Chen Y, Li Z, Zhou Q, Li W
  TITLE     Synergistic engineering of CRISPR-Cas nucleases enables robust 
            mammalian genome editing.
  JOURNAL   Innovation (Camb). 2022 May 26;3(4):100264. doi: 
            10.1016/j.xinn.2022.100264. eCollection 2022 Jul 12.
  PUBMED    35693153
REFERENCE   2  (bases 1 to 8698)
  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: "Innovation 
            (Camb)."; date: "2022-05-26"; volume: "3(4):100264. doi"; pages: " 
            10.1016/j.xinn.2022.100264. eCollection 2022 Jul 12"
COMMENT     Sequence Label: pCAG-AaCas12b-T2A-EGFP
FEATURES             Location/Qualifiers
     source          1..8698
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        8..387
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        389..664
                     /label=chicken beta-actin promoter
     intron          665..1681
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1605..1627
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1689..1708
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     promoter        1742..1760
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1772..1792
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     regulatory      4583..4592
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             5192..5212
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5219..5308
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     CDS             5330..5383
                     /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(5384..5386,5387..5389,5390..6103)
                     /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(5384..5386,5387..5389,5390..6100)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(5429..5450)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(5690..5709)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     6037..6058
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    6146..6267
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6183..6202)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6237..6256
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6310..6326)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6310..6326)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6323..6345)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6334..6350
                     /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(6358..6364,6365..6382,6383..6388))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6403..6424
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6541..6558)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6712..7300)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6792..6811)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7471..8262,8263..8331))
                     /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     8094..8113
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8332..8436)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(8515..8534)
                     /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 ggggggggsg cgcgccaggc ggggcggggc ggggcgaggg
      541 gcggggcggg gcgaggcgga gaggtgcggc ggcagccaat cagagcggcg cgctccgaaa
      601 gtttcctttt atggcgaggc ggcggcggcg gcggccctat aaaaagcgaa gcgcgcggcg
      661 ggcgggagtc gctgcgcgct gccttcgccc cgtgccccgc tccgccgccg cctcgcgccg
      721 cccgccccgg ctctgactga ccgcgttact cccacaggtg agcgggcggg acggcccttc
      781 tcctccgggc tgtaattagc gcttggttta atgacggctt gtttcttttc tgtggctgcg
      841 tgaaagcctt gaggggctcc gggagggccc tttgtgcggg gggagcggct cggggggtgc
      901 gtgcgtgtgt gtgtgcgtgg ggagcgccgc gtgcggctcc gcgctgcccg gcggctgtga
      961 gcgctgcggg cgcggcgcgg ggctttgtgc gctccgcagt gtgcgcgagg ggagcgcggc
     1021 cgggggcggt gccccgcggt gcgggggggg ctgcgagggg aacaaaggct gcgtgcgggg
     1081 tgtgtgcgtg ggggggtgag cagggggtgt gggcgcgtcg gtcgggctgc aaccccccct
     1141 gcacccccct ccccgagttg ctgagcacgg cccggcttcg ggtgcggggc tccgtacggg
     1201 gcgtggcgcg gggctcgccg tgccgggcgg ggggtggcgg caggtggggg tgccgggcgg
     1261 ggcggggccg cctcgggccg gggagggctc gggggagggg cgcggcggcc cccggagcgc
     1321 cggcggctgt cgaggcgcgg cgagccgcag ccattgcctt ttatggtaat cgtgcgagag
     1381 ggcgcaggga cttcctttgt cccaaatctg tgcggagccg aaatctggga ggcgccgccg
     1441 caccccctct agcgggcgcg gggcgaagcg gtgcggcgcc ggcaggaagg aaatgggcgg
     1501 ggagggcctt cgtgcgtcgc cgcgccgccg tccccttctc cctctccagc ctcggggctg
     1561 tccgcggggg gacggctgcc ttcggggggg acggggcagg gcggggttcg gcttctggcg
     1621 tgtgaccggc ggctctagcg cctctgctaa ccatgttcat gccttcttct ttttcctaca
     1681 gctcctgggc aacgtgctgg ttattgtgct gtctcatcat tttggcaaag ctagtgaatt
     1741 ctaatacgac tcactatagg ccgccaccat gcccaagaag aagaggaagg ttcccgggat
     1801 ggccgtgaag tccatgaagg tgaagctgcg gctggataac atgcccgaga tcagagccgg
     1861 cctgtggaag ctgcacaccg aagtgaatgc cggcgtgagg tactatacag agtggctgtc
     1921 tctgctgaga caggagaacc tgtaccggag aagccccaat ggcgacggcg agcaggagtg
     1981 ctataagacc gccgaggagt gtaaggcaga gctgctggag aggctgaggg cacggcaggt
     2041 ggagaacggc cactgcggcc ctgccggctc cgacgatgag ctgctgcagc tggcaaggca
     2101 gctgtacgag ctgctggtgc cacaggcaat cggagcaaag ggcgatgcac agcagatcgc
     2161 cagaaagttc ctgtctccac tggcagacaa ggatgcagtg ggaggactgg gaatcgcaaa
     2221 ggcaggcaat aagcctcggt gggtgagaat gagggaggca ggagagccag gatgggagga
     2281 ggagaaggca aaggcagagg caaggaagag caccgacagg acagcagatg tgctgagggc
     2341 cctggcagac ttcggcctga agccactgat gcgcgtgtac accgactccg atatgagctc
     2401 cgtgcagtgg aagcctctga ggaagggaca ggccgtgcgg acatgggaca gagatatgtt
     2461 tcagcaggcc atcgagcgga tgatgtcctg ggagtcttgg aaccagagag tgggcgaggc
     2521 ctatgccaag ctggtggagc agaagagccg cttcgagcag aagaactttg tgggccagga
     2581 gcacctggtg cagctggtga atcagctgca gcaggatatg aaggaggcct ctcacggcct
     2641 ggagagcaag gagcagaccg cacactacct gacaggacgg gccctgagag gatccgacaa
     2701 ggtgttcgag aagtgggaga agctggaccc cgatgcccct tttgacctgt atgataccga
     2761 gatcaagaac gtgcagaggc gcaatacacg gagattcggc agccacgatc tgtttgccaa
     2821 gctggccgag ccaaagtacc aggccctgtg gagggaggac gcctccttcc tgacccgcta
     2881 cgccgtgtat aactctatcg tgaggaagct gaatcacgcc aagatgttcg ccacctttac
     2941 actgccagat gccaccgccc accccatctg gacacgcttt gacaagctgg gcggcaacct
     3001 gcaccagtat acattcctgt ttaatgagtt cggagagggc aggcacgcaa tcaggtttca
     3061 gaagctgctg accgtggagg acggagtggc aaaggaggtg gacgatgtga cagtgcccat
     3121 ctccatgtct gcccagctgg acgatctgct gccaagggat ccacacgagc tggtggccct
     3181 gtacttccag gactatggcg ccgagcagca cctggcagga gagtttggag gagccaagat
     3241 ccagtacagg cgcgatcagc tgaaccacct gcacgcaagg agaggagcaa gagacgtgta
     3301 tctgaatctg tccgtgcggg tgcagagcca gtccgaggca agaggagaga ggcgcccccc
     3361 ttacgcagcc gtgttccggc tggtgggcga caaccacaga gccttcgtgc actttgacaa
     3421 gctgtccgat tatctggccg agcaccctga cgatggcaag ctgggatccg agggactgct
     3481 gtctggactg agagtgatgt ctgtggatct gggcctgaga acctctgcca gcatctccgt
     3541 gtttagggtg gcccgcaagg acgagctgaa gcctaatagc gagggccggg tgcctttctg
     3601 ctttccaatc gagggcaacg agaatctggt ggccgtgcac gagagatccc agctgctgaa
     3661 gctgccaggc gagacagagt ctaaggatct gagggcaatc agagaggaga ggcagcggac
     3721 cctgaggcag ctgcgcacac agctggccta cctgcggctg ctggtgagat gcggaagcga
     3781 ggacgtgggc cggagagaga ggtcctgggc caagctgatc gagcagccta tggacgccaa
     3841 ccagatgacc ccagattggc gcgaggcctt cgaggacgag ctgcagaagc tgaagagcct
     3901 gtacggcatc tgtggcgata gggagtggac agaggccgtg tatgagtccg tgaggcgcgt
     3961 gtggaggcac atgggcaagc aggtgcggga ctggagaaag gatgtgaggt ctggagagag
     4021 gccaaagatc aggggctatc agaaggacgt ggtgggcggc aatagcatcg agcagatcga
     4081 gtacctggag aggcagtata agttcctgaa gtcttggagc ttctttggca aggtgagcgg
     4141 ccaggtcatc agagccgaga agggctccag gtttgccatc accctgcgcg agcacatcga
     4201 ccacgccaag gaggataggc tgaagaagct ggccgatcgc atcatcatgg aggccctggg
     4261 ctacgtgtat gccctggacg atgagcgcgg caagggcaag tgggtggcca agtacccacc
     4321 ctgtcagctg atcctgctgg aggagctgag cgagtatcag ttcaacaatg accggcctcc
     4381 atctgagaac aatcagctga tgcagtggag ccacagaggc gtgtttcagg agctgctgaa
     4441 ccaggcccag gtgcacgatc tgctggtggg caccatgtac gccgccttct ctagcagatt
     4501 tgacgcaagg acaggagcac caggaatcag gtgcaggaga gtgcctgcaa ggtgtgcaag
     4561 ggagcagaac ccagagcctt tcccatggtg gctgaataag tttgtggccg agcacaagct
     4621 ggacggctgc ccactgagag cagacgatct gatcccaacc ggagagggag agttctttgt
     4681 gagccctttc tccgccgagg agggcgattt tcaccagatc cacgccgacc tgaacgccgc
     4741 ccagaatctg cagaggcgcc tgtggagcga cttcgatatc tcccagatca gactgaggtg
     4801 tgactgggga gaggtggatg gagagcccgt gctgatccct agaaccacag gcaagaggac
     4861 cgccgattct tacggcaaca aggtgttcta taccaagaca ggcgtgacat actatgagcg
     4921 cgagcggggc aagaagcgga gaaaggtgtt tgcacaggag gagctgagcg aggaggaggc
     4981 agagctgctg gtggaggcag acgaggccag ggagaagtcc gtggtgctga tgcgcgatcc
     5041 atctggcatc atcaaccgcg gcgactggac ccggcagaag gagttctgga gcatggtgaa
     5101 tcagagaatc gagggctacc tggtgaagca gatcagatct agggtgcgcc tgcaggagag
     5161 cgcctgtgag aatacaggcg acatcgctag cccaaagaag aagaggaaag tctctagata
     5221 cccttatgat gttccagatt atgccggata cccatacgat gtccctgact atgcaggctc
     5281 ctacccttat gacgtcccag actacgccgg atccaggtcc ggcggcggag agggcagagg
     5341 aagtcttcta acatgcggtg acgtggagga gaatcccggc ccaatggtga gcaagggcga
     5401 ggagctgttc accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca
     5461 caagttcagc gtgtccggcg agggcgaggg cgatgccacc tacggcaagc tgaccctgaa
     5521 gttcatctgc accaccggca agctgcccgt gccctggccc accctcgtga ccaccctgac
     5581 ctacggcgtg cagtgcttca gccgctaccc cgaccacatg aagcagcacg acttcttcaa
     5641 gtccgccatg cccgaaggct acgtccagga gcgcaccatc ttcttcaagg acgacggcaa
     5701 ctacaagacc cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct
     5761 gaagggcatc gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaacta
     5821 caacagccac aacgtctata tcatggccga caagcagaag aacggcatca aggtgaactt
     5881 caagatccgc cacaacatcg aggacggcag cgtgcagctc gccgaccact accagcagaa
     5941 cacccccatc ggcgacggcc ccgtgctgct gcccgacaac cactacctga gcacccagtc
     6001 cgccctgagc aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac
     6061 cgccgccggg atcactctcg gcatggacga gctgtacaag taactgcagc gcggggatct
     6121 catgctggag ttcttcgccc accccaactt gtttattgca gcttataatg gttacaaata
     6181 aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg
     6241 tttgtccaaa ctcatcaatg tatcttatta attaacgtcg acctctagct agagcttggc
     6301 gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa
     6361 catacgagcc ggaagcataa agtgtaaagc ctagggtgcc taatgagtga gctaactcac
     6421 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     6481 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc
     6541 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
     6601 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
     6661 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     6721 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     6781 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     6841 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     6901 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     6961 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     7021 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     7081 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     7141 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     7201 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     7261 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     7321 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
     7381 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
     7441 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
     7501 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     7561 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg
     7621 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
     7681 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     7741 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
     7801 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
     7861 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
     7921 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
     7981 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
     8041 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
     8101 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
     8161 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
     8221 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
     8281 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct
     8341 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
     8401 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc
     8461 tgacgtcgac ggatcgggag atcgatctcc cgatccccta gggtcgactc tcagtacaat
     8521 ctgctctgat gccgcatagt taagccagta tctgctccct gcttgtgtgt tggaggtcgc
     8581 tgagtagtgc gcgagcaaaa tttaagctac aacaaggcaa ggcttgaccg acaattgcat
     8641 gaagaatctg cttagggtta ggcgttttgc gctgcttcgc gatgtacggg ccagatat
//
