LOCUS       sequence-373783-        8245 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 8245)
  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 8245)
  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-PlmCasX(T26R/K610R/K808R)-T2A-EGFP
FEATURES             Location/Qualifiers
     source          1..8245
                     /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"
     CDS             4739..4759
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4766..4855
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     CDS             4877..4930
                     /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(4931..4933,4934..4936,4937..5650)
                     /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(4931..4933,4934..4936,4937..5647)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(4976..4997)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(5237..5256)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5584..5605
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    5693..5814
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5730..5749)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5784..5803
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5857..5873)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5857..5873)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5870..5892)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5881..5897
                     /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(5905..5911,5912..5929,5930..5935))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5950..5971
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6088..6105)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6259..6847)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6339..6358)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7018..7809,7810..7878))
                     /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     7641..7660
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7879..7983)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(8062..8081)
                     /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 ggggggggcg 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 gcaggagatc aagaggatca ataagatccg gagaaggctg gtgaaggaca gcaacaccaa
     1861 gaaggccggc aagagaggcc ccatgaagac actgctggtg agagtgatga cacctgatct
     1921 gcgcgagcgg ctggagaacc tgagaaagaa gcctgagaat atcccacagc ccatctctaa
     1981 caccagccgg gccaacctga ataagctgct gaccgactat acagagatga agaaggccat
     2041 cctgcacgtg tactgggagg agttccagaa ggaccccgtg ggcctgatgt ccagagtggc
     2101 ccagcctgcc ccaaagaata tcgaccagcg gaagctgatc cccgtgaagg atggcaacga
     2161 gagactgacc agctccggct tcgcctgctc tcagtgctgt cagcctctgt acgtgtataa
     2221 gctggagcaa gtgaatgaca agggcaagcc acacacaaac tactttggcc ggtgtaacgt
     2281 gagcgagcac gagagactga tcctgctgtc tccccacaag cctgaggcca acgacgagct
     2341 ggtgacctat tccctgggca agttcggaca gcgggccctg gacttctact ctatccacgt
     2401 gacacgcgag agcaatcacc cagtgaagcc cctggagcag atcggaggca actcctgcgc
     2461 ctctggacca gtgggcaagg ccctgagcga cgcatgtatg ggagcagtgg cctccttcct
     2521 gaccaagtat caggatatca tcctggagca ccagaaagtg atcaagaaga acgagaagcg
     2581 gctggccaat ctgaaggaca tcgcctccgc caacggcctg gcctttccta agatcaccct
     2641 gccccctcag ccacacacaa aggagggcat cgaggcctac aacaatgtgg tggcccagat
     2701 cgtgatctgg gtgaacctga atctgtggca gaagctgaag atcggaaggg acgaggcaaa
     2761 gccactgcag cgcctgaagg gcttcccttc ttttccactg gtggagaggc aggccaatga
     2821 ggtggactgg tgggatatgg tgtgcaacgt gaagaagctg atcaatgaga agaaggagga
     2881 tggcaaggtg ttctggcaga acctggccgg ctataagcgc caggaggccc tgctgcctta
     2941 cctgtctagc gaggaggacc ggaagaaggg caagaagttc gccagatatc agtttggcga
     3001 tctgctgctg cacctggaga agaagcacgg cgaggactgg ggcaaggtgt acgatgaggc
     3061 ctgggagagg atcgacaaga aggtggaggg cctgtctaag cacatcaagc tggaggagga
     3121 gcgccggagc gaggacgccc agtccaaggc cgccctgacc gattggctga gggccaaggc
     3181 cagcttcgtg atcgagggcc tgaaggaggc cgacaaggat gagttttgcc ggtgtgagct
     3241 gaagctgcag aagtggtatg gcgacctgag aggcaagccc tttgccatcg aggccgagaa
     3301 tagcatcctg gatatcagcg gcttctccaa gcagtacaac tgcgccttta tctggcagaa
     3361 ggatggcgtg aagaagctga acctgtacct gatcatcaat tatttcaagg gcggcaagct
     3421 gaggtttaag aagatcaagc ccgaggcctt cgaggccaat cgcttttata cagtgatcaa
     3481 caagaagtcc ggcgagatcg tgcctatgga ggtgaacttc aattttgacg atccaaatct
     3541 gatcatcctg cccctggcct tcggcaagag acagggcagg gagtttatct ggaacgacct
     3601 gctgagcctg gagacaggca gcctgagact ggccaatggc agagtgatcg agaagaccct
     3661 gtacaacaga aggacacgcc aggatgagcc tgccctgttc gtggccctga catttgagcg
     3721 ccgggaggtg ctggactcct ctaacatcaa gccaatgaat ctgatcggca tcgatcgggg
     3781 cgagaatatc ccagccgtga tcgccctgac cgaccccgag ggatgtcctc tgtccagatt
     3841 caaggattct ctgggcaacc caacccacat cctgaggatc ggcgagagct ataaggagaa
     3901 gcagcgcaca atccaggccg ccaaggaggt ggagcagaga agggcaggag gatacagccg
     3961 gaagtatgcc tccaaggcca agaatctggc cgacgatatg gtgagaaaca ccgccaggga
     4021 cctgctgtac tatgccgtga cacaggatgc catgctgatc ttcgagaacc tgtctcgcgg
     4081 ctttggccgg cagggcaaga gaaccttcat ggccgagagg cagtacaccc gcatggagga
     4141 ctggctgaca gccaagctgg cctacgaggg cctgccttct aagacctatc tgagcaagac
     4201 actggcccag tacacctcca gaacatgctc taattgtggc tttaccatca caagcgccga
     4261 ctatgataga gtgctggaga agctgaagaa gaccgccaca ggctggatga ccacaatcaa
     4321 cggcaaggag ctgaaggtgg agggccagat cacatactat aaccgctaca agcggcagaa
     4381 tgtggtgaag gacctgagcg tggagctgga taggctgtct gaggagagcg tgaacaatga
     4441 tatcagctcc tggaccaagg gccgctccgg agaggccctg tctctgctga agaagcggtt
     4501 ctcccacaga ccagtgcagg agaagttcgt gtgcctgaat tgtggctttg agacacacgc
     4561 agacgagcag gccgccctga acatcgccag gagctggctg tttctgcgct cccaggagta
     4621 caagaagtat cagaccaaca agaccacagg caatacagat aagcgggcct tcgtggagac
     4681 atggcagtcc ttttacagaa agaagctgaa ggaagtgtgg aagcccgccg tggctagccc
     4741 aaagaagaag aggaaagtct ctagataccc ttatgatgtt ccagattatg ccggataccc
     4801 atacgatgtc cctgactatg caggctccta cccttatgac gtcccagact acgccggatc
     4861 caggtccggc ggcggagagg gcagaggaag tcttctaaca tgcggtgacg tggaggagaa
     4921 tcccggccca atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt
     4981 cgagctggac ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga
     5041 tgccacctac ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc
     5101 ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga
     5161 ccacatgaag cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg
     5221 caccatcttc ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg
     5281 cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat
     5341 cctggggcac aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa
     5401 gcagaagaac ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt
     5461 gcagctcgcc gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc
     5521 cgacaaccac tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga
     5581 tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct
     5641 gtacaagtaa ctgcagcgcg gggatctcat gctggagttc ttcgcccacc ccaacttgtt
     5701 tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc
     5761 atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttattaatt
     5821 aacgtcgacc tctagctaga gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa
     5881 ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagccta
     5941 gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca
     6001 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     6061 tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg
     6121 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
     6181 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
     6241 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     6301 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     6361 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     6421 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
     6481 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     6541 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     6601 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     6661 gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc
     6721 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     6781 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     6841 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
     6901 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
     6961 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
     7021 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
     7081 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
     7141 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca
     7201 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
     7261 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
     7321 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
     7381 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
     7441 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
     7501 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
     7561 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
     7621 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
     7681 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
     7741 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
     7801 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
     7861 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta
     7921 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
     7981 gcgcacattt ccccgaaaag tgccacctga cgtcgacgga tcgggagatc gatctcccga
     8041 tcccctaggg tcgactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatct
     8101 gctccctgct tgtgtgttgg aggtcgctga gtagtgcgcg agcaaaattt aagctacaac
     8161 aaggcaaggc ttgaccgaca attgcatgaa gaatctgctt agggttaggc gttttgcgct
     8221 gcttcgcgat gtacgggcca gatat
//
