LOCUS       sequence-373777-        8473 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 8473)
  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 8473)
  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-Cas12i2-T2A-EGFP
FEATURES             Location/Qualifiers
     source          1..8473
                     /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             4967..4987
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4994..5083
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     CDS             5105..5158
                     /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(5159..5161,5162..5164,5165..5878)
                     /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(5159..5161,5162..5164,5165..5875)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(5204..5225)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(5465..5484)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5812..5833
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    5921..6042
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5958..5977)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6012..6031
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6085..6101)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6085..6101)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6098..6120)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6109..6125
                     /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(6133..6139,6140..6157,6158..6163))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6178..6199
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6316..6333)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6487..7075)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6567..6586)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7246..8037,8038..8106))
                     /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     7869..7888
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8107..8211)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(8290..8309)
                     /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 gagcagcgcc atcaagagct acaagagcgt gctgcgcccc aacgagcgca agaaccagct
     1861 gctgaagagc accatccagt gcctggagga cggcagcgcc ttcttcttca agatgctgca
     1921 gggcctgttc ggcggcatca cccccgagat cgtgcgcttc agcaccgagc aggagaagca
     1981 gcagcaggac atcgccctgt ggtgcgccgt gaactggttc cgccccgtga gccaggacag
     2041 cctgacccac accatcgcca gcgacaacct ggtggagaag ttcgaggagt actacggcgg
     2101 caccgccagc gacgccatca agcagtactt cagcgccagc atcggcgaga gctactactg
     2161 gaacgactgc cgccagcagt actacgacct gtgccgcgag ctgggcgtgg aggtgagcga
     2221 cctgacccac gacctggaga tcctgtgccg cgagaagtgc ctggccgtgg ccaccgagag
     2281 caaccagaac aacagcatca tcagcgtgct gttcggcacc ggcgagaagg aggaccgcag
     2341 cgtgaagctg cgcatcacca agaagatcct ggaggccatc agcaacctga aggagatccc
     2401 caagaacgtg gcccccatcc aggagatcat cctgaacgtg gccaaggcca ccaaggagac
     2461 cttccgccag gtgtacgccg gcaacctggg cgcccccagc accctggaga agttcatcgc
     2521 caaggacggc cagaaggagt tcgacctgaa gaagctgcag accgacctga agaaggtgat
     2581 ccgcggcaag agcaaggagc gcgactggtg ctgccaggag gagctgcgca gctacgtgga
     2641 gcagaacacc atccagtacg acctgtgggc ctggggcgag atgttcaaca aggcccacac
     2701 cgccctgaag atcaagagca cccgcaacta caacttcgcc aagcagcgcc tggagcagtt
     2761 caaggagatc cagagcctga acaacctgct ggtggtgaag aagctgaacg acttcttcga
     2821 cagcgagttc ttcagcggcg aggagaccta caccatctgc gtgcaccacc tgggcggcaa
     2881 ggacctgagc aagctgtaca aggcctggga ggacgacccc gccgaccccg agaacgccat
     2941 cgtggtgctg tgcgacgacc tgaagaacaa cttcaagaag gagcccatcc gcaacatcct
     3001 gcgctacatc ttcaccatcc gccaggagtg cagcgcccag gacatcctgg ccgccgccaa
     3061 gtacaaccag cagctggacc gctacaagag ccagaaggcc aaccccagcg tgctgggcaa
     3121 ccagggcttc acctggacca acgccgtgat cctgcccgag aaggcccagc gcaacgaccg
     3181 ccccaacagc ctggacctgc gcatctggct gtacctgaag ctgcgccacc ccgacggccg
     3241 ctggaagaag caccacatcc ccttctacga cacccgcttc ttccaggaga tctacgccgc
     3301 cggcaacagc cccgtggaca cctgccagtt ccgcaccccc cgcttcggct accacctgcc
     3361 caagctgacc gaccagaccg ccatccgcgt gaacaagaag cacgtgaagg ccgccaagac
     3421 cgaggcccgc atccgcctgg ccatccagca gggcaccctg cccgtgagca acctgaagat
     3481 caccgagatc agcgccacca tcaacagcaa gggccaggtg cgcatccccg tgaagttcga
     3541 cgtgggccgc cagaagggca ccctgcagat cggcgaccgc ttctgcggct acgaccagaa
     3601 ccagaccgcc agccacgcct acagcctgtg ggaggtggtg aaggagggcc agtaccacaa
     3661 ggagctgggc tgcttcgtgc gcttcatcag cagcggcgac atcgtgagca tcaccgagaa
     3721 ccgcggcaac cagttcgacc agctgagcta cgagggcctg gcctaccccc agtacgccga
     3781 ctggcgcaag aaggccagca agttcgtgag cctgtggcag atcaccaaga agaacaagaa
     3841 gaaggagatc gtgaccgtgg aggccaagga gaagttcgac gccatctgca agtaccagcc
     3901 ccgcctgtac aagttcaaca aggagtacgc ctacctgctg cgcgacatcg tgcgcggcaa
     3961 gagcctggtg gagctgcagc agatccgcca ggagatcttc cgcttcatcg agcaggactg
     4021 cggcgtgacc cgcctgggca gcctgagcct gagcaccctg gagaccgtga aggccgtgaa
     4081 gggcatcatc tacagctact tcagcaccgc cctgaacgcc agcaagaaca accccatcag
     4141 cgacgagcag cgcaaggagt tcgaccccga gctgttcgcc ctgctggaga agctggagct
     4201 gatccgcacc cgcaagaaga agcagaaggt ggagcgcatc gccaacagcc tgatccagac
     4261 ctgcctggag aacaacatca agttcatccg cggcgagggc gacctgagca ccaccaacaa
     4321 cgccaccaag aagaaggcca acagccgcag catggactgg ctggcccgcg gcgtgttcaa
     4381 caagatccgc cagctggccc ccatgcacaa catcaccctg ttcggctgcg gcagcctgta
     4441 caccagccac caggaccccc tggtgcaccg caaccccgac aaggccatga agtgccgctg
     4501 ggccgccatc cccgtgaagg acatcggcga ctgggtgctg cgcaagctga gccagaacct
     4561 gcgcgccaag aacatcggca ccggcgagta ctaccaccag ggcgtgaagg agttcctgag
     4621 ccactacgag ctgcaggacc tggaggagga gctgctgaag tggcgcagcg accgcaagag
     4681 caacatcccc tgctgggtgc tgcagaaccg cctggccgag aagctgggca acaaggaggc
     4741 cgtggtgtac atccccgtgc gcggcggccg catctacttc gccacccaca aggtggccac
     4801 cggcgccgtg agcatcgtgt tcgaccagaa gcaggtgtgg gtgtgcaacg ccgaccacgt
     4861 ggccgccgcc aacatcgccc tgaccgtgaa gggcatcggc gagcagagca gcgacgagga
     4921 gaaccccgac ggcagccgca tcaagctgca gctgaccagc gctagcccaa agaagaagag
     4981 gaaagtctct agataccctt atgatgttcc agattatgcc ggatacccat acgatgtccc
     5041 tgactatgca ggctcctacc cttatgacgt cccagactac gccggatcca ggtccggcgg
     5101 cggagagggc agaggaagtc ttctaacatg cggtgacgtg gaggagaatc ccggcccaat
     5161 ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg
     5221 cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg
     5281 caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct
     5341 cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc taccccgacc acatgaagca
     5401 gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt
     5461 caaggacgac ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt
     5521 gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa
     5581 gctggagtac aactacaaca gccacaacgt ctatatcatg gccgacaagc agaagaacgg
     5641 catcaaggtg aacttcaaga tccgccacaa catcgaggac ggcagcgtgc agctcgccga
     5701 ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta
     5761 cctgagcacc cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct
     5821 gctggagttc gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaagtaact
     5881 gcagcgcggg gatctcatgc tggagttctt cgcccacccc aacttgttta ttgcagctta
     5941 taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact
     6001 gcattctagt tgtggtttgt ccaaactcat caatgtatct tattaattaa cgtcgacctc
     6061 tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct
     6121 cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctagg gtgcctaatg
     6181 agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
     6241 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
     6301 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
     6361 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
     6421 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
     6481 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
     6541 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     6601 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     6661 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     6721 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     6781 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     6841 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     6901 gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc
     6961 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     7021 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
     7081 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
     7141 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag
     7201 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
     7261 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
     7321 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
     7381 accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
     7441 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
     7501 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
     7561 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
     7621 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
     7681 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
     7741 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
     7801 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
     7861 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
     7921 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
     7981 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
     8041 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
     8101 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag
     8161 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc
     8221 ccgaaaagtg ccacctgacg tcgacggatc gggagatcga tctcccgatc ccctagggtc
     8281 gactctcagt acaatctgct ctgatgccgc atagttaagc cagtatctgc tccctgcttg
     8341 tgtgttggag gtcgctgagt agtgcgcgag caaaatttaa gctacaacaa ggcaaggctt
     8401 gaccgacaat tgcatgaaga atctgcttag ggttaggcgt tttgcgctgc ttcgcgatgt
     8461 acgggccaga tat
//
