LOCUS       sequence-373792-        8737 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 8737)
  AUTHORS   Teng F, Cui T, Gao Q, Guo L, Zhou Q, Li W
  TITLE     Artificial sgRNAs engineered for genome editing with new Cas12b 
            orthologs.
  JOURNAL   Cell Discov. 2019 Apr 23;5:23. doi: 10.1038/s41421-019-0091-0. 
            eCollection 2019.
  PUBMED    31016029
REFERENCE   2  (bases 1 to 8737)
  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: "2019-04-23"; volume: "5:23. doi"; pages: " 
            10.1038/s41421-019-0091-0. eCollection 2019"
COMMENT     Sequence Label: pCAG-TcCas12b-T2A-EGFP
FEATURES             Location/Qualifiers
     source          1..8737
                     /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             5231..5251
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5258..5347
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     CDS             5369..5422
                     /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(5423..5425,5426..5428,5429..6142)
                     /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(5423..5425,5426..5428,5429..6139)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(5468..5489)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(5729..5748)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     6076..6097
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    6185..6306
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6222..6241)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6276..6295
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6349..6365)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6349..6365)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6362..6384)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6373..6389
                     /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(6397..6403,6404..6421,6422..6427))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6442..6463
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6580..6597)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6751..7339)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6831..6850)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7510..8301,8302..8370))
                     /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     8133..8152
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8371..8475)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(8554..8573)
                     /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 gaacatccac ctgaaggagc tgatccgcat ggccaccaag agcttcatcc tgaagatgaa
     1861 gaccaagaac aacccccagc tgcgcctgag cctgtggaag acccacgagc tgttcaactt
     1921 cggcgtggcc tactacatgg acctgctgag cctgttccgc cagaaggacc tgtacatgca
     1981 caacgacgag gaccccgacc accccgtggt gctgaagaag gaggagatcc aggagcgcct
     2041 gtggatgaag gtgcgcgaga cccagcagaa gaacggcttc cacggcgagg tgagcaagga
     2101 cgaggtgctg gagaccctgc gcgccctgta cgaggagctg gtgcccagcg ccgtgggcaa
     2161 gagcggcgag gccaaccaga tcagcaacaa gtacctgtac cccctgaccg accccgccag
     2221 ccagagcggc aagggcaccg ccaacagcgg ccgcaagccc cgctggaaga agctgaagga
     2281 ggccggcgac cccagctgga aggacgccta cgagaagtgg gagaaggagc gccaggagga
     2341 ccccaagctg aagatcctgg ccgccctgca gagcttcggc ctgatccccc tgttccgccc
     2401 cttcaccgag aacgaccaca aggccgtgat cagcgtgaag tggatgccca agagcaagaa
     2461 ccagagcgtg cgcaagttcg acaaggacat gttcaaccag gccatcgagc gcttcctgag
     2521 ctgggagagc tggaacgaga aggtggccga ggactacgag aagaccgtga gcatctacga
     2581 gagcctgcag aaggagctga agggcatcag caccaaggcc ttcgagatca tggagcgcgt
     2641 ggagaaggcc tacgaggccc acctgcgcga gatcaccttc agcaacagca cctaccgcat
     2701 cggcaaccgc gccatccgcg gctggaccga gatcgtgaag aagtggatga agctggaccc
     2761 cagcgccccc cagggcaact acctggacgt ggtgaaggac taccagcgcc gccacccccg
     2821 cgagagcggc gacttcaagc tgttcgagct gctgagccgc cccgagaacc aggccgcctg
     2881 gcgcgagtac cccgagttcc tgcccctgta cgtgaagtac cgccacgccg agcagcgcat
     2941 gaagaccgcc aagaagcagg ccaccttcac cctgtgcgac cccatccgcc accccctgtg
     3001 ggtgcgctac gaggagcgca gcggcaccaa cctgaacaag taccgcctga tcatgaacga
     3061 gaaggagaag gtggtgcagt tcgaccgcct gatctgcctg aacgccgacg gccactacga
     3121 ggagcaggag gacgtgaccg tgcccctggc ccccagccag cagttcgacg accagatcaa
     3181 gttcagcagc gaggacaccg gcaagggcaa gcacaacttc agctactacc acaagggcat
     3241 caactacgag ctgaagggca ccctgggcgg cgcccgcatc cagttcgacc gcgagcacct
     3301 gctgcgccgc cagggcgtga aggccggcaa cgtgggccgc atcttcctga acgtgaccct
     3361 gaacatcgag cccatgcagc ccttcagccg cagcggcaac ctgcagacca gcgtgggcaa
     3421 ggccctgaag gtgtacgtgg acggctaccc caaggtggtg aacttcaagc ccaaggagct
     3481 gaccgagcac atcaaggaga gcgagaagaa caccctgacc ctgggcgtgg agagcctgcc
     3541 caccggcctg cgcgtgatga gcgtggacct gggccagcgc caggccgccg ccatcagcat
     3601 cttcgaggtg gtgagcgaga agcccgacga caacaagctg ttctaccccg tgaaggacac
     3661 cgacctgttc gccgtgcacc gcaccagctt caacatcaag ctgcccggcg agaagcgcac
     3721 cgagcgccgc atgctggagc agcagaagcg cgaccaggcc atccgcgacc tgagccgcaa
     3781 gctgaagttc ctgaagaacg tgctgaacat gcagaagctg gagaagaccg acgagcgcga
     3841 gaagcgcgtg aaccgctgga tcaaggaccg cgagcgcgag gaggagaacc ccgtgtacgt
     3901 gcaggagttc gagatgatca gcaaggtgct gtacagcccc cacagcgtgt gggtggacca
     3961 gctgaagagc atccaccgca agctggagga gcagctgggc aaggagatca gcaagtggcg
     4021 ccagagcatc agccagggcc gccagggcgt gtacggcatc agcctgaaga acatcgagga
     4081 catcgagaag acccgccgcc tgctgttccg ctggagcatg cgccccgaga accccggcga
     4141 ggtgaagcag ctgcagcccg gcgagcgctt cgccatcgac cagcagaacc acctgaacca
     4201 cctgaaggac gaccgcatca agaagctggc caaccagatc gtgatgaccg ccctgggcta
     4261 ccgctacgac ggcaagcgca agaagtggat cgccaagcac cccgcctgcc agctggtgct
     4321 gttcgaggac ctgagccgct acgccttcta cgacgagcgc agccgcctgg agaaccgcaa
     4381 cctgatgcgc tggagccgcc gcgagatccc caagcaggtg gcccagatcg gcggcctgta
     4441 cggcctgctg gtgggcgagg tgggcgccca gtacagcagc cgcttccacg ccaagagcgg
     4501 cgcccccggc atccgctgcc gcgtggtgaa ggagcacgag ctgtacatca ccgagggcgg
     4561 ccagaaggtg cgcaaccaga agttcctgga cagcctggtg gagaacaaca tcatcgagcc
     4621 cgacgacgcc cgccgcctgg agcccggcga cctgatccgc gaccagggcg gcgacaagtt
     4681 cgccaccctg gacgagcgcg gcgagctggt gatcacccac gccgacatca acgccgccca
     4741 gaacctgcag aagcgcttct ggacccgcac ccacggcctg taccgcatcc gctgcgagag
     4801 ccgcgagatc aaggacgccg tggtgctggt gcccagcgac aaggaccaga aggagaagat
     4861 ggagaacctg ttcggcatcg gctacctgca gcccttcaag caggagaacg acgtgtacaa
     4921 gtgggtgaag ggcgagaaga tcaagggcaa gaagaccagc agccagagcg acgacaagga
     4981 gctggtgagc gagatcctgc aggaggcgag cgtgatggcc gacgagctga agggcaaccg
     5041 caagaccctg ttccgcgacc ccagcggcta cgtgttcccc aaggaccgct ggtacaccgg
     5101 cggccgctac ttcggcaccc tggagcacct gctgaagcgc aagctggccg agcgccgcct
     5161 gttcgacggc ggcagcagcc gccgcggcct gttcaacggc accgacagca acaccaacgt
     5221 ggaggctagc ccaaagaaga agaggaaagt ctctagatac ccttatgatg ttccagatta
     5281 tgccggatac ccatacgatg tccctgacta tgcaggctcc tacccttatg acgtcccaga
     5341 ctacgccgga tccaggtccg gcggcggaga gggcagagga agtcttctaa catgcggtga
     5401 cgtggaggag aatcccggcc caatggtgag caagggcgag gagctgttca ccggggtggt
     5461 gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga
     5521 gggcgagggc gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa
     5581 gctgcccgtg ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag
     5641 ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta
     5701 cgtccaggag cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt
     5761 gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga
     5821 ggacggcaac atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat
     5881 catggccgac aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga
     5941 ggacggcagc gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc
     6001 cgtgctgctg cccgacaacc actacctgag cacccagtcc gccctgagca aagaccccaa
     6061 cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg
     6121 catggacgag ctgtacaagt aactgcagcg cggggatctc atgctggagt tcttcgccca
     6181 ccccaacttg tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt
     6241 cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt
     6301 atcttattaa ttaacgtcga cctctagcta gagcttggcg taatcatggt catagctgtt
     6361 tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa
     6421 gtgtaaagcc tagggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact
     6481 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc
     6541 ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg
     6601 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
     6661 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
     6721 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     6781 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     6841 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     6901 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     6961 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     7021 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     7081 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     7141 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt
     7201 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     7261 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     7321 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     7381 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     7441 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     7501 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     7561 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     7621 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
     7681 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     7741 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     7801 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
     7861 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     7921 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     7981 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     8041 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     8101 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     8161 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     8221 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     8281 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     8341 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
     8401 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     8461 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtcgacg gatcgggaga
     8521 tcgatctccc gatcccctag ggtcgactct cagtacaatc tgctctgatg ccgcatagtt
     8581 aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg cgagcaaaat
     8641 ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc ttagggttag
     8701 gcgttttgcg ctgcttcgcg atgtacgggc cagatat
//
