LOCUS       sequence-241195-        7560 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  Mammalian expression, Genome editing.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 7560)
  AUTHORS   Teng F, Li J, Cui T, Xu K, Guo L, Gao Q, Feng G, Chen C, Han D, Zhou
            Q, Li W
  TITLE     Enhanced mammalian genome editing by new Cas12a orthologs with 
            optimized crRNA scaffolds.
  JOURNAL   Genome Biol. 2019 Feb 5;20(1):15. doi: 10.1186/s13059-019-1620-8.
  PUBMED    30717767
REFERENCE   2  (bases 1 to 7560)
  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: "Genome 
            Biol."; date: "2019-02-5"; volume: "20(1):15. doi"; pages: " 
            10.1186/s13059-019-1620-8"
COMMENT     Sequence Label: pCAG-HkCas12a-2AeGFP
FEATURES             Location/Qualifiers
     source          1..7560
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     primer_bind     8..27
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     promoter        61..79
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             91..111
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4054..4074
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4081..4170
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     CDS             4192..4245
                     /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(4246..4248,4249..4251,4252..4965)
                     /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(4246..4248,4249..4251,4252..4962)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(4291..4312)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4552..4571)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4899..4920
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    5008..5129
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5045..5064)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5099..5118
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5172..5188)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5172..5188)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5185..5207)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5196..5212
                     /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(5220..5226,5227..5244,5245..5250))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5265..5286
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5403..5420)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5574..6162)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5654..5673)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6333..7124,7125..7193))
                     /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     6956..6975
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7194..7298)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7377..7396)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 ctcctgggca acgtgctggt tattgtgctg tctcatcatt ttggcaaagc tagtgaattc
       61 taatacgact cactataggc cgccaccatg cccaagaaga agaggaaggt tcccgggatg
      121 ttcgagaagc tgagcaacat cgtgagcatc agcaagacca tccgcttcaa gctgatcccc
      181 gtgggcaaga ccctggagaa catcgagaag ctgggcaagc tggagaagga cttcgagcgc
      241 agcgacttct accccatcct gaagaacatc agcgacgact actaccgcca gtacatcaag
      301 gagaagctga gcgacctgaa cctggactgg cagaagctgt acgacgccca cgagctgctg
      361 gacagcagca agaaggagag ccagaagaac ctggagatga tccaggccca gtaccgcaag
      421 gtgctgttca acatcctgag cggcgagctg gacaagagcg gcgagaagaa cagcaaggac
      481 ctgatcaaga acaacaaggc cctgtacggc aagctgttca agaagcagtt catcctggag
      541 gtgctgcccg acttcgtgaa caacaacgac agctacagcg aggaggacct ggagggcctg
      601 aacctgtaca gcaagttcac cacccgcctg aagaacttct gggagacccg caagaacgtg
      661 ttcaccgaca aggacatcgt gaccgccatc cccttccgcg ccgtgaacga gaacttcggc
      721 ttctactacg acaacatcaa gatcttcaac aagaacatcg agtacctgga gaacaagatc
      781 cccaacctgg agaacgagct gaaggaggcc gacatcctgg acgacaaccg cagcgtgaag
      841 gactacttca cccccaacgg cttcaactac gtgatcaccc aggacggcat cgacgtgtac
      901 caggccatcc gcggcggctt caccaaggag aacggcgaga aggtgcaggg catcaacgag
      961 atcctgaacc tgacccagca gcagctgcgc cgcaagcccg agaccaagaa cgtgaagctg
     1021 ggcgtgctga ccaagctgcg caagcagatc ctggagtaca gcgagagcac cagcttcctg
     1081 atcgaccaga tcgaggacga caacgacctg gtggaccgca tcaacaagtt caacgtgagc
     1141 ttcttcgaga gcaccgaggt gagccccagc ctgttcgagc agatcgagcg cctgtacaac
     1201 gccctgaaga gcatcaagaa ggaggaggtg tacatcgacg cccgcaacac ccagaagttc
     1261 agccagatgc tgttcggcca gtgggacgtg atccgccgcg gctacaccgt gaagatcacc
     1321 gagggcagca aggaggagaa gaagaagtac aaggagtacc tggagctgga cgagaccagc
     1381 aaggccaagc gctacctgaa catccgcgag atcgaggagc tggtgaacct ggtggagggc
     1441 ttcgaggagg tggacgtgtt cagcgtgctg ctggagaagt tcaagatgaa caacatcgag
     1501 cgcagcgagt tcgaggcccc catctacggc agccccatca agctggaggc catcaaggag
     1561 tacctggaga agcacctgga ggagtaccac aagtggaagc tgctgctgat cggcaacgac
     1621 gacctggaca ccgacgagac cttctacccc ctgctgaacg aggtgatcag cgactactac
     1681 atcatccccc tgtacaacct gacccgcaac tacctgaccc gcaagcacag cgacaaggac
     1741 aagatcaagg tgaacttcga cttccccacc ctggccgacg gctggagcga gagcaagatc
     1801 agcgacaacc gcagcatcat cctgcgcaag ggcggctact actacctggg catcctgatc
     1861 gacaacaagc tgctgatcaa caagaagaac aagagcaaga agatctacga gatcctgatc
     1921 tacaaccaga tccccgagtt cagcaagagc atccccaact accccttcac caagaaggtg
     1981 aaggagcact tcaagaacaa cgtgagcgac ttccagctga tcgacggcta cgtgagcccc
     2041 ctgatcatca ccaaggagat ctacgacatc aagaaggaga agaagtacaa gaaggacttc
     2101 tacaaggaca acaacaccaa caagaactac ctgtacacca tctacaagtg gatcgagttc
     2161 tgcaagcagt tcctgtacaa gtacaagggc cccaacaagg agagctacaa ggagatgtac
     2221 gacttcagca ccctgaagga caccagcctg tacgtgaacc tgaacgactt ctacgccgac
     2281 gtgaacagct gcgcctaccg cgtgctgttc aacaagatcg acgagaacac catcgacaac
     2341 gccgtggagg acggcaagct gctgctgttc cagatctaca acaaggactt cagccccgag
     2401 agcaagggca agaagaacct gcacaccctg tactggctga gcatgttcag cgaggagaac
     2461 ctgcgcaccc gcaagctgaa gctgaacggc caggccgaga tcttctaccg caagaagctg
     2521 gagaagaagc ccatcatcca caaggagggc agcatcctgc tgaacaagat cgacaaggag
     2581 ggcaacacca tccccgagaa catctaccac gagtgctacc gctacctgaa caagaagatc
     2641 ggccgcgagg acctgagcga cgaggccatc gccctgttca acaaggacgt gctgaagtac
     2701 aaggaggccc gcttcgacat catcaaggac cgccgctaca gcgagagcca gttcttcttc
     2761 cacgtgccca tcaccttcaa ctgggacatc aagaccaaca agaacgtgaa ccagatcgtg
     2821 cagggcatga tcaaggacgg cgagatcaag cacatcatcg gcatcgaccg cggcgagcgc
     2881 cacctgctgt actacagcgt gatcgacctg gagggcaaca tcgtggagca gggcagcctg
     2941 aacaccctgg agcagaaccg cttcgacaac agcaccgtga aggtggacta ccagaacaag
     3001 ctgcgcaccc gcgaggagga ccgcgaccgc gcccgcaaga actggaccaa catcaacaag
     3061 atcaaggagc tgaaggacgg ctacctgagc cacgtggtgc acaagctgag ccgcctgatc
     3121 atcaagtacg aggccatcgt gatcatggag aacctgaacc agggcttcaa gcgcggccgc
     3181 ttcaaggtgg agcgccaggt gtaccagaag ttcgagctgg ccctgatgaa caagctgagc
     3241 gccctgagct tcaaggagaa gtacgacgag cgcaagaacc tggagcccag cggcatcctg
     3301 aaccccatcc aggcctgcta ccccgtggac gcctaccagg agctgcaggg ccagaacggc
     3361 atcgtgttct acctgcccgc cgcctacacc agcgtgatcg accccgtgac cggcttcacc
     3421 aacctgttcc gcctgaagag catcaacagc agcaagtacg aggagttcat caagaagttc
     3481 aagaacatct acttcgacaa cgaggaggag gacttcaagt tcatcttcaa ctacaaggac
     3541 ttcgccaagg ccaacctggt gatcctgaac aacatcaaga gcaaggactg gaagatcagc
     3601 acccgcggcg agcgcatcag ctacaacagc aagaagaagg agtacttcta cgtgcagccc
     3661 accgagttcc tgatcaacaa gctgaaggag ctgaacatcg actacgagaa catcgacatc
     3721 atccccctga tcgacaacct ggaggagaag gccaagcgca agatcctgaa ggccctgttc
     3781 gacaccttca agtacagcgt gcagctgcgc aactacgact tcgagaacga ctacatcatc
     3841 agccccaccg ccgacgacaa cggcaactac tacaacagca acgagatcga catcgacaag
     3901 accaacctgc ccaacaacgg cgacgccaac ggcgccttca acatcgcccg caagggcctg
     3961 ctgctgaagg accgcatcgt gaacagcaac gagagcaagg tggacctgaa gatcaagaac
     4021 gaggactgga tcaacttcat catcagcgct agcccaaaga agaagaggaa agtctctaga
     4081 tacccttatg atgttccaga ttatgccgga tacccatacg atgtccctga ctatgcaggc
     4141 tcctaccctt atgacgtccc agactacgcc ggatccaggt ccggcggcgg agagggcaga
     4201 ggaagtcttc taacatgcgg tgacgtggag gagaatcccg gcccaatggt gagcaagggc
     4261 gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc
     4321 cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa gctgaccctg
     4381 aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccctg
     4441 acctacggcg tgcagtgctt cagccgctac cccgaccaca tgaagcagca cgacttcttc
     4501 aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa ggacgacggc
     4561 aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag
     4621 ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac
     4681 tacaacagcc acaacgtcta tatcatggcc gacaagcaga agaacggcat caaggtgaac
     4741 ttcaagatcc gccacaacat cgaggacggc agcgtgcagc tcgccgacca ctaccagcag
     4801 aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct gagcacccag
     4861 tccgccctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct ggagttcgtg
     4921 accgccgccg ggatcactct cggcatggac gagctgtaca agtaactgca gcgcggggat
     4981 ctcatgctgg agttcttcgc ccaccccaac ttgtttattg cagcttataa tggttacaaa
     5041 taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt
     5101 ggtttgtcca aactcatcaa tgtatcttat taattaacgt cgacctctag ctagagcttg
     5161 gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac
     5221 aacatacgag ccggaagcat aaagtgtaaa gcctagggtg cctaatgagt gagctaactc
     5281 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     5341 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct
     5401 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
     5461 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     5521 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     5581 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     5641 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     5701 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     5761 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     5821 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     5881 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     5941 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     6001 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     6061 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     6121 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     6181 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     6241 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     6301 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     6361 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     6421 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
     6481 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     6541 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     6601 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
     6661 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     6721 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     6781 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     6841 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     6901 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
     6961 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     7021 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     7081 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     7141 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     7201 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     7261 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
     7321 cctgacgtcg acggatcggg agatcgatct cccgatcccc tagggtcgac tctcagtaca
     7381 atctgctctg atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc
     7441 gctgagtagt gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc
     7501 atgaagaatc tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat
//
