LOCUS       sequence-241193-        7262 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 7262)
  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 7262)
  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-PxCas12a-2AeGFP
FEATURES             Location/Qualifiers
     source          1..7262
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     promoter        48..66
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             78..98
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3756..3776
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3783..3872
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     CDS             3894..3947
                     /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(3948..3950,3951..3953,3954..4667)
                     /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(3948..3950,3951..3953,3954..4664)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3993..4014)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4254..4273)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4601..4622
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    4710..4831
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4747..4766)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4801..4820
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4874..4890)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4874..4890)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4887..4909)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4898..4914
                     /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(4922..4928,4929..4946,4947..4952))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4967..4988
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5105..5122)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5276..5864)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5356..5375)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6035..6826,6827..6895))
                     /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     6658..6677
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6896..7000)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7079..7098)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 tgctggttat tgtgctgtct catcattttg gcaaagctag tgaattctaa tacgactcac
       61 tataggccgc caccatgccc aagaagaaga ggaaggttcc cgggatgatc atcggccgcg
      121 acttcaacat gtactaccag aacctgacca agatgtaccc catcagcaag accctgcgca
      181 acgagctgat ccccgtgggc aagaccctgg agaacatccg caagaacggc atcctggagg
      241 ccgacatcca gcgcaaggcc gactacgagc acgtgaagaa gctgatggac aactaccaca
      301 agcagctgat caacgaggcc ctgcagggcg tgcacctgag cgacctgagc gacgcctacg
      361 acctgtactt caacctgagc aaggagaaga acagcgtgga cgccttcagc aagtgccagg
      421 acaagctgcg caaggagatc gtgagcttcc tgaagaacca cgagaacttc cccaagatcg
      481 gcaacaagga gatcatcaag ctgatccaga gcctgaacga caacgacgcc gacaacaacg
      541 ccctggacag cttcagcaac ttctacacct acttcagcag ctacaacgag gtgcgcaaga
      601 acctgtacag cgacgaggag aagagcagca ccgtggccta ccgcctgatc aacgagaacc
      661 tgcccaagag cctggacaac atcaaggcct acgccatcgc caagaaggcc ggcgtgcgcg
      721 ccgagggcct gagcgaggag gagcaggact gcctgttcat catcgagacc ttcgagcgca
      781 ccctgaccca ggacggcatc gacaactaca acgccgacat cggcaagctg aacaccgcca
      841 tcaacctgta caaccagcag aacaagaagc aggagggctt ccgcaaggtg ccccagatga
      901 agtgcctgta caagcagatc ctgagcgacc gcgaggaggc cttcatcgac gagttcagcg
      961 acgacgagga cctgatcacc aacatcgaga gcttcgccga gaacatgaac gtgttcctga
     1021 acagcgagat catcaccgac ttcaagaacg ccctggtgga gagcgacggc agcctggtgt
     1081 acatcaagaa cgacgtgagc aagaccctgt tcagcaacat cgtgttcggc agctggaacg
     1141 ccatcgacga gaagctgagc gacgagtacg acctggccaa cagcaagaag aagaaggacg
     1201 agaagtacta cgagaagcgc cagaaggagc tgaagaagaa caagagctac gacctggaga
     1261 ccatcatcgg cctgttcgac gacagcatcg acgtgatcgg caagtacatc gagaagctgg
     1321 agagcgacat caccgccatc gccgaggcca agaacgactt cgacgagatc gtgctgcgca
     1381 agcacgacaa gaacaagagc ctgcgcaaga acaccaacgc cgtggaggcc atcaagagct
     1441 acctggacac cgtgaaggac ttcgagcgcg acatcaagct gatcaacggc agcggccagg
     1501 aggtggagaa gaacctggtg gtgtacgccg agcaggagaa catcctggcc gagatcaaga
     1561 acgtggacag cctgtacaac atgagccgca actacctgac ccagaagccc ttcagcaccg
     1621 agaagttcaa gctgaacttc gagaacccca ccctgctgaa cggctgggac cgcaacaagg
     1681 agaaggacta cctgggcatc ctgttcgaga aggagggcat gtactacctg ggcatcatca
     1741 acaacaacca ccgcaagatc ttcgagaacg agaagctgtg caccggcaag gagagctgct
     1801 tcaacaagat cgtgtacaag cagatcagca acgccgccaa gtacctgagc agcaagcaga
     1861 tcaaccccca gaaccccccc aaggagatcg ccgagatcct gctgaagcgc aaggccgaca
     1921 gcagcagcct gagccgcaag gagaccgagc tgttcatcga ctacctgaag gacgacttcc
     1981 tggtgaacta ccccatgatc atcaacagcg acggcgagaa cttcttcaac ttccacttca
     2041 agcaggccaa ggactacggc agcctgcagg agttcttcaa ggaggtggag caccaggcct
     2101 acagcctgaa gacccgcccc atcgacgaca gctacatcta ccgcatgatc gacgagggca
     2161 agctgtacct gttccagatc cacaacaagg acttcagccc ctacagcaag ggcaacctga
     2221 acctgcacac catctacctg cagatgctgt tcgaccagcg caacctgaac aacgtggtgt
     2281 acaagctgaa cggcgaggcc gaggtgttct accgccccgc cagcatcaac gacgaggagg
     2341 tgatcatcca caaggccggc gaggagatca agaacaagaa cagcaagcgc gccgtggaca
     2401 agcccaccag caagttcggc tacgacatca tcaaggaccg ccgctacagc aaggacaagt
     2461 tcatgctgca catccccgtg accatgaact tcggcgtgga cgagacccgc cgcttcaacg
     2521 acgtggtgaa cgacgccctg cgcaacgacg agaaggtgcg cgtgatcggc atcgaccgcg
     2581 gcgagcgcaa cctgctgtac gtggtggtgg tggacaccga cggcaccatc ctggagcaga
     2641 tcagcctgaa cagcatcatc aacaacgagt acagcatcga gaccgactac cacaagctgc
     2701 tggacgagaa ggagggcgac cgcgaccgcg cccgcaagaa ctggaccacc atcgagaaca
     2761 tcaaggagct gaaggagggc tacctgagcc aggtggtgaa cgtgatcgcc aagctggtgc
     2821 tgaagtacaa cgccatcatc tgcctggagg acctgaactt cggcttcaag cgcggccgcc
     2881 agaaggtgga gaagcaggtg taccagaagt tcgagaagat gctgatcgac aagctgaact
     2941 acctggtgat cgacaagagc cgcaagcagg agaagcccga ggagttcggc ggcgccctga
     3001 acgccctgca gctgaccagc aagttcacca gcttcaagga catgggcaag cagaccggca
     3061 tcatctacta cgtgcccgcc tacctgacca gcaagatcga ccccaccacc ggcttcgcca
     3121 acctgttcta cgtgaagtac gagaacgtgg agaaggccaa ggagttcttc agccgcttcg
     3181 acagcatcag ctacaacaac gagagcggct acttcgagtt cgccttcgac tacaagaagt
     3241 tcaccgaccg cgcctgcggc gcccgcagcc agtggaccgt gtgcacctac ggcgagcgca
     3301 tcatcaagta ccgcaacgcc gacaagaaca acagcttcga cgacaagacc atcgtgctga
     3361 gcgaggagtt caaggagctg ttcagcatct acggcatcag ctacgaggac ggcgccgagc
     3421 tgaagaacaa gatcatgagc gtggacgagg ccgacttctt ccgctgcctg accggcctgc
     3481 tgcagaagac cctgcagatg cgcaacagca gcaacgacgg cacccgcgac tacatcatca
     3541 gccccatcat gaacgaccgc ggcgagttct tcaacagcga ggcctgcgac gccagcaagc
     3601 ccaaggacgc cgacgccaac ggcgccttca acatcgcccg caagggcctg tgggtgctgg
     3661 agcagatccg caacaccccc agcggcgaca agctgaacct ggccatgagc aacgccgagt
     3721 ggctggagta cgcccagcgc aaccagatcg ctagcccaaa gaagaagagg aaagtctcta
     3781 gataccctta tgatgttcca gattatgccg gatacccata cgatgtccct gactatgcag
     3841 gctcctaccc ttatgacgtc ccagactacg ccggatccag gtccggcggc ggagagggca
     3901 gaggaagtct tctaacatgc ggtgacgtgg aggagaatcc cggcccaatg gtgagcaagg
     3961 gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg
     4021 gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc aagctgaccc
     4081 tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc
     4141 tgacctacgg cgtgcagtgc ttcagccgct accccgacca catgaagcag cacgacttct
     4201 tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc aaggacgacg
     4261 gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg
     4321 agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca
     4381 actacaacag ccacaacgtc tatatcatgg ccgacaagca gaagaacggc atcaaggtga
     4441 acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac cactaccagc
     4501 agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac ctgagcaccc
     4561 agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg
     4621 tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagtaactg cagcgcgggg
     4681 atctcatgct ggagttcttc gcccacccca acttgtttat tgcagcttat aatggttaca
     4741 aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt
     4801 gtggtttgtc caaactcatc aatgtatctt attaattaac gtcgacctct agctagagct
     4861 tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac
     4921 acaacatacg agccggaagc ataaagtgta aagcctaggg tgcctaatga gtgagctaac
     4981 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     5041 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg
     5101 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     5161 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     5221 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     5281 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     5341 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     5401 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     5461 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     5521 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     5581 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     5641 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     5701 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     5761 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     5821 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     5881 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     5941 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     6001 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     6061 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     6121 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     6181 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     6241 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     6301 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     6361 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     6421 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     6481 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     6541 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     6601 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     6661 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     6721 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     6781 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     6841 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     6901 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     6961 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     7021 cacctgacgt cgacggatcg ggagatcgat ctcccgatcc cctagggtcg actctcagta
     7081 caatctgctc tgatgccgca tagttaagcc agtatctgct ccctgcttgt gtgttggagg
     7141 tcgctgagta gtgcgcgagc aaaatttaag ctacaacaag gcaaggcttg accgacaatt
     7201 gcatgaagaa tctgcttagg gttaggcgtt ttgcgctgct tcgcgatgta cgggccagat
     7261 at
//
