LOCUS       sequence-239712-        7193 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 7193)
  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 7193)
  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-BsCas12a-2AeGFP
FEATURES             Location/Qualifiers
     source          1..7193
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     promoter        6..24
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             36..56
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3687..3707
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3714..3803
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     CDS             3825..3878
                     /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(3879..3881,3882..3884,3885..4598)
                     /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(3879..3881,3882..3884,3885..4595)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3924..3945)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4185..4204)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4532..4553
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    4641..4762
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4678..4697)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4732..4751
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4805..4821)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4805..4821)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4818..4840)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4829..4845
                     /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(4853..4859,4860..4877,4878..4883))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4898..4919
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5036..5053)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5207..5795)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5287..5306)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5966..6757,6758..6826))
                     /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     6589..6608
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6827..6931)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7010..7029)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 aattctaata cgactcacta taggccgcca ccatgcccaa gaagaagagg aaggttcccg
       61 ggatgtacta ccagaacctg accaagaagt accccgtgag caagaccatc cgcaacgagc
      121 tgatccccat cggcaagacc ctggagaaca tccgcaagaa caacatcctg gagagcgacg
      181 tgaagcgcaa gcaggactac gagcacgtga agggcatcat ggacgagtac cacaagcagc
      241 tgatcaacga ggccctggac aactacatgc tgcccagcct gaaccaggcc gccgagatct
      301 acctgaagaa gcacgtggac gtggaggacc gcgaggagtt caagaagacc caggacctgc
      361 tgcgccgcga ggtgaccggc cgcctgaagg agcacgagaa ctacaccaag atcggcaaga
      421 aggacatcct ggacctgctg gagaagctgc ccagcatcag cgaggaggac tacaacgccc
      481 tggagagctt ccgcaacttc tacacctact tcaccagcta caacaaggtg cgcgagaacc
      541 tgtacagcga cgaggagaag agcagcaccg tggcctaccg cctgatcaac gagaacctgc
      601 ccaagttcct ggacaacatc aagagctacg ccttcgtgaa ggccgccggc gtgctggccg
      661 actgcatcga ggaggaggag caggacgccc tgttcatggt ggagaccttc aacatgaccc
      721 tgacccagga gggcatcgac atgtacaact accagatcgg caaggtgaac agcgccatca
      781 acctgtacaa ccagaagaac cacaaggtgg aggagttcaa gaagatcccc aagatgaagg
      841 tgctgtacaa gcagatcctg agcgaccgcg aggaggtgtt catcggcgag ttcaaggacg
      901 acgagaccct gctgagcagc atcggcgcct acggcaacgt gctgatgacc tacctgaaga
      961 gcgagaagat caacatcttc ttcgacgccc tgcgcgagag cgagggcaag aacgtgtacg
     1021 tgaagaacga cctgagcaag accaccatga gcaacatcgt gttcggcagc tggagcgcct
     1081 tcgacgagct gctgaaccag gagtacgacc tggccaacga gaacaagaag aaggacgaca
     1141 agtacttcga gaagcgccag aaggagctga agaagaacaa gagctacacc ctggagcaga
     1201 tgagcaacct gagcaaggag gacatcagcc ccatcgagaa ctacatcgag cgcatcagcg
     1261 aggacatcga gaagatctgc atctacaacg gcgagttcga gaagatcgtg gtgaacgagc
     1321 acgacagcag ccgcaagctg agcaagaaca tcaaggccgt gaaggtgatc aaggactacc
     1381 tggacagcat caaggagctg gagcacgaca tcaagctgat caacggcagc ggccaggagc
     1441 tggagaagaa cctggtggtg tacgtgggcc aggaggaggc cctggagcag ctgcgccccg
     1501 tggacagcct gtacaacctg acccgcaact acctgaccaa gaagcccttc agcaccgaga
     1561 aggtgaagct gaacttcaac aagagcaccc tgctgaacgg ctgggacaag aacaaggaga
     1621 ccgacaacct gggcatcctg ttcttcaagg acggcaagta ctacctgggc atcatgaaca
     1681 ccaccgccaa caaggccttc gtgaaccccc ccgccgccaa gaccgagaac gtgttcaaga
     1741 aggtggacta caagctgctg cccggcagca acaagatgct gcccaaggtg ttcttcgcca
     1801 agagcaacat cggctactac aaccccagca ccgagctgta cagcaactac aagaagggca
     1861 cccacaagaa gggccccagc ttcagcatcg acgactgcca caacctgatc gacttcttca
     1921 aggagagcat caagaagcac gaggactgga gcaagttcgg cttcgagttc agcgacaccg
     1981 ccgactaccg cgacatcagc gagttctacc gcgaggtgga gaagcagggc tacaagctga
     2041 ccttcaccga catcgacgag agctacatca acgacctgat cgagaagaac gagctgtacc
     2101 tgttccagat ctacaacaag gacttcagcg agtacagcaa gggcaagctg aacctgcaca
     2161 ccctgtactt catgatgctg ttcgaccagc gcaacctgga caacgtggtg tacaagctga
     2221 acggcgaggc cgaggtgttc taccgccccg ccagcatcgc cgagaacgag ctggtgatcc
     2281 acaaggccgg cgagggcatc aagaacaaga accccaaccg cgccaaggtg aaggagacca
     2341 gcaccttcag ctacgacatc gtgaaggaca agcgctacag caagtacaag ttcaccctgc
     2401 acatccccat caccatgaac ttcggcgtgg acgaggtgcg ccgcttcaac gacgtgatca
     2461 acaacgccct gcgcaccgac gacaacgtga acgtgatcgg catcgaccgc ggcgagcgca
     2521 acctgctgta cgtggtggtg atcaacagcg agggcaagat cctggagcag atcagcctga
     2581 acagcatcat caacaaggag tacgacatcg agaccaacta ccacgccctg ctggacgagc
     2641 gcgaggacga ccgcaacaag gcccgcaagg actggaacac catcgagaac atcaaggagc
     2701 tgaagaccgg ctacctgagc caggtggtga acgtggtggc caagctggtg ctgaagtaca
     2761 acgccatcat ctgcctggag gacctgaact tcggcttcaa gcgcggccgc cagaaggtgg
     2821 agaagcaggt gtaccagaag ttcgagaaga tgctgatcga gaagctgaac tacctggtga
     2881 tcgacaagag ccgcgagcag gtgagccccg agaagatggg cggcgccctg aacgccctgc
     2941 agctgaccag caagttcaag agcttcgccg agctgggcaa gcagagcggc atcatctact
     3001 acgtgcccgc ctacctgacc agcaagatcg accccaccac cggcttcgtg aacctgttct
     3061 acatcaagta cgagaacatc gagaaggcca agcagttctt cgacggcttc gacttcatcc
     3121 gcttcaacaa gaaggacgac atgttcgagt tcagcttcga ctacaagagc ttcacccaga
     3181 aggcctgcgg catccgcagc aagtggatcg tgtacaccaa cggcgagcgc atcatcaagt
     3241 accccaaccc cgagaagaac aacctgttcg acgagaaggt gatcaacgtg accgacgaga
     3301 tcaagggcct gttcaagcag taccgcatcc cctacgagaa cggcgaggac atcaaggaga
     3361 tcatcatcag caaggccgag gccgacttct acaagcgcct gttccgcctg ctgcaccaga
     3421 ccctgcagat gcgcaacagc accagcgacg gcacccgcga ctacatcatc agccccgtga
     3481 agaacgaccg cggcgagttc ttctgcagcg agttcagcga gggcaccatg cccaaggacg
     3541 ccgacgccaa cggcgcctac aacatcgccc gcaagggcct gtgggtgctg gagcagatcc
     3601 gccagaagga cgagggcgag aaggtgaacc tgagcatgac caacgccgag tggctgaagt
     3661 acgcccagct gcacctgctg gctagcccaa agaagaagag gaaagtctct agataccctt
     3721 atgatgttcc agattatgcc ggatacccat acgatgtccc tgactatgca ggctcctacc
     3781 cttatgacgt cccagactac gccggatcca ggtccggcgg cggagagggc agaggaagtc
     3841 ttctaacatg cggtgacgtg gaggagaatc ccggcccaat ggtgagcaag ggcgaggagc
     3901 tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt
     3961 tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc ctgaagttca
     4021 tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ctgacctacg
     4081 gcgtgcagtg cttcagccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg
     4141 ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac ggcaactaca
     4201 agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg
     4261 gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac aactacaaca
     4321 gccacaacgt ctatatcatg gccgacaagc agaagaacgg catcaaggtg aacttcaaga
     4381 tccgccacaa catcgaggac ggcagcgtgc agctcgccga ccactaccag cagaacaccc
     4441 ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagcacc cagtccgccc
     4501 tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc gtgaccgccg
     4561 ccgggatcac tctcggcatg gacgagctgt acaagtaact gcagcgcggg gatctcatgc
     4621 tggagttctt cgcccacccc aacttgttta ttgcagctta taatggttac aaataaagca
     4681 atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt
     4741 ccaaactcat caatgtatct tattaattaa cgtcgacctc tagctagagc ttggcgtaat
     4801 catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac
     4861 gagccggaag cataaagtgt aaagcctagg gtgcctaatg agtgagctaa ctcacattaa
     4921 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat
     4981 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc
     5041 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
     5101 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     5161 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     5221 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     5281 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     5341 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     5401 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     5461 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     5521 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     5581 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     5641 ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     5701 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     5761 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     5821 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     5881 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     5941 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
     6001 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
     6061 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
     6121 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
     6181 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
     6241 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
     6301 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
     6361 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
     6421 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
     6481 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
     6541 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
     6601 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
     6661 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
     6721 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
     6781 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
     6841 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     6901 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg
     6961 tcgacggatc gggagatcga tctcccgatc ccctagggtc gactctcagt acaatctgct
     7021 ctgatgccgc atagttaagc cagtatctgc tccctgcttg tgtgttggag gtcgctgagt
     7081 agtgcgcgag caaaatttaa gctacaacaa ggcaaggctt gaccgacaat tgcatgaaga
     7141 atctgcttag ggttaggcgt tttgcgctgc ttcgcgatgt acgggccaga tat
//
