LOCUS       sequence-254835-        8046 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses ErCas12a and an sgRNA to target a region of interest.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8046)
  AUTHORS   Wierson WA, Simone BW, WareJoncas Z, Mann C, Welker JM, Kar B, Emch 
            MJ, Friedberg I, Gendron WAC, Barry MA, Clark KJ, Dobbs DL, McGrail 
            MA, Ekker SC, Essner JJ
  TITLE     Expanding the CRISPR Toolbox with ErCas12a in Zebrafish and Human 
            Cells.
  JOURNAL   CRISPR J. 2019 Dec;2(6):417-433. doi: 10.1089/crispr.2019.0026. Epub
            2019 Nov 19.
  PUBMED    31742435
REFERENCE   2  (bases 1 to 8046)
  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"; doi: 
            "10.1089/crispr.2019.0026"; journalName: "CRISPR J"; date: 
            "2019-12"; volume: "2"; issue: "6"; pages: "417-433"
COMMENT     Sequence Label: pErCas12a EZ Clone
FEATURES             Location/Qualifiers
     source          1..8046
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     enhancer        154..533
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        534..737
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     688..708
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     5'UTR           756..798
                     /label=Xenopus globin 5'-UTR
                     /note="translational enhancer from the 5'-UTR of the major 
                     beta-globin gene of Xenopus laevis"
     regulatory      814..823
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             832..852
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4639..4659
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4666..4746
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     primer_bind     complement(4774..4791)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4780..4987
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        4994..5242
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     4994..5014
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     5165..5184
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     repeat_region   5309..5449
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5309..5438
                     /label=AAV2 ITR
     rep_origin      5524..5979
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5611..5630)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5821..5842
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5996..6015)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6115..6137
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6175..6193)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6261..6365
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6366..6434,6435..7226)
                     /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     complement(6584..6603)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7397..7985
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7886..7905
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gcggcctcta gactcgaggc gttgacattg attattgact agttattaat
      181 agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac
      241 ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa
      301 tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt
      361 atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc
      421 ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat
      481 gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc atggtgatgc
      541 ggttttggca gtacatcaat gggcgtggat agcggtttga ctcacgggga tttccaagtc
      601 tccaccccat tgacgtcaat gggagtttgt tttggcacca aaatcaacgg gactttccaa
      661 aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg taggcgtgta cggtgggagg
      721 tctatataag cagagctctc tggctaacta ccggtcttgt tctttttgca gaagctcaga
      781 ataaacgctc aactttggca gatctaactc gacgccacca tggcttctcc acctaagaag
      841 aagagaaagg tgaacaacgg aactaataat tttcaaaact tcattgggat tagttctctg
      901 cagaagaccc ttcggaatgc cctcattccc actgagacga ctcaacagtt tatcgtaaaa
      961 aatggaatta ttaaggagga tgagttgcgg ggggagaata ggcagatttt gaaagacatc
     1021 atggatgact attaccgggg ttttatctcc gagacgctgt cctctatcga tgatattgat
     1081 tggacgtctc tttttgagaa gatggagatt cagctgaaaa atggtgataa caaagatacc
     1141 ctcattaagg agcaaaccga gtaccggaag gcgatccaca agaagttcgc caacgatgat
     1201 cgttttaaga atatgttctc agccaaactc atcagtgaca tccttccaga atttgtaatt
     1261 cataataata actactctgc gtctgagaaa gaagaaaaaa ctcaagtcat caagctcttt
     1321 tcacggtttg caacgagctt taaggattac tttaaaaacc gcgctaattg tttttctgcg
     1381 gacgacatca gctcatccag ctgccacaga atcgtcaatg acaatgcgga gatcttcttc
     1441 tccaatgctc tggtatatag gcgcattgta aagtccttgt ccaatgacga tattaataag
     1501 ataagtggtg atatgaagga ttctctcaag gaaatgtcat tggaggagat ctacagctat
     1561 gagaaatacg gtgaatttat tacacaagaa ggaatatcct tttataatga catctgtggg
     1621 aaggtgaatt ctttcatgaa tttgtactgt caaaaaaaca aggagaacaa aaacctctac
     1681 aaattgcaaa aactgcataa gcaaattctt tgtatagcgg acactagcta tgaggtgccg
     1741 tataaatttg agtcagatga ggaggtctac caaagcgtaa acggcttcct cgacaacatt
     1801 tccagtaagc atatcgtgga acggctcagg aagatagggg ataactataa tggctataac
     1861 cttgacaaga tctatatcgt gagtaaattc tatgaaagtg tatctcaaaa gacctatcga
     1921 gattgggaaa ccataaacac agctcttgag attcattaca ataatattct tcctggtaac
     1981 gggaaaagta aagccgataa agtgaagaag gccgtcaaaa acgacctgca gaagagcata
     2041 acggaaatca atgaattggt gtctaactac aagctgtgct cagatgacaa cataaaagct
     2101 gagacatata tccatgagat cagccacata ctgaataact ttgaggcgca agagctgaaa
     2161 tataatcctg agatccacct tgtagagtct gaactcaagg cttccgaact gaaaaatgta
     2221 cttgacgtaa tcatgaatgc ttttcactgg tgtagtgtat tcatgactga ggaactggtt
     2281 gataaggata ataattttta tgcggaactt gaagaaatat acgatgagat ttatcccgtt
     2341 atctcactct ataatttggt ccgaaattat gtaactcaaa aaccatactc cacaaagaaa
     2401 atcaagctca atttcggtat cccgaccttg gctgacggat ggtctaaaag caaggagtac
     2461 tccaataacg cgataatctt gatgcgagat aatctttact atctcggaat ttttaatgct
     2521 aaaaataagc ccgataaaaa gattattgaa ggaaacacat ctgagaacaa aggcgattat
     2581 aaaaagatga tttataattt gctccctgga ccaaacaaaa tgatccctaa agttttcctc
     2641 agttccaaga ccggggttga gacgtacaag cctagtgcat atatcttgga aggttataag
     2701 caaaacaagc acatcaaaag ttctaaggac tttgacatca ctttttgtca tgatttgatt
     2761 gactatttta aaaactgtat tgcaattcac ccagagtgga agaattttgg atttgacttc
     2821 tcagacacgt ctacctatga agatatatca ggattttatc gcgaggttga gctccagggt
     2881 tacaagattg attggactta tatcagcgag aaggatattg atcttttgca ggaaaaaggc
     2941 caactttatt tgttccaaat ctacaacaag gacttttcta agaaatcaac tggcaacgat
     3001 aaccttcata ctatgtacct caaaaatctc ttttccgaag agaatcttaa ggatatcgtg
     3061 ctcaagctga acggtgaggc agagatattt ttccgaaaga gttctatcaa aaacccaatt
     3121 atccacaaaa aaggcagcat cctggttaac aggacgtacg aggccgaaga gaaagatcag
     3181 ttcggcaaca tacagatagt gcggaagaat ataccagaga atatctacca agagctttat
     3241 aagtatttta atgataagtc agacaaagag ctttcagacg aggcggcgaa gttgaaaaat
     3301 gtagtgggac atcacgaagc cgccacaaac atcgtgaagg actatcggta tacctatgat
     3361 aagtacttcc ttcacatgcc aatcacgatc aattttaaag cgaataagac cgggttcata
     3421 aatgaccgga ttctgcagta catagcaaag gagaaagatc ttcatgttat aggcattgat
     3481 cgcggcgaaa gaaaccttat ttatgtctcc gttatagaca catgtgggaa catcgttgaa
     3541 caaaaatcct ttaatatcgt taatggatac gactatcaga taaagctcaa acaacaggag
     3601 ggggcgcgcc agattgctcg taaagaatgg aaggaaatag gaaaaataaa agaaatcaag
     3661 gagggttacc tgagccttgt aattcatgaa atctccaaaa tggttataaa gtacaacgcg
     3721 attattgtca tggaagattt gagctacggt tttaaaaaag ggaggttcaa ggtggagcga
     3781 caggtgtacc aaaagtttga aacgatgctt attaataaac tcaattacct cgtgttcaag
     3841 gatataagca taacagaaaa tggagggctc cttaagggat accagctcac atacataccg
     3901 gacaagctta aaaacgtggg acaccagtgc gggtgtatat tttacgttcc tgccgcgtat
     3961 acatcaaaga tagaccccac cacagggttc gtgaatatct tcaagtttaa ggacttgaca
     4021 gtcgatgcaa aacgtgagtt catcaagaaa ttcgattcaa tccggtacga ttcagaaaag
     4081 aatctgttct gttttacgtt cgattataac aactttatta cgcaaaatac agtgatgtca
     4141 aagagctcat ggagtgtcta cacatacggg gttaggataa agcgcaggtt cgttaacggt
     4201 cggttctcaa acgaatcaga cacgattgac attacgaagg atatggaaaa gactctggag
     4261 atgaccgaca taaattggcg agacggccac gacctccgac aagatatcat tgactacgag
     4321 atcgtccaac acatttttga aatcttccgg ttgaccgtcc agatgcgaaa cagtctttct
     4381 gaattggaag accgggatta cgacagattg atcagtcctg tattgaacga aaacaacata
     4441 ttctatgatt ccgccaaagc tggcgatgct ttgccaaaag acgccgacgc gaatggagca
     4501 tattgtatcg cccttaaagg cctttacgaa atcaaacaaa taacagagaa ctggaaagag
     4561 gatgggaaat ttagccgaga taagctcaag atcagcaaca aagactggtt tgactttatt
     4621 caaaacaaac ggtacttgcc gaaaaagaaa cgcaaagtag gatcctaccc atacgatgtt
     4681 ccagattacg cttacccata cgatgttcca gattacgctt acccatacga tgttccagat
     4741 tacgcttaag aattcctaga gctcgctgat cagcctcgac tgtgccttct agttgccagc
     4801 catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg
     4861 tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc
     4921 tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagagaat agcaggcatg
     4981 ctggggaggt accgagggcc tatttcccat gattccttca tatttgcata tacgatacaa
     5041 ggctgttaga gagataattg gaattaattt gactgtaaac acaaagatat tagtacaaaa
     5101 tacgtgacgt agaaagtaat aatttcttgg gtagtttgca gttttaaaat tatgttttaa
     5161 aatggactat catatgctta ccgtaacttg aaagtatttc gatttcttgg ctttatatat
     5221 cttgtggaaa ggacgaaaca ccgtcaaaag acctttttaa tttctactct tgtagataga
     5281 gaccgggggt ggtctctttt ttggccgcag gaacccctag tgatggagtt ggccactccc
     5341 tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc
     5401 tttgcccggg cggcctcagt gagcgagcga gcgcgcagct gcctgcaggg gcgcctgatg
     5461 cggtattttc tccttacgca tctgtgcggt atttcacacc gcatacgtca aagcaaccat
     5521 agtacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
     5581 ccgctacact tgccagcgcc ttagcgcccg ctcctttcgc tttcttccct tcctttctcg
     5641 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
     5701 ttagtgcttt acggcacctc gaccccaaaa aacttgattt gggtgatggt tcacgtagtg
     5761 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
     5821 gtggactctt gttccaaact ggaacaacac tcaactctat ctcgggctat tcttttgatt
     5881 tataagggat tttgccgatt tcggtctatt ggttaaaaaa tgagctgatt taacaaaaat
     5941 ttaacgcgaa ttttaacaaa atattaacgt ttacaatttt atggtgcact ctcagtacaa
     6001 tctgctctga tgccgcatag ttaagccagc cccgacaccc gccaacaccc gctgacgcgc
     6061 cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga
     6121 gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgagacga aagggcctcg
     6181 tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg
     6241 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
     6301 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
     6361 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc
     6421 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg
     6481 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
     6541 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat
     6601 tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg
     6661 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag
     6721 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa
     6781 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc
     6841 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca
     6901 cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc
     6961 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc
     7021 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg
     7081 gaagccgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta
     7141 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag
     7201 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga
     7261 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc
     7321 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     7381 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     7441 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     7501 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt
     7561 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     7621 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     7681 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     7741 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     7801 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     7861 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     7921 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     7981 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     8041 acatgt
//
