LOCUS       Exported                8042 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Expresses ErCas12a and an sgRNA to target a region of interest.
ACCESSION   .
VERSION     .
KEYWORDS    pErCas12a EZ Clone
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8042)
  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 8042)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8042
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     repeat_region   38..167
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     enhancer        191..570
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        571..774
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     725..745
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      847..856
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             865..885
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4672..4692
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4699..4779
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     primer_bind     complement(4807..4824)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4813..5020
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        5027..5267
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     primer_bind     5027..5047
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     5198..5217
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     repeat_region   5342..5482
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5342..5471
                     /label=AAV2 ITR
     rep_origin      5557..6012
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5644..5663)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5854..5875
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6029..6048)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6148..6170
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6208..6226)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6294..6398
                     /gene="bla"
                     /label=AmpR promoter
     CDS             6399..7259
                     /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(6617..6636)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7430..8018
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7919..7938
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cggttcctgg ccttttgctg gccttttgct cacatgtcct gcaggcagct gcgcgctcgc
       61 tcgctcactg aggccgcccg ggcgtcgggc gacctttggt cgcccggcct cagtgagcga
      121 gcgagcgcgc agagagggag tggccaactc catcactagg ggttcctgcg gcctctagac
      181 tcgaggcgtt gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta
      241 gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc
      301 tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg
      361 ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg
      421 gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa
      481 tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac
      541 atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg
      601 cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg
      661 agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca
      721 ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg
      781 ctaactaccg gtttcttttt gcagaagctc agaataaacg ctcaactttg gcagatctaa
      841 ctcgacgcca ccatggcttc tccacctaag aagaagagaa aggtgaacaa cggaactaat
      901 aattttcaaa acttcattgg gattagttct ctgcagaaga cccttcggaa tgccctcatt
      961 cccactgaga cgactcaaca gtttatcgta aaaaatggaa ttattaagga ggatgagttg
     1021 cggggggaga ataggcagat tttgaaagac atcatggatg actattaccg gggttttatc
     1081 tccgagacgc tgtcctctat cgatgatatt gattggacgt ctctttttga gaagatggag
     1141 attcagctga aaaatggtga taacaaagat accctcatta aggagcaaac cgagtaccgg
     1201 aaggcgatcc acaagaagtt cgccaacgat gatcgtttta agaatatgtt ctcagccaaa
     1261 ctcatcagtg acatccttcc agaatttgta attcataata ataactactc tgcgtctgag
     1321 aaagaagaaa aaactcaagt catcaagctc ttttcacggt ttgcaacgag ctttaaggat
     1381 tactttaaaa accgcgctaa ttgtttttct gcggacgaca tcagctcatc cagctgccac
     1441 agaatcgtca atgacaatgc ggagatcttc ttctccaatg ctctggtata taggcgcatt
     1501 gtaaagtcct tgtccaatga cgatattaat aagataagtg gtgatatgaa ggattctctc
     1561 aaggaaatgt cattggagga gatctacagc tatgagaaat acggtgaatt tattacacaa
     1621 gaaggaatat ccttttataa tgacatctgt gggaaggtga attctttcat gaatttgtac
     1681 tgtcaaaaaa acaaggagaa caaaaacctc tacaaattgc aaaaactgca taagcaaatt
     1741 ctttgtatag cggacactag ctatgaggtg ccgtataaat ttgagtcaga tgaggaggtc
     1801 taccaaagcg taaacggctt cctcgacaac atttccagta agcatatcgt ggaacggctc
     1861 aggaagatag gggataacta taatggctat aaccttgaca agatctatat cgtgagtaaa
     1921 ttctatgaaa gtgtatctca aaagacctat cgagattggg aaaccataaa cacagctctt
     1981 gagattcatt acaataatat tcttcctggt aacgggaaaa gtaaagccga taaagtgaag
     2041 aaggccgtca aaaacgacct gcagaagagc ataacggaaa tcaatgaatt ggtgtctaac
     2101 tacaagctgt gctcagatga caacataaaa gctgagacat atatccatga gatcagccac
     2161 atactgaata actttgaggc gcaagagctg aaatataatc ctgagatcca ccttgtagag
     2221 tctgaactca aggcttccga actgaaaaat gtacttgacg taatcatgaa tgcttttcac
     2281 tggtgtagtg tattcatgac tgaggaactg gttgataagg ataataattt ttatgcggaa
     2341 cttgaagaaa tatacgatga gatttatccc gttatctcac tctataattt ggtccgaaat
     2401 tatgtaactc aaaaaccata ctccacaaag aaaatcaagc tcaatttcgg tatcccgacc
     2461 ttggctgacg gatggtctaa aagcaaggag tactccaata acgcgataat cttgatgcga
     2521 gataatcttt actatctcgg aatttttaat gctaaaaata agcccgataa aaagattatt
     2581 gaaggaaaca catctgagaa caaaggcgat tataaaaaga tgatttataa tttgctccct
     2641 ggaccaaaca aaatgatccc taaagttttc ctcagttcca agaccggggt tgagacgtac
     2701 aagcctagtg catatatctt ggaaggttat aagcaaaaca agcacatcaa aagttctaag
     2761 gactttgaca tcactttttg tcatgatttg attgactatt ttaaaaactg tattgcaatt
     2821 cacccagagt ggaagaattt tggatttgac ttctcagaca cgtctaccta tgaagatata
     2881 tcaggatttt atcgcgaggt tgagctccag ggttacaaga ttgattggac ttatatcagc
     2941 gagaaggata ttgatctttt gcaggaaaaa ggccaacttt atttgttcca aatctacaac
     3001 aaggactttt ctaagaaatc aactggcaac gataaccttc atactatgta cctcaaaaat
     3061 ctcttttccg aagagaatct taaggatatc gtgctcaagc tgaacggtga ggcagagata
     3121 tttttccgaa agagttctat caaaaaccca attatccaca aaaaaggcag catcctggtt
     3181 aacaggacgt acgaggccga agagaaagat cagttcggca acatacagat agtgcggaag
     3241 aatataccag agaatatcta ccaagagctt tataagtatt ttaatgataa gtcagacaaa
     3301 gagctttcag acgaggcggc gaagttgaaa aatgtagtgg gacatcacga agccgccaca
     3361 aacatcgtga aggactatcg gtatacctat gataagtact tccttcacat gccaatcacg
     3421 atcaatttta aagcgaataa gaccgggttc ataaatgacc ggattctgca gtacatagca
     3481 aaggagaaag atcttcatgt tataggcatt gatcgcggcg aaagaaacct tatttatgtc
     3541 tccgttatag acacatgtgg gaacatcgtt gaacaaaaat cctttaatat cgttaatgga
     3601 tacgactatc agataaagct caaacaacgg gagggggcgc gccagattgc tcgtaaagaa
     3661 tggaaggaaa taggaaaaat aaaagaaatc aaggagggtt acctgagcct tgtaattcat
     3721 gaaatctcca aaatggttat aaagtacaac gcgattattg tcatggaaga tttgagctac
     3781 ggttttaaaa aagggaggtt caaggtggag cgacaggtgt accaaaagtt tgaaacgatg
     3841 cttattaata aactcaatta cctcgtgttc aaggatataa gcataacaga aaatggaggg
     3901 ctccttaagg gataccagct cacatacata ccggacaagc ttaaaaacgt gggacaccag
     3961 tgcgggtgta tattttacgt tcctgccgcg tatacatcaa agatagaccc caccacaggg
     4021 ttcgtgaata tcttcaagtt taaggacttg acagtcgatg caaaacgtga gttcatcaag
     4081 aaattcgatt caatccggta cgattcagaa aagaatctgt tctgttttac gttcgattat
     4141 aacaacttta ttacgcaaaa tacagtgatg tcaaagagct catggagtgt ctacacatac
     4201 ggggttagga taaagcgcag gttcgttaac ggtcggttct caaacgaatc agacacgatt
     4261 gacattacga aggatatgga aaagactctg gagatgaccg acataaattg gcgagacggc
     4321 cacgacctcc gacaagatat cattgactac gagatcgtcc aacacatttt tgaaatcttc
     4381 cggttgaccg tccagatgcg aaacagtctt tctgaattgg aagaccggga ttacgacaga
     4441 ttgatcagtc ctgtattgaa cgaaaacaac atattctatg attccgccaa agctggcgat
     4501 gctttgccaa aagacgccga cgcgaatgga gcatattgta tcgcccttaa aggcctttac
     4561 gaaatcaaac aaataacaga gaactggaaa gaggatggga aatttagccg agataagctc
     4621 aagatcagca acaaagactg gtttgacttt attcaaaaca aacggtactt gccgaaaaag
     4681 aaacgcaaag taggatccta cccatacgat gttccagatt acgcttaccc atacgatgtt
     4741 ccagattacg cttacccata cgatgttcca gattacgctt aagaattcct agagctcgct
     4801 gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc
     4861 cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg
     4921 catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca
     4981 agggggagga ttgggaagag aatagcaggc atgctgggga ggtaccgagg gcctatttcc
     5041 catgattcct tcatatttgc atatacgata caaggctgtt agagagataa ttggaattaa
     5101 tttgactgta aacacaaaga tattagtaca aaatacgtga cgtagaaagt aataatttct
     5161 tgggtagttt gcagttttaa aattatgttt taaaatggac tatcatatgc ttaccgtaac
     5221 ttgaaagtat ttcgatttct tggctttata tatcttgtgg aaaggacgaa acaccgtcaa
     5281 aagacctttt taatttctac tcttgtagat agagaccggg ggtggtctct tttttggccg
     5341 caggaacccc tagtgatgga gttggccact ccctctctgc gcgctcgctc gctcactgag
     5401 gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc agtgagcgag
     5461 cgagcgcgca gctgcctgca ggggcgcctg atgcggtatt ttctccttac gcatctgtgc
     5521 ggtatttcac accgcatacg tcaaagcaac catagtacgc gccctgtagc ggcgcattaa
     5581 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccttagcgc
     5641 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
     5701 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
     5761 aaaaacttga tttgggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc
     5821 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa
     5881 cactcaactc tatctcgggc tattcttttg atttataagg gattttgccg atttcggtct
     5941 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa
     6001 cgtttacaat tttatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
     6061 agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat
     6121 ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt
     6181 catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg cctattttta taggttaatg
     6241 tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa
     6301 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
     6361 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg
     6421 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc
     6481 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg
     6541 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga
     6601 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc
     6661 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag
     6721 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga
     6781 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg
     6841 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga
     6901 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt
     6961 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact
     7021 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt
     7081 ttattgctga taaatctgga gccggtgagc gtggaagccg cggtatcatt gcagcactgg
     7141 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta
     7201 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac
     7261 tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta
     7321 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
     7381 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     7441 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     7501 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     7561 agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     7621 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     7681 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     7741 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     7801 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     7861 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     7921 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     7981 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     8041 ta
//
