LOCUS pCAG-henAsCas12a 8893 bp ds-DNA circular SYN 06-JUL-2018 DEFINITION CAG promoter expression plasmid for human codon optimized enCas12a-HF1(E174R/N282A/S542R/K548R) with C-terminal NLS and HA tag . ACCESSION VERSION . KEYWORDS pCAG-enCas12a-HF1(E174R/N282A/S542R/K548R)-NLS(nuc)-3xHA (AAS1.. SOURCE synthetic DNA construct ORGANISM synthetic DNA construct. REFERENCE 1 (bases 1 to 8893) TITLE Joung lab unpublished Cas12a plasmids JOURNAL Unpublished REFERENCE 2 (bases 1 to 8893) AUTHORS . TITLE Direct Submission JOURNAL Exported Jun 12, 2018 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8893 /organism="synthetic DNA construct" /mol_type="other DNA" enhancer 5..384 /note="human cytomegalovirus immediate early enhancer" /label="CMV enhancer" promoter 387..662 /label="chicken beta-actin promoter" intron 663..1671 /note="chimera between introns from chicken beta-actin and rabbit beta-globin" /label="chimeric intron" source <1770..>5690 /organism="synthetic DNA construct" /mol_type="other DNA" CDS 1770..5690 /codon_start=1 /product="Engineered CRISPR-associated protein from Acidaminococcus sp. BV3L6 with residue swaps E174R/S542RK/548R and additional specificity enhancing mutation N282A" /modified_by="User" /label="Human codon optimized enAsCas12a(E174R/N282A/S542R/K548R)" misc_marker 2289..2291 /created_by="User" /label="E174R" misc_marker 2613..2615 /created_by="User" /Color="#0000ff" /modified_by="User" /label="N282A" misc_marker 3393..3395 /created_by="User" /label="S542R" misc_marker 3411..3413 /created_by="User" /label="K548R" regulatory 5410..5419 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 5691..5738 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /translation="KRPAATKKAGQAKKKK" /label="nucleoplasmin NLS" CDS 5745..5825 /codon_start=1 /product="three tandem HA epitope tags" /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA" /label="3xHA" polyA_site 5980..6035 /note="rabbit beta-globin polyadenylation signal" /label="-beta-globin poly(A) signal" protein_bind 6420..6436 /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)." /label="lac operator" promoter complement(6444..6474) /note="promoter for the E. coli lac operon" /label="lac promoter" protein_bind 6489..6510 /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." /label="CAP binding site" promoter 6569..6764 /note="SV40 early promoter" /label="SV40 promoter" rep_origin 6615..6750 /note="SV40 origin of replication" /label="SV40 ori" polyA_signal 6770..6904 /note="SV40 polyadenylation signal" /label="SV40 poly(A) signal" rep_origin complement(7143..7731) /direction="LEFT" /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" /label="ori" CDS complement(7902..8762) /codon_start=1 /gene="bla" /product="beta-lactamase" /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGY IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVE YSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRL DRWEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPL LRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIA EIGASLIKHW" /label="AmpR" promoter complement(8763..8867) /gene="bla" /label="AmpR promoter" ORIGIN 1 ggtcgacatt gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat 61 agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg 121 cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata 181 gggactttcc attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta 241 catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc 301 gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca gtacatctac 361 gtattagtca tcgctattac catgggtcga ggtgagcccc acgttctgct tcactctccc 421 catctccccc ccctccccac ccccaatttt gtatttattt attttttaat tattttgtgc 481 agcgatgggg gcgggggggg ggggggcgcg cgccaggcgg ggcggggcgg ggcgaggggc 541 ggggcggggc gaggcggaga ggtgcggcgg cagccaatca gagcggcgcg ctccgaaagt 601 ttccttttat ggcgaggcgg cggcggcggc ggccctataa aaagcgaagc gcgcggcggg 661 cgggagtcgc tgcgttgcct tcgccccgtg ccccgctccg cgccgcctcg cgccgcccgc 721 cccggctctg actgaccgcg ttactcccac aggtgagcgg gcgggacggc ccttctcctc 781 cgggctgtaa ttagcgcttg gtttaatgac ggctcgtttc ttttctgtgg ctgcgtgaaa 841 gccttaaagg gctccgggag ggccctttgt gcggggggga gcggctcggg gggtgcgtgc 901 gtgtgtgtgt gcgtggggag cgccgcgtgc ggcccgcgct gcccggcggc tgtgagcgct 961 gcgggcgcgg cgcggggctt tgtgcgctcc gcgtgtgcgc gaggggagcg cggccggggg 1021 cggtgccccg cggtgcgggg gggctgcgag gggaacaaag gctgcgtgcg gggtgtgtgc 1081 gtgggggggt gagcaggggg tgtgggcgcg gcggtcgggc tgtaaccccc ccctgcaccc 1141 ccctccccga gttgctgagc acggcccggc ttcgggtgcg gggctccgtg cggggcgtgg 1201 cgcggggctc gccgtgccgg gcggggggtg gcggcaggtg ggggtgccgg gcggggcggg 1261 gccgcctcgg gccggggagg gctcggggga ggggcgcggc ggccccggag cgccggcggc 1321 tgtcgaggcg cggcgagccg cagccattgc cttttatggt aatcgtgcga gagggcgcag 1381 ggacttcctt tgtcccaaat ctggcggagc cgaaatctgg gaggcgccgc cgcaccccct 1441 ctagcgggcg cgggcgaagc ggtgcggcgc cggcaggaag gaaatgggcg gggagggcct 1501 tcgtgcgtcg ccgcgccgcc gtccccttct ccatctccag cctcggggct gccgcagggg 1561 gacggctgcc ttcggggggg acggggcagg gcggggttcg gcttctggcg tgtgaccggc 1621 ggctctagag cctctgctaa ccatgttcat gccttcttct ttttcctaca gctcctgggc 1681 aacgtgctgg ttattgtgct gtctcatcat tttggcaaag aattctgcag tcgacggtac 1741 cgcgggcccg ggatccaccg gtcgccacca tgacacagtt cgagggcttt accaacctgt 1801 atcaggtgag caagacactg cggtttgagc tgatcccaca gggcaagacc ctgaagcaca 1861 tccaggagca gggcttcatc gaggaggaca aggcccgcaa tgatcactac aaggagctga 1921 agcccatcat cgatcggatc tacaagacct atgccgacca gtgcctgcag ctggtgcagc 1981 tggattggga gaacctgagc gccgccatcg actcctatag aaaggagaaa accgaggaga 2041 caaggaacgc cctgatcgag gagcaggcca catatcgcaa tgccatccac gactacttca 2101 tcggccggac agacaacctg accgatgcca tcaataagag acacgccgag atctacaagg 2161 gcctgttcaa ggccgagctg tttaatggca aggtgctgaa gcagctgggc accgtgacca 2221 caaccgagca cgagaacgcc ctgctgcgga gcttcgacaa gtttacaacc tacttctccg 2281 gcttttatag aaacaggaag aacgtgttca gcgccgagga tatcagcaca gccatcccac 2341 accgcatcgt gcaggacaac ttccccaagt ttaaggagaa ttgtcacatc ttcacacgcc 2401 tgatcaccgc cgtgcccagc ctgcgggagc actttgagaa cgtgaagaag gccatcggca 2461 tcttcgtgag cacctccatc gaggaggtgt tttccttccc tttttataac cagctgctga 2521 cacagaccca gatcgacctg tataaccagc tgctgggagg aatctctcgg gaggcaggca 2581 ccgagaagat caagggcctg aacgaggtgc tggccctggc catccagaag aatgatgaga 2641 cagcccacat catcgcctcc ctgccacaca gattcatccc cctgtttaag cagatcctgt 2701 ccgataggaa caccctgtct ttcatcctgg aggagtttaa gagcgacgag gaagtgatcc 2761 agtccttctg caagtacaag acactgctga gaaacgagaa cgtgctggag acagccgagg 2821 ccctgtttaa cgagctgaac agcatcgacc tgacacacat cttcatcagc cacaagaagc 2881 tggagacaat cagcagcgcc ctgtgcgacc actgggatac actgaggaat gccctgtatg 2941 agcggagaat ctccgagctg acaggcaaga tcaccaagtc tgccaaggag aaggtgcagc 3001 gcagcctgaa gcacgaggat atcaacctgc aggagatcat ctctgccgca ggcaaggagc 3061 tgagcgaggc cttcaagcag aaaaccagcg agatcctgtc ccacgcacac gccgccctgg 3121 atcagccact gcctacaacc ctgaagaagc aggaggagaa ggagatcctg aagtctcagc 3181 tggacagcct gctgggcctg taccacctgc tggactggtt tgccgtggat gagtccaacg 3241 aggtggaccc cgagttctct gcccggctga ccggcatcaa gctggagatg gagccttctc 3301 tgagcttcta caacaaggcc agaaattatg ccaccaagaa gccctactcc gtggagaagt 3361 tcaagctgaa ctttcagatg cctacactgg ccagaggctg ggacgtgaat agagagaaga 3421 acaatggcgc catcctgttt gtgaagaacg gcctgtacta tctgggcatc atgccaaagc 3481 agaagggcag gtataaggcc ctgagcttcg agcccacaga gaaaaccagc gagggctttg 3541 ataagatgta ctatgactac ttccctgatg ccgccaagat gatcccaaag tgcagcaccc 3601 agctgaaggc cgtgacagcc cactttcaga cccacacaac ccccatcctg ctgtccaaca 3661 atttcatcga gcctctggag atcacaaagg agatctacga cctgaacaat cctgagaagg 3721 agccaaagaa gtttcagaca gcctacgcca agaaaaccgg cgaccagaag ggctacagag 3781 aggccctgtg caagtggatc gacttcacaa gggattttct gtccaagtat accaagacaa 3841 cctctatcga tctgtctagc ctgcggccat cctctcagta taaggacctg ggcgagtact 3901 atgccgagct gaatcccctg ctgtaccaca tcagcttcca gagaatcgcc gagaaggaga 3961 tcatggatgc cgtggagaca ggcaagctgt acctgttcca gatctataac aaggactttg 4021 ccaagggcca ccacggcaag cctaatctgc acacactgta ttggaccggc ctgttttctc 4081 cagagaacct ggccaagaca agcatcaagc tgaatggcca ggccgagctg ttctaccgcc 4141 ctaagtccag gatgaagagg atggcacacc ggctgggaga gaagatgctg aacaagaagc 4201 tgaaggatca gaaaacccca atccccgaca ccctgtacca ggagctgtac gactatgtga 4261 atcacagact gtcccacgac ctgtctgatg aggccagggc cctgctgccc aacgtgatca 4321 ccaaggaggt gtctcacgag atcatcaagg ataggcgctt taccagcgac aagttctttt 4381 tccacgtgcc tatcacactg aactatcagg ccgccaattc cccatctaag ttcaaccaga 4441 gggtgaatgc ctacctgaag gagcaccccg agacacctat catcggcatc gatcggggcg 4501 agagaaacct gatctatatc acagtgatcg actccaccgg caagatcctg gagcagcgga 4561 gcctgaacac catccagcag tttgattacc agaagaagct ggacaacagg gagaaggaga 4621 gggtggcagc aaggcaggcc tggtctgtgg tgggcacaat caaggatctg aagcagggct 4681 atctgagcca ggtcatccac gagatcgtgg acctgatgat ccactaccag gccgtggtgg 4741 tgctggagaa cctgaatttc ggctttaaga gcaagaggac cggcatcgcc gagaaggccg 4801 tgtaccagca gttcgagaag atgctgatcg ataagctgaa ttgcctggtg ctgaaggact 4861 atccagcaga gaaagtggga ggcgtgctga acccatacca gctgacagac cagttcacct 4921 cctttgccaa gatgggcacc cagtctggct tcctgtttta cgtgcctgcc ccatatacat 4981 ctaagatcga tcccctgacc ggcttcgtgg accccttcgt gtggaaaacc atcaagaatc 5041 acgagagccg caagcacttc ctggagggct tcgactttct gcactacgac gtgaaaaccg 5101 gcgacttcat cctgcacttt aagatgaaca gaaatctgtc cttccagagg ggcctgcccg 5161 gctttatgcc tgcatgggat atcgtgttcg agaagaacga gacacagttt gacgccaagg 5221 gcaccccttt catcgccggc aagagaatcg tgccagtgat cgagaatcac agattcaccg 5281 gcagataccg ggacctgtat cctgccaacg agctgatcgc cctgctggag gagaagggca 5341 tcgtgttcag ggatggctcc aacatcctgc caaagctgct ggagaatgac gattctcacg 5401 ccatcgacac catggtggcc ctgatccgca gcgtgctgca gatgcggaac tccaatgccg 5461 ccacaggcga ggactatatc aacagccccg tgcgcgatct gaatggcgtg tgcttcgact 5521 cccggtttca gaacccagag tggcccatgg acgccgatgc caatggcgcc taccacatcg 5581 ccctgaaggg ccagctgctg ctgaatcacc tgaaggagag caaggatctg aagctgcaga 5641 acggcatctc caatcaggac tggctggcct acatccagga gctgcgcaac aaaaggccgg 5701 cggccacgaa aaaggccggc caggcaaaaa agaaaaaggg atcctaccca tacgatgttc 5761 cagattacgc ttatccctac gacgtgcctg attatgcata cccatatgat gtccccgact 5821 atgcctaaag cggccgcact cctcaggtgc aggctgccta tcagaaggtg gtggctggtg 5881 tggccaatgc cctggctcac aaataccact gagatctttt tccctctgcc aaaaattatg 5941 gggacatcat gaagcccctt gagcatctga cttctggcta ataaaggaaa tttattttca 6001 ttgcaatagt gtgttggaat tttttgtgtc tctcactcgg aaggacatat gggagggcaa 6061 atcatttaaa acatcagaat gagtatttgg tttagagttt ggcaacatat gcccatatgc 6121 tggctgccat gaacaaaggt tggctataaa gaggtcatca gtatatgaaa cagccccctg 6181 ctgtccattc cttattccat agaaaagcct tgacttgagg ttagattttt tttatatttt 6241 gttttgtgtt atttttttct ttaacatccc taaaattttc cttacatgtt ttactagcca 6301 gatttttcct cctctcctga ctactcccag tcatagctgt ccctcttctc ttatggagat 6361 ccctcgacct gcagcccaag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat 6421 tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg 6481 ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag 6541 tcgggaaacc tgtcgtgcca gcggatccgc atctcaatta gtcagcaacc atagtcccgc 6601 ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg 6661 gctgactaat tttttttatt tatgcagagg ccgaggccgc ctcggcctct gagctattcc 6721 agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagcta acttgtttat 6781 tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt 6841 tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg 6901 gatccgctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc 6961 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta 7021 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag 7081 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg 7141 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg 7201 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg 7261 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga 7321 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc 7381 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt 7441 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact 7501 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg 7561 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt 7621 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt 7681 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct 7741 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg 7801 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt 7861 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt 7921 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc 7981 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg 8041 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc 8101 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg 8161 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca 8221 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga 8281 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct 8341 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg 8401 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca 8461 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata 8521 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct 8581 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact 8641 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa 8701 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc 8761 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga 8821 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga 8881 aaagtgccac ctg //