LOCUS pCAG-henAsCas12a 8890 bp ds-DNA circular SYN 06-JUL-2018 DEFINITION CAG promoter expression plasmid for human codon optimized enCas12a(E174R/S542R/K548R) nuclease with C-terminal NLS and HA tag . ACCESSION VERSION . KEYWORDS SQT1659. SOURCE ORGANISM . REFERENCE 1 (bases 1 to 8890) AUTHORS Kleinstiver, BP, Tsai, SQ, Prew, MS, Nguyen, NT, Welch, MM, Lopez, JM, McCaw, ZR, Aryee, MJ, Joung and JK TITLE Genome-wide specificities of CRISPR-Cas Cpf1 nucleases in human cells. JOURNAL Nat Biotechnol. 2016 Jun 27. doi: 10.1038/nbt.3620. PUBMED 27347757 REFERENCE 2 (bases 1 to 8890) AUTHORS . TITLE Direct Submission JOURNAL Exported Jan 10, 2018 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8890 /organism="synthetic DNA construct" /mol_type="other DNA" enhancer 4..383 /note="human cytomegalovirus immediate early enhancer" /label="CMV enhancer" promoter 386..661 /label="chicken beta-actin promoter" intron 662..1670 /note="chimera between introns from chicken beta-actin and rabbit beta-globin" /label="chimeric intron" source <1769..>5689 /organism="synthetic DNA construct" /mol_type="other DNA" CDS 1769..5689 /codon_start=1 /product="Engineered CRISPR-associated protein from Acidaminococcus sp. BV3L6 with residue substitutions E174R/S542RK/548R" /modified_by="User" /label="Human codon optimized AsCas12a(E174R/S542R/K548R)" misc_marker 2288..2290 /created_by="User" /label="E174R" misc_marker 3392..3394 /created_by="User" /label="S542R" misc_marker 3410..3412 /created_by="User" /label="K548R" regulatory 5409..5418 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 5690..5737 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /translation="KRPAATKKAGQAKKKK" /label="nucleoplasmin NLS" CDS 5744..5824 /codon_start=1 /product="three tandem HA epitope tags" /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA" /label="3xHA" polyA_site 5979..6034 /note="rabbit beta-globin polyadenylation signal" /label="-beta-globin poly(A) signal" protein_bind 6417..6433 /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(6441..6471) /note="promoter for the E. coli lac operon" /label="lac promoter" protein_bind 6486..6507 /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." /label="CAP binding site" promoter 6566..6761 /note="SV40 early promoter" /label="SV40 promoter" rep_origin 6612..6747 /note="SV40 origin of replication" /label="SV40 ori" polyA_signal 6767..6901 /note="SV40 polyadenylation signal" /label="SV40 poly(A) signal" rep_origin complement(7140..7728) /direction="LEFT" /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" /label="ori" CDS complement(7899..8759) /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(8760..8864) /gene="bla" /label="AmpR promoter" ORIGIN 1 gtcgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata 61 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc 121 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag 181 ggactttcca ttgacgtcaa tgggtggact atttacggta aactgcccac ttggcagtac 241 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg 301 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg 361 tattagtcat cgctattacc atgggtcgag gtgagcccca cgttctgctt cactctcccc 421 atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca 481 gcgatggggg cggggggggg gggggcgcgc gccaggcggg gcggggcggg gcgaggggcg 541 gggcggggcg aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt 601 tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc 661 gggagtcgct gcgttgcctt cgccccgtgc cccgctccgc gccgcctcgc gccgcccgcc 721 ccggctctga ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc cttctcctcc 781 gggctgtaat tagcgcttgg tttaatgacg gctcgtttct tttctgtggc tgcgtgaaag 841 ccttaaaggg ctccgggagg gccctttgtg cgggggggag cggctcgggg ggtgcgtgcg 901 tgtgtgtgtg cgtggggagc gccgcgtgcg gcccgcgctg cccggcggct gtgagcgctg 961 cgggcgcggc gcggggcttt gtgcgctccg cgtgtgcgcg aggggagcgc ggccgggggc 1021 ggtgccccgc ggtgcggggg ggctgcgagg ggaacaaagg ctgcgtgcgg ggtgtgtgcg 1081 tgggggggtg agcagggggt gtgggcgcgg cggtcgggct gtaacccccc cctgcacccc 1141 cctccccgag ttgctgagca cggcccggct tcgggtgcgg ggctccgtgc ggggcgtggc 1201 gcggggctcg ccgtgccggg cggggggtgg cggcaggtgg gggtgccggg cggggcgggg 1261 ccgcctcggg ccggggaggg ctcgggggag gggcgcggcg gccccggagc gccggcggct 1321 gtcgaggcgc ggcgagccgc agccattgcc ttttatggta atcgtgcgag agggcgcagg 1381 gacttccttt gtcccaaatc tggcggagcc gaaatctggg aggcgccgcc gcaccccctc 1441 tagcgggcgc gggcgaagcg gtgcggcgcc ggcaggaagg aaatgggcgg ggagggcctt 1501 cgtgcgtcgc cgcgccgccg tccccttctc catctccagc ctcggggctg ccgcaggggg 1561 acggctgcct tcggggggga cggggcaggg cggggttcgg cttctggcgt gtgaccggcg 1621 gctctagagc ctctgctaac catgttcatg ccttcttctt tttcctacag ctcctgggca 1681 acgtgctggt tattgtgctg tctcatcatt ttggcaaaga attctgcagt cgacggtacc 1741 gcgggcccgg gatccaccgg tcgccaccat gacacagttc gagggcttta ccaacctgta 1801 tcaggtgagc aagacactgc ggtttgagct gatcccacag ggcaagaccc tgaagcacat 1861 ccaggagcag ggcttcatcg aggaggacaa ggcccgcaat gatcactaca aggagctgaa 1921 gcccatcatc gatcggatct acaagaccta tgccgaccag tgcctgcagc tggtgcagct 1981 ggattgggag aacctgagcg ccgccatcga ctcctataga aaggagaaaa ccgaggagac 2041 aaggaacgcc ctgatcgagg agcaggccac atatcgcaat gccatccacg actacttcat 2101 cggccggaca gacaacctga ccgatgccat caataagaga cacgccgaga tctacaaggg 2161 cctgttcaag gccgagctgt ttaatggcaa ggtgctgaag cagctgggca ccgtgaccac 2221 aaccgagcac gagaacgccc tgctgcggag cttcgacaag tttacaacct acttctccgg 2281 cttttataga aacaggaaga acgtgttcag cgccgaggat atcagcacag ccatcccaca 2341 ccgcatcgtg caggacaact tccccaagtt taaggagaat tgtcacatct tcacacgcct 2401 gatcaccgcc gtgcccagcc tgcgggagca ctttgagaac gtgaagaagg ccatcggcat 2461 cttcgtgagc acctccatcg aggaggtgtt ttccttccct ttttataacc agctgctgac 2521 acagacccag atcgacctgt ataaccagct gctgggagga atctctcggg aggcaggcac 2581 cgagaagatc aagggcctga acgaggtgct gaatctggcc atccagaaga atgatgagac 2641 agcccacatc atcgcctccc tgccacacag attcatcccc ctgtttaagc agatcctgtc 2701 cgataggaac accctgtctt tcatcctgga ggagtttaag agcgacgagg aagtgatcca 2761 gtccttctgc aagtacaaga cactgctgag aaacgagaac gtgctggaga cagccgaggc 2821 cctgtttaac gagctgaaca gcatcgacct gacacacatc ttcatcagcc acaagaagct 2881 ggagacaatc agcagcgccc tgtgcgacca ctgggataca ctgaggaatg ccctgtatga 2941 gcggagaatc tccgagctga caggcaagat caccaagtct gccaaggaga aggtgcagcg 3001 cagcctgaag cacgaggata tcaacctgca ggagatcatc tctgccgcag gcaaggagct 3061 gagcgaggcc ttcaagcaga aaaccagcga gatcctgtcc cacgcacacg ccgccctgga 3121 tcagccactg cctacaaccc tgaagaagca ggaggagaag gagatcctga agtctcagct 3181 ggacagcctg ctgggcctgt accacctgct ggactggttt gccgtggatg agtccaacga 3241 ggtggacccc gagttctctg cccggctgac cggcatcaag ctggagatgg agccttctct 3301 gagcttctac aacaaggcca gaaattatgc caccaagaag ccctactccg tggagaagtt 3361 caagctgaac tttcagatgc ctacactggc cagaggctgg gacgtgaata gagagaagaa 3421 caatggcgcc atcctgtttg tgaagaacgg cctgtactat ctgggcatca tgccaaagca 3481 gaagggcagg tataaggccc tgagcttcga gcccacagag aaaaccagcg agggctttga 3541 taagatgtac tatgactact tccctgatgc cgccaagatg atcccaaagt gcagcaccca 3601 gctgaaggcc gtgacagccc actttcagac ccacacaacc cccatcctgc tgtccaacaa 3661 tttcatcgag cctctggaga tcacaaagga gatctacgac ctgaacaatc ctgagaagga 3721 gccaaagaag tttcagacag cctacgccaa gaaaaccggc gaccagaagg gctacagaga 3781 ggccctgtgc aagtggatcg acttcacaag ggattttctg tccaagtata ccaagacaac 3841 ctctatcgat ctgtctagcc tgcggccatc ctctcagtat aaggacctgg gcgagtacta 3901 tgccgagctg aatcccctgc tgtaccacat cagcttccag agaatcgccg agaaggagat 3961 catggatgcc gtggagacag gcaagctgta cctgttccag atctataaca aggactttgc 4021 caagggccac cacggcaagc ctaatctgca cacactgtat tggaccggcc tgttttctcc 4081 agagaacctg gccaagacaa gcatcaagct gaatggccag gccgagctgt tctaccgccc 4141 taagtccagg atgaagagga tggcacaccg gctgggagag aagatgctga acaagaagct 4201 gaaggatcag aaaaccccaa tccccgacac cctgtaccag gagctgtacg actatgtgaa 4261 tcacagactg tcccacgacc tgtctgatga ggccagggcc ctgctgccca acgtgatcac 4321 caaggaggtg tctcacgaga tcatcaagga taggcgcttt accagcgaca agttcttttt 4381 ccacgtgcct atcacactga actatcaggc cgccaattcc ccatctaagt tcaaccagag 4441 ggtgaatgcc tacctgaagg agcaccccga gacacctatc atcggcatcg atcggggcga 4501 gagaaacctg atctatatca cagtgatcga ctccaccggc aagatcctgg agcagcggag 4561 cctgaacacc atccagcagt ttgattacca gaagaagctg gacaacaggg agaaggagag 4621 ggtggcagca aggcaggcct ggtctgtggt gggcacaatc aaggatctga agcagggcta 4681 tctgagccag gtcatccacg agatcgtgga cctgatgatc cactaccagg ccgtggtggt 4741 gctggagaac ctgaatttcg gctttaagag caagaggacc ggcatcgccg agaaggccgt 4801 gtaccagcag ttcgagaaga tgctgatcga taagctgaat tgcctggtgc tgaaggacta 4861 tccagcagag aaagtgggag gcgtgctgaa cccataccag ctgacagacc agttcacctc 4921 ctttgccaag atgggcaccc agtctggctt cctgttttac gtgcctgccc catatacatc 4981 taagatcgat cccctgaccg gcttcgtgga ccccttcgtg tggaaaacca tcaagaatca 5041 cgagagccgc aagcacttcc tggagggctt cgactttctg cactacgacg tgaaaaccgg 5101 cgacttcatc ctgcacttta agatgaacag aaatctgtcc ttccagaggg gcctgcccgg 5161 ctttatgcct gcatgggata tcgtgttcga gaagaacgag acacagtttg acgccaaggg 5221 cacccctttc atcgccggca agagaatcgt gccagtgatc gagaatcaca gattcaccgg 5281 cagataccgg gacctgtatc ctgccaacga gctgatcgcc ctgctggagg agaagggcat 5341 cgtgttcagg gatggctcca acatcctgcc aaagctgctg gagaatgacg attctcacgc 5401 catcgacacc atggtggccc tgatccgcag cgtgctgcag atgcggaact ccaatgccgc 5461 cacaggcgag gactatatca acagccccgt gcgcgatctg aatggcgtgt gcttcgactc 5521 ccggtttcag aacccagagt ggcccatgga cgccgatgcc aatggcgcct accacatcgc 5581 cctgaagggc cagctgctgc tgaatcacct gaaggagagc aaggatctga agctgcagaa 5641 cggcatctcc aatcaggact ggctggccta catccaggag ctgcgcaaca aaaggccggc 5701 ggccacgaaa aaggccggcc aggcaaaaaa gaaaaaggga tcctacccat acgatgttcc 5761 agattacgct tatccctacg acgtgcctga ttatgcatac ccatatgatg tccccgacta 5821 tgcctaaagc ggccgcactc ctcaggtgca ggctgcctat cagaaggtgg tggctggtgt 5881 ggccaatgcc ctggctcaca aataccactg agatcttttt ccctctgcca aaaattatgg 5941 ggacatcatg aagccccttg agcatctgac ttctggctaa taaaggaaat ttattttcat 6001 tgcaatagtg tgttggaatt ttttgtgtct ctcactcgga aggacatatg ggagggcaaa 6061 tcatttaaaa catcagaatg agtatttggt ttagagtttg gcaacatatg ccatatgctg 6121 gctgccatga acaaaggtgg ctataaagag gtcatcagta tatgaaacag ccccctgctg 6181 tccattcctt attccataga aaagccttga cttgaggtta gatttttttt atattttgtt 6241 ttgtgttatt tttttcttta acatccctaa aattttcctt acatgtttta ctagccagat 6301 ttttcctcct ctcctgacta ctcccagtca tagctgtccc tcttctctta tgaagatccc 6361 tcgacctgca gcccaagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt 6421 tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt 6481 gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg 6541 ggaaacctgt cgtgccagcg gatccgcatc tcaattagtc agcaaccata gtcccgcccc 6601 taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct 6661 gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag ctattccaga 6721 agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagctaact tgtttattgc 6781 agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt 6841 ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctggat 6901 ccgctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct 6961 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca 7021 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac 7081 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt 7141 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg 7201 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc 7261 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc 7321 gtggcgcttt ctcaatgctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc 7381 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac 7441 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt 7501 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct 7561 aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc 7621 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt 7681 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg 7741 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc 7801 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa 7861 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag 7921 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg 7981 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga 8041 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag 8101 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa 8161 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc 8221 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca 8281 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg 8341 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat 8401 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc 8461 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg 8521 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg 8581 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt 8641 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca 8701 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata 8761 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac 8821 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa 8881 gtgccacctg //