LOCUS Exported 8250 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION CMV and T7 promoter expression plasmid for human codon optimized AsCas12a with a c-terminal NLS(nucleoplasmin), 3x HA tag, and P2A-EGFP. ACCESSION . VERSION . KEYWORDS pCMV-T7-AsCas12a-P2A-EGFP (RTW2861) SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8250) AUTHORS Walton RT, Hsu JY, Joung JK, Kleinstiver BP TITLE Scalable characterization of the PAM requirements of CRISPR-Cas enzymes using HT-PAMDA. JOURNAL Nat Protoc. 2021 Feb 5. pii: 10.1038/s41596-020-00465-2. doi: 10.1038/s41596-020-00465-2. PUBMED 33547443 REFERENCE 2 (bases 1 to 8250) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8250 /organism="synthetic DNA construct" /mol_type="other DNA" enhancer 1..380 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 381..584 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 535..555 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" primer_bind 626..645 /label=T7 /note="T7 promoter, forward primer" promoter 626..644 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" CDS 658..4578 /codon_start=1 /product="CRISPR-associated protein from Acidaminococcus sp. BV3L6 (Zetsche et al., 2015)" /label=AsCpf1 /translation="MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARND HYKELKPIIDRIYKTYADQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRN AIHDYFIGRTDNLTDAINKRHAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFD KFTTYFSGFYENRKNVFSAEDISTAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHF ENVKKAIGIFVSTSIEEVFSFPFYNQLLTQTQIDLYNQLLGGISREAGTEKIKGLNEVL NLAIQKNDETAHIIASLPHRFIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKTLL RNENVLETAEALFNELNSIDLTHIFISHKKLETISSALCDHWDTLRNALYERRISELTG KITKSAKEKVQRSLKHEDINLQEIISAAGKELSEAFKQKTSEILSHAHAALDQPLPTTL KKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEFSARLTGIKLEMEPSLSFYNKA RNYATKKPYSVEKFKLNFQMPTLASGWDVNKEKNNGAILFVKNGLYYLGIMPKQKGRYK ALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQTHTTPILLSNNFIEP LEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRDFLSKYTKTTSID LSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQIYNKDFAKG HHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAHRLGEKMLNKKLK DQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDKFFF HVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQR SLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAV VVLENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQ FTSFAKMGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYD VKTGDFILHFKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIEN HRFTGRYRDLYPANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQM RNSNAATGEDYINSPVRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKES KDLKLQNGISNQDWLAYIQELRN" regulatory 4298..4307 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 4579..4626 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /label=nucleoplasmin NLS /translation="KRPAATKKAGQAKKKK" CDS 4633..4713 /codon_start=1 /product="three tandem HA epitope tags" /label=3xHA /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA" CDS 4732..4788 /codon_start=1 /product="2A peptide from porcine teschovirus-1 polyprotein" /label=P2A /note="Eukaryotic ribosomes fail to insert a peptide bond between the Gly and Pro residues, yielding separate polypeptides." /translation="ATNFSLLKQAGDVEENPGP" CDS 4789..5505 /codon_start=1 /product="enhanced GFP" /label=EGFP /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" primer_bind complement(4834..4855) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(5095..5114) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 5442..5463 /label=EGFP-C /note="EGFP, forward primer" CDS 5514..5531 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" primer_bind complement(5554..5571) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 5560..5784 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" primer_bind complement(5855..5871) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(5855..5871) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(5868..5890) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 5879..5895 /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(5903..5933) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 5948..5969 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(6086..6103) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(6257..6845) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(6337..6356) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(7016..7876) /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 7639..7658 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(7877..7981) /gene="bla" /label=AmpR promoter primer_bind complement(8060..8079) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" ORIGIN 1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc 61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca 121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga 181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc 241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct 301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat 361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc 421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt 481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa 541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctggttta gtgaaccgtc 601 agatccgcta gagatccgcg gccgctaata cgactcacta tagggagagc cgccaccatg 661 acacagttcg agggctttac caacctgtat caggtgagca agacactgcg gtttgagctg 721 atcccacagg gcaagaccct gaagcacatc caggagcagg gcttcatcga ggaggacaag 781 gcccgcaatg atcactacaa ggagctgaag cccatcatcg atcggatcta caagacctat 841 gccgaccagt gcctgcagct ggtgcagctg gattgggaga acctgagcgc cgccatcgac 901 tcctatagaa aggagaaaac cgaggagaca aggaacgccc tgatcgagga gcaggccaca 961 tatcgcaatg ccatccacga ctacttcatc ggccggacag acaacctgac cgatgccatc 1021 aataagagac acgccgagat ctacaagggc ctgttcaagg ccgagctgtt taatggcaag 1081 gtgctgaagc agctgggcac cgtgaccaca accgagcacg agaacgccct gctgcggagc 1141 ttcgacaagt ttacaaccta cttctccggc ttttatgaga acaggaagaa cgtgttcagc 1201 gccgaggata tcagcacagc catcccacac cgcatcgtgc aggacaactt ccccaagttt 1261 aaggagaatt gtcacatctt cacacgcctg atcaccgccg tgcccagcct gcgggagcac 1321 tttgagaacg tgaagaaggc catcggcatc ttcgtgagca cctccatcga ggaggtgttt 1381 tccttccctt tttataacca gctgctgaca cagacccaga tcgacctgta taaccagctg 1441 ctgggaggaa tctctcggga ggcaggcacc gagaagatca agggcctgaa cgaggtgctg 1501 aatctggcca tccagaagaa tgatgagaca gcccacatca tcgcctccct gccacacaga 1561 ttcatccccc tgtttaagca gatcctgtcc gataggaaca ccctgtcttt catcctggag 1621 gagtttaaga gcgacgagga agtgatccag tccttctgca agtacaagac actgctgaga 1681 aacgagaacg tgctggagac agccgaggcc ctgtttaacg agctgaacag catcgacctg 1741 acacacatct tcatcagcca caagaagctg gagacaatca gcagcgccct gtgcgaccac 1801 tgggatacac tgaggaatgc cctgtatgag cggagaatct ccgagctgac aggcaagatc 1861 accaagtctg ccaaggagaa ggtgcagcgc agcctgaagc acgaggatat caacctgcag 1921 gagatcatct ctgccgcagg caaggagctg agcgaggcct tcaagcagaa aaccagcgag 1981 atcctgtccc acgcacacgc cgccctggat cagccactgc ctacaaccct gaagaagcag 2041 gaggagaagg agatcctgaa gtctcagctg gacagcctgc tgggcctgta ccacctgctg 2101 gactggtttg ccgtggatga gtccaacgag gtggaccccg agttctctgc ccggctgacc 2161 ggcatcaagc tggagatgga gccttctctg agcttctaca acaaggccag aaattatgcc 2221 accaagaagc cctactccgt ggagaagttc aagctgaact ttcagatgcc tacactggcc 2281 tctggctggg acgtgaataa ggagaagaac aatggcgcca tcctgtttgt gaagaacggc 2341 ctgtactatc tgggcatcat gccaaagcag aagggcaggt ataaggccct gagcttcgag 2401 cccacagaga aaaccagcga gggctttgat aagatgtact atgactactt ccctgatgcc 2461 gccaagatga tcccaaagtg cagcacccag ctgaaggccg tgacagccca ctttcagacc 2521 cacacaaccc ccatcctgct gtccaacaat ttcatcgagc ctctggagat cacaaaggag 2581 atctacgacc tgaacaatcc tgagaaggag ccaaagaagt ttcagacagc ctacgccaag 2641 aaaaccggcg accagaaggg ctacagagag gccctgtgca agtggatcga cttcacaagg 2701 gattttctgt ccaagtatac caagacaacc tctatcgatc tgtctagcct gcggccatcc 2761 tctcagtata aggacctggg cgagtactat gccgagctga atcccctgct gtaccacatc 2821 agcttccaga gaatcgccga gaaggagatc atggatgccg tggagacagg caagctgtac 2881 ctgttccaga tctataacaa ggactttgcc aagggccacc acggcaagcc taatctgcac 2941 acactgtatt ggaccggcct gttttctcca gagaacctgg ccaagacaag catcaagctg 3001 aatggccagg ccgagctgtt ctaccgccct aagtccagga tgaagaggat ggcacaccgg 3061 ctgggagaga agatgctgaa caagaagctg aaggatcaga aaaccccaat ccccgacacc 3121 ctgtaccagg agctgtacga ctatgtgaat cacagactgt cccacgacct gtctgatgag 3181 gccagggccc tgctgcccaa cgtgatcacc aaggaggtgt ctcacgagat catcaaggat 3241 aggcgcttta ccagcgacaa gttctttttc cacgtgccta tcacactgaa ctatcaggcc 3301 gccaattccc catctaagtt caaccagagg gtgaatgcct acctgaagga gcaccccgag 3361 acacctatca tcggcatcga tcggggcgag agaaacctga tctatatcac agtgatcgac 3421 tccaccggca agatcctgga gcagcggagc ctgaacacca tccagcagtt tgattaccag 3481 aagaagctgg acaacaggga gaaggagagg gtggcagcaa ggcaggcctg gtctgtggtg 3541 ggcacaatca aggatctgaa gcagggctat ctgagccagg tcatccacga gatcgtggac 3601 ctgatgatcc actaccaggc cgtggtggtg ctggagaacc tgaatttcgg ctttaagagc 3661 aagaggaccg gcatcgccga gaaggccgtg taccagcagt tcgagaagat gctgatcgat 3721 aagctgaatt gcctggtgct gaaggactat ccagcagaga aagtgggagg cgtgctgaac 3781 ccataccagc tgacagacca gttcacctcc tttgccaaga tgggcaccca gtctggcttc 3841 ctgttttacg tgcctgcccc atatacatct aagatcgatc ccctgaccgg cttcgtggac 3901 cccttcgtgt ggaaaaccat caagaatcac gagagccgca agcacttcct ggagggcttc 3961 gactttctgc actacgacgt gaaaaccggc gacttcatcc tgcactttaa gatgaacaga 4021 aatctgtcct tccagagggg cctgcccggc tttatgcctg catgggatat cgtgttcgag 4081 aagaacgaga cacagtttga cgccaagggc acccctttca tcgccggcaa gagaatcgtg 4141 ccagtgatcg agaatcacag attcaccggc agataccggg acctgtatcc tgccaacgag 4201 ctgatcgccc tgctggagga gaagggcatc gtgttcaggg atggctccaa catcctgcca 4261 aagctgctgg agaatgacga ttctcacgcc atcgacacca tggtggccct gatccgcagc 4321 gtgctgcaga tgcggaactc caatgccgcc acaggcgagg actatatcaa cagccccgtg 4381 cgcgatctga atggcgtgtg cttcgactcc cggtttcaga acccagagtg gcccatggac 4441 gccgatgcca atggcgccta ccacatcgcc ctgaagggcc agctgctgct gaatcacctg 4501 aaggagagca aggatctgaa gctgcagaac ggcatctcca atcaggactg gctggcctac 4561 atccaggagc tgcgcaacaa aaggccggcg gccacgaaaa aggccggcca ggcaaaaaag 4621 aaaaagggat cctacccata cgatgttcca gattacgctt atccctacga cgtgcctgat 4681 tatgcatacc catatgatgt ccccgactat gccggaggcg gtggaagcgg cgcaacaaac 4741 ttctctctgc tgaaacaagc cggagatgtc gaagagaatc ctggaccgat ggtgagcaag 4801 ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac 4861 ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc 4921 ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc 4981 ctgacctacg gcgtgcagtg cttcagccgc taccccgacc acatgaagca gcacgacttc 5041 ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac 5101 ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc 5161 gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac 5221 aactacaaca gccacaacgt ctatatcatg gccgacaagc agaagaacgg catcaaggtg 5281 aacttcaaga tccgccacaa catcgaggac ggcagcgtgc agctcgccga ccactaccag 5341 cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagcacc 5401 cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc 5461 gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaagtgacc ggtcatcatc 5521 accatcacca ttgagtttaa acccgctgat cagcctcgac tgtgccttct agttgccagc 5581 catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg 5641 tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc 5701 tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg 5761 ctggggatgc ggtgggctct atggcttctg aggcggaaag aaccagctgg ggctcgatac 5821 cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt 5881 gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctagg 5941 gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt 6001 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt 6061 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc 6121 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg 6181 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg 6241 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac 6301 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg 6361 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct 6421 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg 6481 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct 6541 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac 6601 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt 6661 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc 6721 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca 6781 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat 6841 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac 6901 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt 6961 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc 7021 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg 7081 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg 7141 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc 7201 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta 7261 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg 7321 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct 7381 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta 7441 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg 7501 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga 7561 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt 7621 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca 7681 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt 7741 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt 7801 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga 7861 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt 7921 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc 7981 gcacatttcc ccgaaaagtg ccacctgacg tcgacggatc gggagatcga tctcccgatc 8041 ccctagggtc gactctcagt acaatctgct ctgatgccgc atagttaagc cagtatctgc 8101 tccctgcttg tgtgttggag gtcgctgagt agtgcgcgag caaaatttaa gctacaacaa 8161 ggcaaggctt gaccgacaat tgcatgaaga atctgcttag ggttaggcgt tttgcgctgc 8221 ttcgcgatgt acgggccaga tatacgcgtt //