LOCUS Exported 9187 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Express both AsCas12a and CRISPR arrays on single transcripts. ACCESSION . VERSION . KEYWORDS pCE048-SiT-Cas12a SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 9187) AUTHORS Campa CC, Weisbach NR, Santinha AJ, Incarnato D, Platt RJ TITLE Multiplexed genome engineering by Cas12a and CRISPR arrays encoded on single transcripts. JOURNAL Nat Methods. 2019 Aug 12. pii: 10.1038/s41592-019-0508-6. doi: 10.1038/s41592-019-0508-6. PUBMED 31406383 REFERENCE 2 (bases 1 to 9187) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..9187 /organism="synthetic DNA construct" /mol_type="other DNA" repeat_region 1..130 /label=AAV2 ITR (alternate) /note="Functional equivalent of wild-type AAV2 ITR" promoter 233..1411 /label=EF-1-alpha promoter /note="strong constitutive promoter for human elongation factor EF-1-alpha" intron 464..1402 /label=EF-1-alpha intron A /note="intron upstream of the start codon of human EF-1-alpha" primer_bind 1359..1379 /label=EF1a-F /note="Human elongation factor-1a promoter, forward primer" CDS 1446..5366 /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 5086..5095 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 5367..5414 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /label=nucleoplasmin NLS /translation="KRPAATKKAGQAKKKK" CDS 5421..5501 /codon_start=1 /product="three tandem HA epitope tags" /label=3xHA /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA" primer_bind 5698..5714 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 5698..5714 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" misc_feature 5775..6363 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(5828..5848) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(6246..6257) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" repeat_region 6450..6590 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 6450..6579 /label=AAV2 ITR rep_origin 6665..7120 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(6752..6771) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 6962..6983 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(7137..7156) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 7256..7278 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(7316..7334) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 7402..7506 /gene="bla" /label=AmpR promoter CDS 7507..8367 /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(7725..7744) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 8538..9126 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 9027..9046 /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 gcggccgcac gcgtttaatt aatgcaaaga tggataaagt tttaaacaga 181 gaggaatctt tgcagctaat ggaccttcta ggtcttgaaa ggagtgggaa ttggctccgg 241 tgcccgtcag tgggcagagc gcacatcgcc cacagtcccc gagaagttgg ggggaggggt 301 cggcaattga accggtgcct agagaaggtg gcgcggggta aactgggaaa gtgatgtcgt 361 gtactggctc cgcctttttc ccgagggtgg gggagaaccg tatataagtg cagtagtcgc 421 cgtgaacgtt ctttttcgca acgggtttgc cgccagaaca caggtaagtg ccgtgtgtgg 481 ttcccgcggg cctggcctct ttacgggtta tggcccttgc gtgccttgaa ttacttccac 541 ctggctgcag tacgtgattc ttgatcccga gcttcgggtt ggaagtgggt gggagagttc 601 gaggccttgc gcttaaggag ccccttcgcc tcgtgcttga gttgaggcct ggcctgggcg 661 ctggggccgc cgcgtgcgaa tctggtggca ccttcgcgcc tgtctcgctg ctttcgataa 721 gtctctagcc atttaaaatt tttgatgacc tgctgcgacg ctttttttct ggcaagatag 781 tcttgtaaat gcgggccaag atctgcacac tggtatttcg gtttttgggg ccgcgggcgg 841 cgacggggcc cgtgcgtccc agcgcacatg ttcggcgagg cggggcctgc gagcgcggcc 901 accgagaatc ggacgggggt agtctcaagc tggccggcct gctctggtgc ctggcctcgc 961 gccgccgtgt atcgccccgc cctgggcggc aaggctggcc cggtcggcac cagttgcgtg 1021 agcggaaaga tggccgcttc ccggccctgc tgcagggagc tcaaaatgga ggacgcggcg 1081 ctcgggagag cgggcgggtg agtcacccac acaaaggaaa agggcctttc cgtcctcagc 1141 cgtcgcttca tgtgactcca cggagtaccg ggcgccgtcc aggcacctcg attagttctc 1201 gagcttttgg agtacgtcgt ctttaggttg gggggagggg ttttatgcga tggagtttcc 1261 ccacactgag tgggtggaga ctgaagttag gccagcttgg cacttgatgt aattctcctt 1321 ggaatttgcc ctttttgagt ttggatcttg gttcattctc aagcctcaga cagtggttca 1381 aagttttttt cttccatttc aggtgtcgtg agctagcgtt taaacttaag cttggtaccg 1441 ccaccatgac acagttcgag ggctttacca acctgtatca ggtgagcaag acactgcggt 1501 ttgagctgat cccacagggc aagaccctga agcacatcca ggagcagggc ttcatcgagg 1561 aggacaaggc ccgcaatgat cactacaagg agctgaagcc catcatcgat cggatctaca 1621 agacctatgc cgaccagtgc ctgcagctgg tgcagctgga ttgggagaac ctgagcgccg 1681 ccatcgactc ctatagaaag gagaaaaccg aggagacaag gaacgccctg atcgaggagc 1741 aggccacata tcgcaatgcc atccacgact acttcatcgg ccggacagac aacctgaccg 1801 atgccatcaa taagagacac gccgagatct acaagggcct gttcaaggcc gagctgttta 1861 atggcaaggt gctgaagcag ctgggcaccg tgaccacaac cgagcacgag aacgccctgc 1921 tgcggagctt cgacaagttt acaacctact tctccggctt ttatgagaac aggaagaacg 1981 tgttcagcgc cgaggatatc agcacagcca tcccacaccg catcgtgcag gacaacttcc 2041 ccaagtttaa ggagaattgt cacatcttca cacgcctgat caccgccgtg cccagcctgc 2101 gggagcactt tgagaacgtg aagaaggcca tcggcatctt cgtgagcacc tccatcgagg 2161 aggtgttttc cttccctttt tataaccagc tgctgacaca gacccagatc gacctgtata 2221 accagctgct gggaggaatc tctcgggagg caggcaccga gaagatcaag ggcctgaacg 2281 aggtgctgaa tctggccatc cagaagaatg atgagacagc ccacatcatc gcctccctgc 2341 cacacagatt catccccctg tttaagcaga tcctgtccga taggaacacc ctgtctttca 2401 tcctggagga gtttaagagc gacgaggaag tgatccagtc cttctgcaag tacaagacac 2461 tgctgagaaa cgagaacgtg ctggagacag ccgaggccct gtttaacgag ctgaacagca 2521 tcgacctgac acacatcttc atcagccaca agaagctgga gacaatcagc agcgccctgt 2581 gcgaccactg ggatacactg aggaatgccc tgtatgagcg gagaatctcc gagctgacag 2641 gcaagatcac caagtctgcc aaggagaagg tgcagcgcag cctgaagcac gaggatatca 2701 acctgcagga gatcatctct gccgcaggca aggagctgag cgaggccttc aagcagaaaa 2761 ccagcgagat cctgtcccac gcacacgccg ccctggatca gccactgcct acaaccctga 2821 agaagcagga ggagaaggag atcctgaagt ctcagctgga cagcctgctg ggcctgtacc 2881 acctgctgga ctggtttgcc gtggatgagt ccaacgaggt ggaccccgag ttctctgccc 2941 ggctgaccgg catcaagctg gagatggagc cttctctgag cttctacaac aaggccagaa 3001 attatgccac caagaagccc tactccgtgg agaagttcaa gctgaacttt cagatgccta 3061 cactggcctc tggctgggac gtgaataagg agaagaacaa tggcgccatc ctgtttgtga 3121 agaacggcct gtactatctg ggcatcatgc caaagcagaa gggcaggtat aaggccctga 3181 gcttcgagcc cacagagaaa accagcgagg gctttgataa gatgtactat gactacttcc 3241 ctgatgccgc caagatgatc ccaaagtgca gcacccagct gaaggccgtg acagcccact 3301 ttcagaccca cacaaccccc atcctgctgt ccaacaattt catcgagcct ctggagatca 3361 caaaggagat ctacgacctg aacaatcctg agaaggagcc aaagaagttt cagacagcct 3421 acgccaagaa aaccggcgac cagaagggct acagagaggc cctgtgcaag tggatcgact 3481 tcacaaggga ttttctgtcc aagtatacca agacaacctc tatcgatctg tctagcctgc 3541 ggccatcctc tcagtataag gacctgggcg agtactatgc cgagctgaat cccctgctgt 3601 accacatcag cttccagaga atcgccgaga aggagatcat ggatgccgtg gagacaggca 3661 agctgtacct gttccagatc tataacaagg actttgccaa gggccaccac ggcaagccta 3721 atctgcacac actgtattgg accggcctgt tttctccaga gaacctggcc aagacaagca 3781 tcaagctgaa tggccaggcc gagctgttct accgccctaa gtccaggatg aagaggatgg 3841 cacaccggct gggagagaag atgctgaaca agaagctgaa ggatcagaaa accccaatcc 3901 ccgacaccct gtaccaggag ctgtacgact atgtgaatca cagactgtcc cacgacctgt 3961 ctgatgaggc cagggccctg ctgcccaacg tgatcaccaa ggaggtgtct cacgagatca 4021 tcaaggatag gcgctttacc agcgacaagt tctttttcca cgtgcctatc acactgaact 4081 atcaggccgc caattcccca tctaagttca accagagggt gaatgcctac ctgaaggagc 4141 accccgagac acctatcatc ggcatcgatc ggggcgagag aaacctgatc tatatcacag 4201 tgatcgactc caccggcaag atcctggagc agcggagcct gaacaccatc cagcagtttg 4261 attaccagaa gaagctggac aacagggaga aggagagggt ggcagcaagg caggcctggt 4321 ctgtggtggg cacaatcaag gatctgaagc agggctatct gagccaggtc atccacgaga 4381 tcgtggacct gatgatccac taccaggccg tggtggtgct ggagaacctg aatttcggct 4441 ttaagagcaa gaggaccggc atcgccgaga aggccgtgta ccagcagttc gagaagatgc 4501 tgatcgataa gctgaattgc ctggtgctga aggactatcc agcagagaaa gtgggaggcg 4561 tgctgaaccc ataccagctg acagaccagt tcacctcctt tgccaagatg ggcacccagt 4621 ctggcttcct gttttacgtg cctgccccat atacatctaa gatcgatccc ctgaccggct 4681 tcgtggaccc cttcgtgtgg aaaaccatca agaatcacga gagccgcaag cacttcctgg 4741 agggcttcga ctttctgcac tacgacgtga aaaccggcga cttcatcctg cactttaaga 4801 tgaacagaaa tctgtccttc cagaggggcc tgcccggctt tatgcctgca tgggatatcg 4861 tgttcgagaa gaacgagaca cagtttgacg ccaagggcac ccctttcatc gccggcaaga 4921 gaatcgtgcc agtgatcgag aatcacagat tcaccggcag ataccgggac ctgtatcctg 4981 ccaacgagct gatcgccctg ctggaggaga agggcatcgt gttcagggat ggctccaaca 5041 tcctgccaaa gctgctggag aatgacgatt ctcacgccat cgacaccatg gtggccctga 5101 tccgcagcgt gctgcagatg cggaactcca atgccgccac aggcgaggac tatatcaaca 5161 gccccgtgcg cgatctgaat ggcgtgtgct tcgactcccg gtttcagaac ccagagtggc 5221 ccatggacgc cgatgccaat ggcgcctacc acatcgccct gaagggccag ctgctgctga 5281 atcacctgaa ggagagcaag gatctgaagc tgcagaacgg catctccaat caggactggc 5341 tggcctacat ccaggagctg cgcaacaaaa ggccggcggc cacgaaaaag gccggccagg 5401 caaaaaagaa aaagggatcc tacccatacg atgttccaga ttacgcttat ccctacgacg 5461 tgcctgatta tgcataccca tatgatgtcc ccgactatgc ctaagaattc gattcgtcag 5521 tagggttgta aaggtttttc ttttcctgag aaaacaacct tttgttttct caggttttgc 5581 tttttggcct ttccctagct ttaaaaaaaa aaaagcaaaa ctcaccgagg cagttccata 5641 ggatggcaag atcctggtat tggtctgcga aatttctact cttgtagatt gaagagccag 5701 gaaacagcta tgacgctctt ctaatttcta ctcttgtaga ttctagaaaa gaattcgata 5761 tcaagcttat cgataatcaa cctctggatt acaaaatttg tgaaagattg actggtattc 5821 ttaactatgt tgctcctttt acgctatgtg gatacgctgc tttaatgcct ttgtatcatg 5881 ctattgcttc ccgtatggct ttcattttct cctccttgta taaatcctgg ttgctgtctc 5941 tttatgagga gttgtggccc gttgtcaggc aacgtggcgt ggtgtgcact gtgtttgctg 6001 acgcaacccc cactggttgg ggcattgcca ccacctgtca gctcctttcc gggactttcg 6061 ctttccccct ccctattgcc acggcggaac tcatcgccgc ctgccttgcc cgctgctgga 6121 caggggctcg gctgttgggc actgacaatt ccgtggtgtt gtcggggaaa tcatcgtcct 6181 ttccttggct gctcgcctat gttgccacct ggattctgcg cgggacgtcc ttctgctacg 6241 tcccttcggc cctcaatcca gcggaccttc cttcccgcgg cctgctgccg gctctgcggc 6301 ctcttccgcg tcttcgcctt cgccctcaga cgagtcggat ctccctttgg gccgcctccc 6361 cgcatcgata ccgagcgcta ataaaagatc tttattttca ttagatctgt gtgttggttt 6421 tttgtgtcac gtgcggaccg agcggccgca ggaaccccta gtgatggagt tggccactcc 6481 ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca aaggtcgccc gacgcccggg 6541 ctttgcccgg gcggcctcag tgagcgagcg agcgcgcagc tgcctgcagg ggcgcctgat 6601 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatacgtc aaagcaacca 6661 tagtacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg 6721 accgctacac ttgccagcgc cttagcgccc gctcctttcg ctttcttccc ttcctttctc 6781 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga 6841 tttagtgctt tacggcacct cgaccccaaa aaacttgatt tgggtgatgg ttcacgtagt 6901 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat 6961 agtggactct tgttccaaac tggaacaaca ctcaactcta tctcgggcta ttcttttgat 7021 ttataaggga ttttgccgat ttcggtctat tggttaaaaa atgagctgat ttaacaaaaa 7081 tttaacgcga attttaacaa aatattaacg tttacaattt tatggtgcac tctcagtaca 7141 atctgctctg atgccgcata gttaagccag ccccgacacc cgccaacacc cgctgacgcg 7201 ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac cttctccggg 7261 agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgatacg aaagggcctc 7321 gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt 7381 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca 7441 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg 7501 aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc 7561 cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg 7621 ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt 7681 cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta 7741 ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat 7801 gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga 7861 gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca 7921 acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact 7981 cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc 8041 acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact 8101 ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt 8161 ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt 8221 gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt 8281 atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata 8341 ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag 8401 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat 8461 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa 8521 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca 8581 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt 8641 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg 8701 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc 8761 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga 8821 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc 8881 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc 8941 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca 9001 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg 9061 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta 9121 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct 9181 cacatgt //