LOCUS Exported 8163 bp ds-DNA circular SYN 10-APR-2019 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS . SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8163) AUTHORS locusliu TITLE Direct Submission JOURNAL Exported Apr 19, 2019 from SnapGene Viewer 4.2.5 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8163 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 217..235 /label=SP6 promoter /note="promoter for bacteriophage SP6 RNA polymerase" CDS 282..4202 /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 3922..3931 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 4203..4250 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /label=nucleoplasmin NLS /translation="KRPAATKKAGQAKKKK" CDS 4257..4337 /codon_start=1 /product="three tandem HA epitope tags" /label=3xHA /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA" CDS 4338..4358 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" promoter complement(4371..4389) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" polyA_signal 4394..4528 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" promoter complement(4641..4659) /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" primer_bind complement(4680..4696) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" protein_bind 4704..4720 /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(4728..4758) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 4773..4794 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." rep_origin complement(5082..5670) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" CDS complement(5841..6701) /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" promoter complement(6702..6806) /gene="bla" /label=AmpR promoter rep_origin complement(6832..7287) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" promoter join(7361..8163,1..182) /label=CMV IE94 promoter /note="enhancer/promoter region of simian cytomegalovirus major immediate early transcription unit IE94" ORIGIN 1 gggtacattg gcagtactcc cattgacgtc aatggcggta aatggcccgc gatggctgcc 61 aagtacatcc ccattgacgt caatggggag gggcaatgac gcaaatgggc gttccattga 121 cgtaaatggg cggtaggcgt gcctaatggg aggtctatat aagcaatgct cgtttaggga 181 accgccattc tgcctgggga cgtcggagca agcttgattt aggtgacact atagaataca 241 agctacttgt tctttttgca ggatcccatc gattcgaatt catgacacag ttcgagggct 301 ttaccaacct gtatcaggtg agcaagacac tgcggtttga gctgatccca cagggcaaga 361 ccctgaagca catccaggag cagggcttca tcgaggagga caaggcccgc aatgatcact 421 acaaggagct gaagcccatc atcgatcgga tctacaagac ctatgccgac cagtgcctgc 481 agctggtgca gctggattgg gagaacctga gcgccgccat cgactcctat agaaaggaga 541 aaaccgagga gacaaggaac gccctgatcg aggagcaggc cacatatcgc aatgccatcc 601 acgactactt catcggccgg acagacaacc tgaccgatgc catcaataag agacacgccg 661 agatctacaa gggcctgttc aaggccgagc tgtttaatgg caaggtgctg aagcagctgg 721 gcaccgtgac cacaaccgag cacgagaacg ccctgctgcg gagcttcgac aagtttacaa 781 cctacttctc cggcttttat gagaacagga agaacgtgtt cagcgccgag gatatcagca 841 cagccatccc acaccgcatc gtgcaggaca acttccccaa gtttaaggag aattgtcaca 901 tcttcacacg cctgatcacc gccgtgccca gcctgcggga gcactttgag aacgtgaaga 961 aggccatcgg catcttcgtg agcacctcca tcgaggaggt gttttccttc cctttttata 1021 accagctgct gacacagacc cagatcgacc tgtataacca gctgctggga ggaatctctc 1081 gggaggcagg caccgagaag atcaagggcc tgaacgaggt gctgaatctg gccatccaga 1141 agaatgatga gacagcccac atcatcgcct ccctgccaca cagattcatc cccctgttta 1201 agcagatcct gtccgatagg aacaccctgt ctttcatcct ggaggagttt aagagcgacg 1261 aggaagtgat ccagtccttc tgcaagtaca agacactgct gagaaacgag aacgtgctgg 1321 agacagccga ggccctgttt aacgagctga acagcatcga cctgacacac atcttcatca 1381 gccacaagaa gctggagaca atcagcagcg ccctgtgcga ccactgggat acactgagga 1441 atgccctgta tgagcggaga atctccgagc tgacaggcaa gatcaccaag tctgccaagg 1501 agaaggtgca gcgcagcctg aagcacgagg atatcaacct gcaggagatc atctctgccg 1561 caggcaagga gctgagcgag gccttcaagc agaaaaccag cgagatcctg tcccacgcac 1621 acgccgccct ggatcagcca ctgcctacaa ccctgaagaa gcaggaggag aaggagatcc 1681 tgaagtctca gctggacagc ctgctgggcc tgtaccacct gctggactgg tttgccgtgg 1741 atgagtccaa cgaggtggac cccgagttct ctgcccggct gaccggcatc aagctggaga 1801 tggagccttc tctgagcttc tacaacaagg ccagaaatta tgccaccaag aagccctact 1861 ccgtggagaa gttcaagctg aactttcaga tgcctacact ggcctctggc tgggacgtga 1921 ataaggagaa gaacaatggc gccatcctgt ttgtgaagaa cggcctgtac tatctgggca 1981 tcatgccaaa gcagaagggc aggtataagg ccctgagctt cgagcccaca gagaaaacca 2041 gcgagggctt tgataagatg tactatgact acttccctga tgccgccaag atgatcccaa 2101 agtgcagcac ccagctgaag gccgtgacag cccactttca gacccacaca acccccatcc 2161 tgctgtccaa caatttcatc gagcctctgg agatcacaaa ggagatctac gacctgaaca 2221 atcctgagaa ggagccaaag aagtttcaga cagcctacgc caagaaaacc ggcgaccaga 2281 agggctacag agaggccctg tgcaagtgga tcgacttcac aagggatttt ctgtccaagt 2341 ataccaagac aacctctatc gatctgtcta gcctgcggcc atcctctcag tataaggacc 2401 tgggcgagta ctatgccgag ctgaatcccc tgctgtacca catcagcttc cagagaatcg 2461 ccgagaagga gatcatggat gccgtggaga caggcaagct gtacctgttc cagatctata 2521 acaaggactt tgccaagggc caccacggca agcctaatct gcacacactg tattggaccg 2581 gcctgttttc tccagagaac ctggccaaga caagcatcaa gctgaatggc caggccgagc 2641 tgttctaccg ccctaagtcc aggatgaaga ggatggcaca ccggctggga gagaagatgc 2701 tgaacaagaa gctgaaggat cagaaaaccc caatccccga caccctgtac caggagctgt 2761 acgactatgt gaatcacaga ctgtcccacg acctgtctga tgaggccagg gccctgctgc 2821 ccaacgtgat caccaaggag gtgtctcacg agatcatcaa ggataggcgc tttaccagcg 2881 acaagttctt tttccacgtg cctatcacac tgaactatca ggccgccaat tccccatcta 2941 agttcaacca gagggtgaat gcctacctga aggagcaccc cgagacacct atcatcggca 3001 tcgatcgggg cgagagaaac ctgatctata tcacagtgat cgactccacc ggcaagatcc 3061 tggagcagcg gagcctgaac accatccagc agtttgatta ccagaagaag ctggacaaca 3121 gggagaagga gagggtggca gcaaggcagg cctggtctgt ggtgggcaca atcaaggatc 3181 tgaagcaggg ctatctgagc caggtcatcc acgagatcgt ggacctgatg atccactacc 3241 aggccgtggt ggtgctggag aacctgaatt tcggctttaa gagcaagagg accggcatcg 3301 ccgagaaggc cgtgtaccag cagttcgaga agatgctgat cgataagctg aattgcctgg 3361 tgctgaagga ctatccagca gagaaagtgg gaggcgtgct gaacccatac cagctgacag 3421 accagttcac ctcctttgcc aagatgggca cccagtctgg cttcctgttt tacgtgcctg 3481 ccccatatac atctaagatc gatcccctga ccggcttcgt ggaccccttc gtgtggaaaa 3541 ccatcaagaa tcacgagagc cgcaagcact tcctggaggg cttcgacttt ctgcactacg 3601 acgtgaaaac cggcgacttc atcctgcact ttaagatgaa cagaaatctg tccttccaga 3661 ggggcctgcc cggctttatg cctgcatggg atatcgtgtt cgagaagaac gagacacagt 3721 ttgacgccaa gggcacccct ttcatcgccg gcaagagaat cgtgccagtg atcgagaatc 3781 acagattcac cggcagatac cgggacctgt atcctgccaa cgagctgatc gccctgctgg 3841 aggagaaggg catcgtgttc agggatggct ccaacatcct gccaaagctg ctggagaatg 3901 acgattctca cgccatcgac accatggtgg ccctgatccg cagcgtgctg cagatgcgga 3961 actccaatgc cgccacaggc gaggactata tcaacagccc cgtgcgcgat ctgaatggcg 4021 tgtgcttcga ctcccggttt cagaacccag agtggcccat ggacgccgat gccaatggcg 4081 cctaccacat cgccctgaag ggccagctgc tgctgaatca cctgaaggag agcaaggatc 4141 tgaagctgca gaacggcatc tccaatcagg actggctggc ctacatccag gagctgcgca 4201 acaaaaggcc ggcggccacg aaaaaggccg gccaggcaaa aaagaaaaag ggatcctacc 4261 catacgatgt tccagattac gcttatccct acgacgtgcc tgattatgca tacccatatg 4321 atgtccccga ctatgcccca aagaagaagc ggaaggtcta atgatctaga actatagtga 4381 gtcgtattac gtagatccag acatgataag atacattgat gagtttggac aaaccacaac 4441 tagaatgcag tgaaaaaaat gctttatttg tgaaatttgt gatgctattg ctttatttgt 4501 aaccattata agctgcaata aacaagttaa caacaacaat tgcattcatt ttatgtttca 4561 ggttcagggg gaggtgtggg aggtttttta attcgcggcc gcggcgccaa tgcattgggc 4621 ccggtaccca gcttttgttc cctttagtga gggttaattg cgcgcttggc gtaatcatgg 4681 tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc 4741 ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg 4801 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc 4861 ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact 4921 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta 4981 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag 5041 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc 5101 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta 5161 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg 5221 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc 5281 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac 5341 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac 5401 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg 5461 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga 5521 aggacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt 5581 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag 5641 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct 5701 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg 5761 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat 5821 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc 5881 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg 5941 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct 6001 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca 6061 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg 6121 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg 6181 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc 6241 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag 6301 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg 6361 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag 6421 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat 6481 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg 6541 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca 6601 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca 6661 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat 6721 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag 6781 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc taaattgtaa 6841 gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc 6901 aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga 6961 gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag 7021 ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc taatcaagtt 7081 ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta 7141 gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag 7201 cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg 7261 cgcttaatgc gccgctacag ggcgcgtccc attcgccatt caggctgcgc aactgttggg 7321 aagggcgatc ggtgcgggcc tcttcgctat tacgccagtc gaccatagcc aattcaatat 7381 ggcgtatatg gactcatgcc aattcaatat ggtggatctg gacctgtgcc aattcaatat 7441 ggcgtatatg gactcgtgcc aattcaatat ggtggatctg gaccccagcc aattcaatat 7501 ggcggacttg gcaccatgcc aattcaatat ggcggacttg gcactgtgcc aactggggag 7561 gggtctactt ggcacggtgc caagtttgag gaggggtctt ggccctgtgc caagtccgcc 7621 atattgaatt ggcatggtgc caataatggc ggccatattg gctatatgcc aggatcaata 7681 tataggcaat atccaatatg gccctatgcc aatatggcta ttggccaggt tcaatactat 7741 gtattggccc tatgccatat agtattccat atatgggttt tcctattgac gtagatagcc 7801 cctcccaatg ggcggtccca tataccatat atggggcttc ctaataccgc ccatagccac 7861 tcccccattg acgtcaatgg tctctatata tggtctttcc tattgacgtc atatgggcgg 7921 tcctattgac gtatatggcg cctcccccat tgacgtcaat tacggtaaat ggcccgcctg 7981 gctcaatgcc cattgacgtc aataggacca cccaccattg acgtcaatgg gatggctcat 8041 tgcccattca tatccgttct cacgccccct attgacgtca atgacggtaa atggcccact 8101 tggcagtaca tcaatatcta ttaatagtaa cttggcaagt acattactat tggaaggacg 8161 cca //