LOCUS Exported 7165 bp ds-DNA circular SYN 20-JUN-2018 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS 1255_pAAV-U6-SA-BbsI-MluI-gRNA-CB-SACas9-HA-OLLAS-spA SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7165) AUTHORS . TITLE Direct Submission JOURNAL Exported Wednesday, Jun 20, 2018 from SnapGene 2.8.3 http://www.snapgene.com COMMENT Component Fragments #1: FRAGMENT of pZAC2.1rhEPO.36.Q-L parent position: from 1309 to 239 original length: 3894 molecule position: from 1 to 3894 Left Terminus XhoI site #1 Right Terminus BglII site #1 #2: FRAGMENT of pAAVNEWTBG-BI-aGAL parent position: from 5114 to 316 original length: 896 molecule position: from 3895 to 4790 Left Terminus BamHI site #1 Right Terminus XhoI site #1 http://www.informaxinc.com/ FEATURES Location/Qualifiers source 1..7165 /organism="synthetic DNA construct" /mol_type="other DNA" misc_feature 1..185 /label=ITR /note="5'ITR" misc_feature complement(1..173) /label=ITR /note="3'ITR" promoter 255..495 /label=U6 Promoter /note="U6 promoter" /note="RNA polymerase III promoter for human U6 snRNA" misc_feature 276..310 /note="U6ForSeq Primer" misc_feature 504..525 /note="NEW CLONING_SITE_FOR_GRNA" misc_feature 524..525 /note="nt leftover from previous site" misc_feature 526..606 /note="sgRNA scaffold" misc_feature 526..539 /note="crRNA Direct Repeat Portion +1 to +14" misc_feature 540..543 /note="synthetic tetraloop" misc_feature 544..606 /note="TracrRNA" misc_feature 607 /note="Additional ""T"" introduced in gblock" misc_feature 608..629 /note="Cloning Site for Additional Guide RNA cassettes" misc_feature 630..1078 /note="Promoter cassette from pAAV-CB-EGFP" enhancer 685..810 /note="Partial CMV enhancer" misc_feature complement(698..719) /note="CBP Rev3 Primer" promoter 811..1031 /note="CB promoter" misc_feature 905..906 /note="5 bp deletion between these two bases" TATA_signal 1007..1014 /note="TATA" primer_bind complement(1053..1079) /note="CLONE_CB_R_NcoI" CDS 1088..1108 /codon_start=1 /product="nuclear localization signal of SV40 large T antigen" /note="SV40 NLS" /translation="PKKKRKV" misc_feature 1133..4288 /note="hSaCas9" misc_feature 1532..1534 /note="Ub high (AAA = WT, AGA = deUb)" misc_feature 1532..1534 /note="K151R" misc_feature 3335..3337 /note="K752R" CDS 4289..4336 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /note="nucleoplasmin NLS" /translation="KRPAATKKAGQAKKKK" CDS 4343..4369 /codon_start=1 /product="HA (human influenza hemagglutinin) epitope tag" /note="HA" /translation="YPYDVPDYA" misc_feature 4370..4411 /note="OLLAS tag" polyA_signal 4427..4484 /note="Syn PolyA" misc_feature 4485..4657 /label=ITR /note="3'ITR" misc_feature complement(4682..7097) /label=pEMBL8 Plasmid Backbone /note="pEMBL8 Plasmid Backbone" rep_origin complement(4891..5479) /direction=LEFT /note="ori" /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" CDS complement(5650..6510) /codon_start=1 /gene="bla" /product="beta-lactamase" /note="AmpR" /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVVMVTTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" promoter complement(6511..6615) /gene="bla" /note="AmpR promoter" rep_origin complement(6642..7097) /direction=LEFT /note="f1 ori" /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" ORIGIN 1 cagctgcgcg ctcgctcgct cactgaggcc gcccgggcaa agcccgggcg tcgggcgacc 61 tttggtcgcc cggcctcagt gagcgagcga gcgcgcagag agggagtggc caactccatc 121 actaggggtt ccttgtagtt aatgattaac ccgccatgct acttatctac gtagccatgc 181 tctggtgtac aaaaaagcag gctttaaagg aaccaattca gtcgactgga tccggtacca 241 aggtcgggca ggaagagggc ctatttccca tgattccttc atatttgcat atacgataca 301 aggctgttag agagataatt agaattaatt tgactgtaaa cacaaagata ttagtacaaa 361 atacgtgacg tagaaagtaa taatttcttg ggtagtttgc agttttaaaa ttatgtttta 421 aaatggacta tcatatgctt accgtaactt gaaagtattt cgatttcttg gctttatata 481 tcttgtggaa aggacgaaac accgggtctt cacgcgtgaa gaccagtttt agtactctgg 541 aaacagaatc tactaaaaca aggcaaaatg ccgtgtttat ctcgtcaact tgttggcgag 601 atttttttct agacccagct ttcaagcttg attaacccgc catgctactt atctacgtag 661 ccatgctcta ggaagatcgg aattcgccct taagctagta tgccaagtac gccccctatt 721 gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac cttatgggac 781 tttcctactt ggcagtacat ctactcgagg ccacgttctg cttcactctc cccatctccc 841 ccccctcccc acccccaatt ttgtatttat ttatttttta attattttgt gcagcgatgg 901 gggcgagggg cggggcgggg cgaggcggag aggtgcggcg gcagccaatc agagcggcgc 961 gctccgaaag tttcctttta tggcgaggcg gcggcggcgg cggccctata aaaagcgaag 1021 cgcgcggcgg gcgggagcgg gatcagaatg atctgatatc atcgatgaat tcgagctcac 1081 catggcccca aagaagaagc ggaaggtcgg tatccacgga gtcccagcag ccaagcggaa 1141 ctacatcctg ggcctggaca tcggcatcac cagcgtgggc tacggcatca tcgactacga 1201 gacacgggac gtgatcgatg ccggcgtgcg gctgttcaaa gaggccaacg tggaaaacaa 1261 cgagggcagg cggagcaaga gaggcgccag aaggctgaag cggcggaggc ggcatagaat 1321 ccagagagtg aagaagctgc tgttcgacta caacctgctg accgaccaca gcgagctgag 1381 cggcatcaac ccctacgagg ccagagtgaa gggcctgagc cagaagctga gcgaggaaga 1441 gttctctgcc gccctgctgc acctggccaa gagaagaggc gtgcacaacg tgaacgaggt 1501 ggaagaggac accggcaacg agctgtccac cagagagcag atcagccgga acagcaaggc 1561 cctggaagag aaatacgtgg ccgaactgca gctggaacgg ctgaagaaag acggcgaagt 1621 gcggggcagc atcaacagat tcaagaccag cgactacgtg aaagaagcca aacagctgct 1681 gaaggtgcag aaggcctacc accagctgga ccagagcttc atcgacacct acatcgacct 1741 gctggaaacc cggcggacct actatgaggg acctggcgag ggcagcccct tcggctggaa 1801 ggacatcaaa gaatggtacg agatgctgat gggccactgc acctacttcc ccgaggaact 1861 gcggagcgtg aagtacgcct acaacgccga cctgtacaac gccctgaacg acctgaacaa 1921 tctcgtgatc accagggacg agaacgagaa gctggaatat tacgagaagt tccagatcat 1981 cgagaacgtg ttcaagcaga agaagaagcc caccctgaag cagatcgcca aagaaatcct 2041 cgtgaacgaa gaggatatta agggctacag agtgaccagc accggcaagc ccgagttcac 2101 caacctgaag gtgtaccacg acatcaagga cattaccgcc cggaaagaga ttattgagaa 2161 cgccgagctg ctggatcaga ttgccaagat cctgaccatc taccagagca gcgaggacat 2221 ccaggaagaa ctgaccaatc tgaactccga gctgacccag gaagagatcg agcagatctc 2281 taatctgaag ggctataccg gcacccacaa cctgagcctg aaggccatca acctgatcct 2341 ggacgagctg tggcacacca acgacaacca gatcgctatc ttcaaccggc tgaagctggt 2401 gcccaagaag gtggacctgt cccagcagaa agagatcccc accaccctgg tggacgactt 2461 catcctgagc cccgtcgtga agagaagctt catccagagc atcaaagtga tcaacgccat 2521 catcaagaag tacggcctgc ccaacgacat cattatcgag ctggcccgcg agaagaactc 2581 caaggacgcc cagaaaatga tcaacgagat gcagaagcgg aaccggcaga ccaacgagcg 2641 gatcgaggaa atcatccgga ccaccggcaa agagaacgcc aagtacctga tcgagaagat 2701 caagctgcac gacatgcagg aaggcaagtg cctgtacagc ctggaagcca tccctctgga 2761 agatctgctg aacaacccct tcaactatga ggtggaccac atcatcccca gaagcgtgtc 2821 cttcgacaac agcttcaaca acaaggtgct cgtgaagcag gaagaaaaca gcaagaaggg 2881 caaccggacc ccattccagt acctgagcag cagcgacagc aagatcagct acgaaacctt 2941 caagaagcac atcctgaatc tggccaaggg caagggcaga atcagcaaga ccaagaaaga 3001 gtatctgctg gaagaacggg acatcaacag gttctccgtg cagaaagact tcatcaaccg 3061 gaacctggtg gataccagat acgccaccag aggcctgatg aacctgctgc ggagctactt 3121 cagagtgaac aacctggacg tgaaagtgaa gtccatcaat ggcggcttca ccagctttct 3181 gcggcggaag tggaagttta agaaagagcg gaacaagggg tacaagcacc acgccgagga 3241 cgccctgatc attgccaacg ccgatttcat cttcaaagag tggaagaaac tggacaaggc 3301 caaaaaagtg atggaaaacc agatgttcga ggaaaggcag gccgagagca tgcccgagat 3361 cgaaaccgag caggagtaca aagagatctt catcaccccc caccagatca agcacattaa 3421 ggacttcaag gactacaagt acagccaccg ggtggacaag aagcctaata gagagctgat 3481 taacgacacc ctgtactcca cccggaagga cgacaagggc aacaccctga tcgtgaacaa 3541 tctgaacggc ctgtacgaca aggacaatga caagctgaaa aagctgatca acaagagccc 3601 cgaaaagctg ctgatgtacc accacgaccc ccagacctac cagaaactga agctgattat 3661 ggaacagtac ggcgacgaga agaatcccct gtacaagtac tacgaggaaa ccgggaacta 3721 cctgaccaag tactccaaaa aggacaacgg ccccgtgatc aagaagatta agtattacgg 3781 caacaaactg aacgcccatc tggacatcac cgacgactac cccaacagca gaaacaaggt 3841 cgtgaagctg tccctgaagc cctacagatt cgacgtgtac ctggacaatg gcgtgtacaa 3901 gttcgtgacc gtgaagaatc tggatgtgat caaaaaagaa aactactacg aagtgaatag 3961 caagtgctat gaggaagcta agaagctgaa gaagatcagc aaccaggccg agtttatcgc 4021 ctccttctac aacaacgatc tgatcaagat caacggcgag ctgtatagag tgatcggcgt 4081 gaacaacgac ctgctgaacc ggatcgaagt gaacatgatc gacatcacct accgcgagta 4141 cctggaaaac atgaacgaca agaggccccc caggatcatt aagacaatcg cctccaagac 4201 ccagagcatt aagaagtaca gcacagacat tctgggcaac ctgtatgaag tgaaatctaa 4261 gaagcaccct cagatcatca aaaagggcaa aaggccggcg gccacgaaaa aggccggcca 4321 ggcaaaaaag aaaaagggat cctacccata cgatgttcca gattacgcta gcggcttcgc 4381 caacgagctt ggacccaggt tgatgggaaa gtaagaattc ctagagcgaa taaaagatct 4441 ttattttcat tagatctgtg tgttggtttt ttgtgtgatg cagctacgta gataagtagc 4501 atggcgggtt aatcattaac tacaaggaac ccctagtgat ggagttggcc actccctctc 4561 tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc ccgggctttg 4621 cccgggcggc ctcagtgagc gagcgagcgc gcagctgcat taatgaatcg gccaacgcgc 4681 ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg 4741 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc 4801 cacagaatca ggggataacg caggaaagaa catgtgagca aaaccgcagc aaaaggccag 4861 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca 4921 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca 4981 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg 5041 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag 5101 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt 5161 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca 5221 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg 5281 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt 5341 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc 5401 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg 5461 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg 5521 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta 5581 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg 5641 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg 5701 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc 5761 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc 5821 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc 5881 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag 5941 tttgcgcaac gttgttacca ttactacagg catcgtggtg tcacgctcgt cgtttggtat 6001 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg 6061 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt 6121 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag 6181 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg 6241 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt 6301 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccact 6361 attgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac 6421 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat 6481 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat 6541 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca 6601 aataggggtt ccgcgcacat ttccccgaaa gatgccacct gaaattataa acgttaatat 6661 tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga 6721 aatcggaaaa atcccttata aatcaaaaga atagaccgag atagggttga gtgttgttcc 6781 agtttggaac aagagtccac tattgaggaa cgtgaactcc agcgtcaaag ggcgaaaaac 6841 cgtctatcgg ggcgatggcc cactacgtga accatcaccc taatcaagtt ttttggggtc 6901 gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg 6961 gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctag 7021 ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc 7081 gccgctacag ggcgcgtccc attcgccatt caggctgcgc aactgttggg aagggcgatc 7141 ggtgcgggcc tcttcgctat tacgc //