LOCUS Exported 5211 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION AAV vector expressing non-targeting AsCpf1 crRNA. ACCESSION . VERSION . KEYWORDS pU6-crRNA(Non-targeting) SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5211) AUTHORS Tekin, Halil, Sean Simmons, Beryl Cummings, Linyi Gao, Xian Adiconis, Cynthia C. Hession, Ayan Ghoshal, Danielle Dionne, Sourav R. Choudhury, Volkan Yesilyurt, Neville E. Sanjana, Xi Shi, Congyi TITLE Effects of 3D culturing conditions on the transcriptomic profile of stem-cell-derived neurons JOURNAL Nature Biomedical Engineering (Epub 09 April 2018) REFERENCE 2 (bases 1 to 5211) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5211 /organism="synthetic DNA construct" /mol_type="other DNA" repeat_region 1..141 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 12..141 /label=AAV2 ITR promoter 158..398 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 158..178 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 329..348 /label=LKO.1 5' /note="Human U6 promoter, forward primer" promoter 494..941 /label=hSyn promoter /note="human synapsin I promoter; confers neuron-specific expression (Kugler et al., 2003)" CDS 979..1683 /codon_start=1 /product="monomeric derivative of DsRed fluorescent protein (Shaner et al., 2004)" /label=mCherry /note="mammalian codon-optimized" /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE GRHSTGGMDELYK" primer_bind complement(1121..1139) /label=mCherry-R /note="mCherry, reverse primer" primer_bind 1389..1408 /label=mCherry-F /note="mCherry, forward primer" polyA_signal 2000..2476 /label=hGH poly(A) signal /note="human growth hormone polyadenylation signal" primer_bind complement(2199..2218) /label=hGH-PA-R /note="Human growth hormone terminator, reverse primer" repeat_region 2485..2614 /label=AAV2 ITR (alternate) /note="Functional equivalent of wild-type AAV2 ITR" rep_origin complement(2676..3264) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(2756..2775) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(3435..4295) /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 4058..4077 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(4296..4400) /gene="bla" /label=AmpR promoter primer_bind 4468..4486 /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" primer_bind complement(4524..4546) /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind 4646..4665 /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" rep_origin complement(4682..5137) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(4819..4840) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 5031..5050 /label=F1ori-R /note="F1 origin, reverse primer" ORIGIN 1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc 61 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca 121 actccatcac taggggttcc tgcggccgcg ctagcctgag ggcctatttc ccatgattcc 181 ttcatatttg catatacgat acaaggctgt tagagagata attggaatta atttgactgt 241 aaacacaaag atattagtac aaaatacgtg acgtagaaag taataatttc ttgggtagtt 301 tgcagtttta aaattatgtt ttaaaatgga ctatcatatg cttaccgtaa cttgaaagta 361 tttcgatttc ttggctttat atatcttgtg gaaaggacga aacaccgtaa tttctactct 421 tgtagattat ctgtgtagcc ggtgtggtag tttttcgacg cgtgtgtcta gactgcagag 481 ggccctgcgt atgagtgcaa gtgggtttta ggaccaggat gaggcggggt gggggtgcct 541 acctgacgac cgaccccgac ccactggaca agcacccaac ccccattccc caaattgcgc 601 atcccctatc agagaggggg aggggaaaca ggatgcggcg aggcgcgtgc gcactgccag 661 cttcagcacc gcggacagtg ccttcgcccc cgcctggcgg cgcgcgccac cgccgcctca 721 gcactgaagg cgcgctgacg tcactcgccg gtcccccgca aactcccctt cccggccacc 781 ttggtcgcgt ccgcgccgcc gccggcccag ccggaccgca ccacgcgagg cgcgagatag 841 gggggcacgg gcgcgaccat ctgcgctgcg gcgccggcga ctcagcgctg cctcagtctg 901 cggtgggcag cggaggagtc gtgtcgtgcc tgagagcgca gtcgagaagg taccggatcc 961 tctagagtcg acgccaccgt gagcaagggc gaggaggata acatggccat catcaaggag 1021 ttcatgcgct tcaaggtgca catggagggc tccgtgaacg gccacgagtt cgagatcgag 1081 ggcgagggcg agggccgccc ctacgagggc acccagaccg ccaagctgaa ggtgaccaag 1141 ggtggccccc tgcccttcgc ctgggacatc ctgtcccctc agttcatgta cggctccaag 1201 gcctacgtga agcaccccgc cgacatcccc gactacttga agctgtcctt ccccgagggc 1261 ttcaagtggg agcgcgtgat gaacttcgag gacggcggcg tggtgaccgt gacccaggac 1321 tcctccctgc aggacggcga gttcatctac aaggtgaagc tgcgcggcac caacttcccc 1381 tccgacggcc ccgtaatgca gaagaagacc atgggctggg aggcctcctc cgagcggatg 1441 taccccgagg acggcgccct gaagggcgag atcaagcaga ggctgaagct gaaggacggc 1501 ggccactacg acgctgaggt caagaccacc tacaaggcca agaagcccgt gcagctgccc 1561 ggcgcctaca acgtcaacat caagttggac atcacctccc acaacgagga ctacaccatc 1621 gtggaacagt acgaacgcgc cgagggccgc cactccaccg gcggcatgga cgagctgtac 1681 aagtccggac tcagatctcg agaggaggag gaggagacag acagcaggat gccccacctc 1741 gacagccccg gcagctccca gccgagacgc tccttcctct caagggtgat cagggcagcg 1801 ctaccgttgc agctgcttct gctgctgctg ctgctcctgg cctgcctgct acctgcctct 1861 gaagatgact acagctgcac ccaggccaac aactttgccc gatccttcta ccccatgctg 1921 cggtacacca acgggccacc tcccacctag gaattcgata tcaagcttat cgataccgag 1981 cgctgctcga gagatctacg ggtggcatcc ctgtgacccc tccccagtgc ctctcctggc 2041 cctggaagtt gccactccag tgcccaccag ccttgtccta ataaaattaa gttgcatcat 2101 tttgtctgac taggtgtcct tctataatat tatggggtgg aggggggtgg tatggagcaa 2161 ggggcaagtt gggaagacaa cctgtagggc ctgcggggtc tattgggaac caagctggag 2221 tgcagtggca caatcttggc tcactgcaat ctccgcctcc tgggttcaag cgattctcct 2281 gcctcagcct cccgagttgt tgggattcca ggcatgcatg accaggctca gctaattttt 2341 gtttttttgg tagagacggg gtttcaccat attggccagg ctggtctcca actcctaatc 2401 tcaggtgatc tacccacctt ggcctcccaa attgctggga ttacaggcgt gaaccactgc 2461 tcccttccct gtccttgcgg ccgcaggaac ccctagtgat ggagttggcc actccctctc 2521 tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc ccgggcggcc 2581 tcagtgagcg agcgagcgcg cagctgcctg caggacatgt gagcaaaagg ccagcaaaag 2641 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac 2701 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga 2761 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt 2821 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc 2881 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc 2941 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta 3001 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat 3061 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca 3121 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct 3181 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt 3241 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct 3301 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc 3361 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa 3421 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta 3481 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc 3541 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat 3601 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta 3661 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt 3721 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt 3781 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg 3841 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc 3901 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc 3961 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg 4021 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga 4081 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta 4141 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct 4201 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag 4261 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga 4321 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat 4381 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc 4441 attattatca tgacattaac ctataaaaat aggcgtatca cgaggccctt tcgtctcgcg 4501 cgtttcggtg atgacggtga aaacctctga cacatgcagc tcccggagac ggtcacagct 4561 tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc 4621 gggtgtcggg gctggcttaa ctatgcggca tcagagcaga ttgtactgag agtgcaccat 4681 aaaattgtaa acgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca 4741 ttttttaacc aatagaccga aatcggcaaa atcccttata aatcaaaaga atagcccgag 4801 atagagttga gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc 4861 aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc 4921 aaatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc 4981 ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa 5041 gcgaaaggag cgggcgctaa ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc 5101 acacccgccg cgcttaatgc gccgctacag ggcgcgtact atggttgctt tgacgtatgc 5161 ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg catcaggcgc c //