LOCUS Exported 4354 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Plasmid for liver-specific expression of EmGFP with a BbsI cloning site for a gRNA. ACCESSION . VERSION . KEYWORDS 1530_pAAV-U6-SA-BbsI-MluI-gRNA-HLP-EmGFP-spA SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 4354) AUTHORS Li A, Lee CM, Hurley AE, Jarrett KE, De Giorgi M, Lu W, Balderrama KS, Doerfler AM, Deshmukh H, Ray A, Bao G, Lagor WR TITLE A Self-Deleting AAV-CRISPR System for In Vivo Genome Editing. JOURNAL Mol Ther Methods Clin Dev. 2018 Dec 6;12:111-122. doi: 10.1016/j.omtm.2018.11.009. eCollection 2019 Mar 15. PUBMED 30619914 REFERENCE 2 (bases 1 to 4354) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..4354 /organism="synthetic DNA construct" /mol_type="other DNA" repeat_region 4..133 /label=AAV2 ITR promoter 255..495 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 255..275 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 426..445 /label=LKO.1 5' /note="Human U6 promoter, forward primer" misc_RNA 526..601 /label=Sa gRNA scaffold /note="guide RNA scaffold for the Staphylococcus aureus CRISPR/Cas9 system" regulatory 878..887 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 884..1603 /codon_start=1 /product="Emerald GFP" /label=EmGFP /note="mammalian codon-optimized" /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTFTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHKVYITADKQKNGIK VNFKTRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" primer_bind complement(929..950) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(1190..1209) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 1537..1558 /label=EGFP-C /note="EGFP, forward primer" polyA_signal 1618..1666 /note="synthetic polyadenylation signal" repeat_region 1714..1843 /label=AAV2 ITR primer_bind complement(1909..1926) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(2080..2668) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(2160..2179) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(2839..3699) /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVVMVTTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind 3462..3481 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(3700..3804) /gene="bla" /label=AmpR promoter rep_origin complement(3831..4286) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(3968..3989) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 4180..4199 /label=F1ori-R /note="F1 origin, reverse primer" 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 agaaagcttt gtttgctgct tgcaatgttt gcccatttta gggtggacac 661 aggacgctgt ggtttctgag ccagggggcg actcagatcc cagccagtgg acttagcccc 721 tgtttgctcc tccgataact ggggtgacct tggttaatat tcaccagcag cctcccccgt 781 tgcccctctg gatccactgc ttaaatacgg acgaggacag ggccctgtct cctcagcttc 841 aggcaccacc actgacctgg gacagtgaat caccggtgcc accatggtga gcaagggcga 901 ggagctgttc accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca 961 caagttcagc gtgtccggcg agggcgaggg cgatgccacc tacggcaagc tgaccctgaa 1021 gttcatctgc accaccggca agctgcccgt gccctggccc accctcgtga ccaccttcac 1081 ctacggcgtg cagtgcttcg cccgctaccc cgaccacatg aagcagcacg acttcttcaa 1141 gtccgccatg cccgaaggct acgtccagga gcgcaccatc ttcttcaagg acgacggcaa 1201 ctacaagacc cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct 1261 gaagggcatc gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaacta 1321 caacagccac aaggtctata tcaccgccga caagcagaag aacggcatca aggtgaactt 1381 caagacccgc cacaacatcg aggacggcag cgtgcagctc gccgaccact accagcagaa 1441 cacccccatc ggcgacggcc ccgtgctgct gcccgacaac cactacctga gcacccagtc 1501 cgccctgagc aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac 1561 cgccgccggg atcactctcg gcatggacga gctgtacaag taagaattcc tagagcgaat 1621 aaaagatctt tattttcatt agatctgtgt gttggttttt tgtgtgatgc agctacgtag 1681 ataagtagca tggcgggtta atcattaact acaaggaacc cctagtgatg gagttggcca 1741 ctccctctct gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc 1801 cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg cagctgcatt aatgaatcgg 1861 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga 1921 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat 1981 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaccgcagca 2041 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc 2101 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata 2161 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc 2221 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc 2281 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga 2341 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc 2401 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag 2461 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag 2521 gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag 2581 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca 2641 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga 2701 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat 2761 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga 2821 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg 2881 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga 2941 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc 3001 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac 3061 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc 3121 agttaatagt ttgcgcaacg ttgttaccat tactacaggc atcgtggtgt cacgctcgtc 3181 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc 3241 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt 3301 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc 3361 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg 3421 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag 3481 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat 3541 cttaccacta ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc 3601 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa 3661 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta 3721 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa 3781 aaataaacaa ataggggttc cgcgcacatt tccccgaaag atgccacctg aaattataaa 3841 cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca 3901 ataggccgaa atcggaaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag 3961 tgttgttcca gtttggaaca agagtccact attgaggaac gtgaactcca gcgtcaaagg 4021 gcgaaaaacc gtctatcggg gcgatggccc actacgtgaa ccatcaccct aatcaagttt 4081 tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag 4141 agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc 4201 gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc 4261 gcttaatgcg ccgctacagg gcgcgtccca ttcgccattc aggctgcgca actgttggga 4321 agggcgatcg gtgcgggcct cttcgctatt acgc //