LOCUS       Exported                4353 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  Plasmid for liver-specific expression of EmGFP with a gRNA against 
            SaCas9.
ACCESSION   .
VERSION     .
KEYWORDS    1531_pAAV-U6-SA-Self1-gRNA-HLP-EmGFP-spA
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4353)
  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 4353)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 13, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..4353
                     /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        525..600
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     regulatory      877..886
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             883..1602
                     /codon_start=1
                     /product="Emerald GFP"
                     /label=EmGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHKVYITADKQKNGIK
                     VNFKTRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(928..949)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1189..1208)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1536..1557
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    1617..1665
                     /note="synthetic polyadenylation signal"
     repeat_region   1713..1842
                     /label=AAV2 ITR
     primer_bind     complement(1908..1925)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2079..2667)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2159..2178)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2838..3698)
                     /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     3461..3480
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3699..3803)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(3830..4285)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3967..3988)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4179..4198
                     /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 accggtgatg ccgatgtcca ggccgtttta gtactctgga
      541 aacagaatct actaaaacaa ggcaaaatgc cgtgtttatc tcgtcaactt gttggcgaga
      601 tttttttcta gaaagctttg tttgctgctt gcaatgtttg cccattttag ggtggacaca
      661 ggacgctgtg gtttctgagc cagggggcga ctcagatccc agccagtgga cttagcccct
      721 gtttgctcct ccgataactg gggtgacctt ggttaatatt caccagcagc ctcccccgtt
      781 gcccctctgg atccactgct taaatacgga cgaggacagg gccctgtctc ctcagcttca
      841 ggcaccacca ctgacctggg acagtgaatc accggtgcca ccatggtgag caagggcgag
      901 gagctgttca ccggggtggt gcccatcctg gtcgagctgg acggcgacgt aaacggccac
      961 aagttcagcg tgtccggcga gggcgagggc gatgccacct acggcaagct gaccctgaag
     1021 ttcatctgca ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac caccttcacc
     1081 tacggcgtgc agtgcttcgc ccgctacccc gaccacatga agcagcacga cttcttcaag
     1141 tccgccatgc ccgaaggcta cgtccaggag cgcaccatct tcttcaagga cgacggcaac
     1201 tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg catcgagctg
     1261 aagggcatcg acttcaagga ggacggcaac atcctggggc acaagctgga gtacaactac
     1321 aacagccaca aggtctatat caccgccgac aagcagaaga acggcatcaa ggtgaacttc
     1381 aagacccgcc acaacatcga ggacggcagc gtgcagctcg ccgaccacta ccagcagaac
     1441 acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag cacccagtcc
     1501 gccctgagca aagaccccaa cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc
     1561 gccgccggga tcactctcgg catggacgag ctgtacaagt aagaattcct agagcgaata
     1621 aaagatcttt attttcatta gatctgtgtg ttggtttttt gtgtgatgca gctacgtaga
     1681 taagtagcat ggcgggttaa tcattaacta caaggaaccc ctagtgatgg agttggccac
     1741 tccctctctg cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg cccgacgccc
     1801 gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc agctgcatta atgaatcggc
     1861 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
     1921 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     1981 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa accgcagcaa
     2041 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     2101 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     2161 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     2221 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     2281 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     2341 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     2401 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     2461 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg
     2521 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     2581 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     2641 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     2701 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     2761 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     2821 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     2881 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     2941 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     3001 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     3061 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     3121 gttaatagtt tgcgcaacgt tgttaccatt actacaggca tcgtggtgtc acgctcgtcg
     3181 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     3241 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     3301 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     3361 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     3421 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     3481 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     3541 ttaccactat tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     3601 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     3661 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     3721 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     3781 aataaacaaa taggggttcc gcgcacattt ccccgaaaga tgccacctga aattataaac
     3841 gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa
     3901 taggccgaaa tcggaaaaat cccttataaa tcaaaagaat agaccgagat agggttgagt
     3961 gttgttccag tttggaacaa gagtccacta ttgaggaacg tgaactccag cgtcaaaggg
     4021 cgaaaaaccg tctatcgggg cgatggccca ctacgtgaac catcacccta atcaagtttt
     4081 ttggggtcga ggtgccgtaa agcactaaat cggaacccta aagggagccc ccgatttaga
     4141 gcttgacggg gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg
     4201 ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg taaccaccac acccgccgcg
     4261 cttaatgcgc cgctacaggg cgcgtcccat tcgccattca ggctgcgcaa ctgttgggaa
     4321 gggcgatcgg tgcgggcctc ttcgctatta cgc
//
