LOCUS       Exported                4353 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 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 12, 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        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
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     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(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 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 aaggccagca
     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 aacagtattt 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 ttgttgccat tgctacaggc 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 cttaccgctg 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 tccccgaaaa gtgccaccta aattgtaagc
     3841 gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa
     3901 taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agaccgagat agggttgagt
     3961 gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa cgtcaaaggg
     4021 cgaaaaaccg tctatcaggg 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
//
