LOCUS       Exported                4595 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  AAV-gRNA targeting the murine Ldlr gene.
ACCESSION   .
VERSION     .
KEYWORDS    1183_pAAV-U6-Ex14-gRNAd-CB-EmGFP
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4595)
  AUTHORS   Jarrett KE, Lee CM, Yeh YH, Hsu RH, Gupta R, Zhang M, Rodriguez PJ, 
            Lee CS, Gillard BK, Bissig KD, Pownall HJ, Martin JF, Bao G, Lagor 
            WR
  TITLE     Somatic genome editing with CRISPR/Cas9 generates and corrects a 
            metabolic disease.
  JOURNAL   Sci Rep. 2017 Mar 16;7:44624. doi: 10.1038/srep44624.
  PUBMED    28300165
REFERENCE   2  (bases 1 to 4595)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..4595
                     /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        524..599
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             1090..1809
                     /codon_start=1
                     /product="Emerald GFP"
                     /label=EmGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHKVYITADKQKNGIK
                     VNFKTRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1135..1156)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1396..1415)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1743..1764
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    1860..1908
                     /note="synthetic polyadenylation signal"
     repeat_region   1956..2085
                     /label=AAV2 ITR
     primer_bind     complement(2151..2168)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2322..2910)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2402..2421)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3081..3941)
                     /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     3704..3723
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3942..4046)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4072..4527)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4209..4230)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4421..4440
                     /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 acctgctgct ggccaaggac atggttttag agctagaaat
      541 agcaagttaa aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtgct
      601 ttttttctag acccagcttt caagcttgat taacccgcca tgctacttat ctacgtagcc
      661 atgctctagg aagatcggaa ttcgccctta agctagtatg ccaagtacgc cccctattga
      721 cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt
      781 tcctacttgg cagtacatct actcgaggcc acgttctgct tcactctccc catctccccc
      841 ccctccccac ccccaatttt gtatttattt attttttaat tattttgtgc agcgatgggg
      901 gcggggcggg gcgaggggcg gggcggggcg aggcggagag gtgcggcggc agccaatcag
      961 agcggcgcgc tccgaaagtt tccttttatg gcgaggcggc ggcggcggcg gccctataaa
     1021 aagcgaagcg cgcggcgggc gggagcggga tcagaatgat ctgatatcat cgatgaattc
     1081 gagctcacca tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc
     1141 gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat
     1201 gccacctacg gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc
     1261 tggcccaccc tcgtgaccac cttcacctac ggcgtgcagt gcttcgcccg ctaccccgac
     1321 cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc
     1381 accatcttct tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc
     1441 gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc
     1501 ctggggcaca agctggagta caactacaac agccacaagg tctatatcac cgccgacaag
     1561 cagaagaacg gcatcaaggt gaacttcaag acccgccaca acatcgagga cggcagcgtg
     1621 cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc
     1681 gacaaccact acctgagcac ccagtccgcc ctgagcaaag accccaacga gaagcgcgat
     1741 cacatggtcc tgctggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg
     1801 tacaagtaag aattctgcag tcgacgcggg cccgggatcc accggtgcgg ccgctcgcga
     1861 ataaaagatc tttattttca ttagatctgt gtgttggttt tttgtgtgat gcagctacgt
     1921 agataagtag catggcgggt taatcattaa ctacaaggaa cccctagtga tggagttggc
     1981 cactccctct ctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg
     2041 cccgggcttt gcccgggcgg cctcagtgag cgagcgagcg cgcagctgca ttaatgaatc
     2101 ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact
     2161 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
     2221 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
     2281 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     2341 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     2401 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     2461 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     2521 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     2581 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     2641 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     2701 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     2761 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     2821 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     2881 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     2941 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
     3001 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     3061 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     3121 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     3181 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
     3241 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     3301 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     3361 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
     3421 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     3481 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     3541 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     3601 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     3661 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
     3721 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
     3781 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
     3841 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
     3901 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
     3961 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     4021 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc taaattgtaa
     4081 gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc
     4141 aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga
     4201 gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag
     4261 ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc taatcaagtt
     4321 ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta
     4381 gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag
     4441 cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg
     4501 cgcttaatgc gccgctacag ggcgcgtccc attcgccatt caggctgcgc aactgttggg
     4561 aagggcgatc ggtgcgggcc tcttcgctat tacgc
//
