LOCUS       pAAV-u6-gRNA(DRD1.2)-hS 5282 bp ds-DNA     circular     27-NOV-2024
DEFINITION  sgRNA expressing AAV construct with a mCherry reporter driven by
            hSyn promoter. (replaced the GFP in pX552 from Zhang lab with
            mCherry).
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
COMMENT     Imported using the Genbank importer. File name:
            addgene-plasmid-87916-sequence-174844.gbk SGRef: number: 1; type:
            "Journal Article"; doi: "10.1167/iovs"; journalName: "Invest
            Ophthalmol Vis Sci"; date: "2016-06-1- 1"; volume: "57"; issue:
            "7"; pages: "3470-6"
FEATURES             Location/Qualifiers
     source          1..5282
                     /label="source"
                     /ApEinfo_revcolor="#c6c9d1"
                     /ApEinfo_fwdcolor="#c6c9d1"
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..21
                     /label="hU6-F"
                     /ApEinfo_revcolor="#d59687"
                     /ApEinfo_fwdcolor="#d59687"
                     /note="Human U6 promoter, forward primer"
     promoter        1..249
                     /label="U6 promoter"
                     /ApEinfo_revcolor="#b7e6d7"
                     /ApEinfo_fwdcolor="#b7e6d7"
                     /note="RNA polymerase III promoter for human U6 snRNA (Domitrovich & Kunkel, 2003)"
     primer_bind     172..191
                     /label="LKO.1 5'"
                     /ApEinfo_revcolor="#d59687"
                     /ApEinfo_fwdcolor="#d59687"
                     /note="Human U6 promoter, forward primer"
     misc_RNA        270..345
                     /label="gRNA scaffold"
                     /ApEinfo_revcolor="#f58a5e"
                     /ApEinfo_fwdcolor="#f58a5e"
                     /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system"
     promoter        379..826
                     /label="hSyn promoter"
                     /ApEinfo_revcolor="#ffef86"
                     /ApEinfo_fwdcolor="#ffef86"
                     /note="human synapsin I promoter; confers neuron-specific expression (Kugler et al., 2003)"
     CDS             858..1568
                     /label="mCherry"
                     /ApEinfo_revcolor="#84b0dc"
                     /ApEinfo_fwdcolor="#84b0dc"
                     /note="mammalian codon-optimized"
                     /product="monomeric derivative of DsRed fluorescent protein(Shaner et al., 2004)"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYK*"
     primer_bind     complement(1003..1021)
                     /label="mCherry-R"
                     /ApEinfo_revcolor="#f58a5e"
                     /ApEinfo_fwdcolor="#f58a5e"
                     /note="mCherry, reverse primer"
     primer_bind     1271..1290
                     /label="mCherry-F"
                     /ApEinfo_revcolor="#84b0dc"
                     /ApEinfo_fwdcolor="#84b0dc"
                     /note="mCherry, forward primer"
     polyA_signal    1885..2361
                     /label="hGH poly(A) signal"
                     /ApEinfo_revcolor="#b4abac"
                     /ApEinfo_fwdcolor="#b4abac"
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(2084..2103)
                     /label="hGH-PA-R"
                     /ApEinfo_revcolor="#9eafd2"
                     /ApEinfo_fwdcolor="#9eafd2"
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   2401..2530
                     /label="AAV2 ITR"
                     /ApEinfo_revcolor="#9eafd2"
                     /ApEinfo_fwdcolor="#9eafd2"
     repeat_region   2401..2541
                     /label="AAV2 ITR"
                     /ApEinfo_revcolor="#f58a5e"
                     /ApEinfo_fwdcolor="#f58a5e"
                     /note="inverted terminal repeat of adeno-associated virus serotype 2"
     rep_origin      2616..3071
                     /label="f1 ori"
                     /ApEinfo_revcolor="#ff9ccd"
                     /ApEinfo_fwdcolor="#ff9ccd"
                     /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis"
                     /direction="RIGHT"
     primer_bind     complement(2703..2722)
                     /label="F1ori-R"
                     /ApEinfo_revcolor="#b7e6d7"
                     /ApEinfo_fwdcolor="#b7e6d7"
                     /note="F1 origin, reverse primer"
     primer_bind     2913..2934
                     /label="F1ori-F"
                     /ApEinfo_revcolor="#85dae9"
                     /ApEinfo_fwdcolor="#85dae9"
                     /note="F1 origin, forward primer"
     primer_bind     complement(3088..3107)
                     /label="pRS-marker"
                     /ApEinfo_revcolor="#faac61"
                     /ApEinfo_fwdcolor="#faac61"
                     /note="pRS vectors, use to sequence yeast selectable marker"
     primer_bind     3207..3229
                     /label="pGEX 3'"
                     /ApEinfo_revcolor="#84b0dc"
                     /ApEinfo_fwdcolor="#84b0dc"
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3267..3285)
                     /label="pBRforEco"
                     /ApEinfo_revcolor="#b7e6d7"
                     /ApEinfo_fwdcolor="#b7e6d7"
                     /note="pBR322 vectors, upsteam of EcoRI site, forward primer"
     promoter        3353..3457
                     /label="AmpR promoter"
                     /ApEinfo_revcolor="#f8d3a9"
                     /ApEinfo_fwdcolor="#f8d3a9"
                     /gene="bla"
     CDS             3458..4318
                     /label="AmpR"
                     /ApEinfo_revcolor="#84b0dc"
                     /ApEinfo_fwdcolor="#84b0dc"
                     /note="confers resistance to ampicillin, carbenicillin, andrelated antibiotics"
                     /gene="bla"
                     /product="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYIELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRWEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGASLIKHW*"
     primer_bind     complement(3676..3695)
                     /label="Amp-R"
                     /ApEinfo_revcolor="#b4abac"
                     /ApEinfo_fwdcolor="#b4abac"
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4489..5077
                     /label="ori"
                     /ApEinfo_revcolor="#b7e6d7"
                     /ApEinfo_fwdcolor="#b7e6d7"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication"
                     /direction="RIGHT"
     primer_bind     4978..4997
                     /label="pBR322ori-F"
                     /ApEinfo_revcolor="#85dae9"
                     /ApEinfo_fwdcolor="#85dae9"
                     /note="pBR322 origin, forward primer"
     repeat_region   5139..5268
                     /label="AAV2 ITR (alternate)"
                     /ApEinfo_revcolor="#c6c9d1"
                     /ApEinfo_fwdcolor="#c6c9d1"
                     /note="Functional equivalent of wild-type AAV2 ITR"
ORIGIN
        1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
       61 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
      121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
      181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
      241 cgaaacaccc tgggcaatcc tgtagatacg ttttagagct agaaatagca agttaaaata
      301 aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt ttctagactg
      361 cagagggccc tgcgtatgag tgcaagtggg ttttaggacc aggatgaggc ggggtggggg
      421 tgcctacctg acgaccgacc ccgacccact ggacaagcac ccaaccccca ttccccaaat
      481 tgcgcatccc ctatcagaga gggggagggg aaacaggatg cggcgaggcg cgtgcgcact
      541 gccagcttca gcaccgcgga cagtgccttc gcccccgcct ggcggcgcgc gccaccgccg
      601 cctcagcact gaaggcgcgc tgacgtcact cgccggtccc ccgcaaactc cccttcccgg
      661 ccaccttggt cgcgtccgcg ccgccgccgg cccagccgga ccgcaccacg cgaggcgcga
      721 gatagggggg cacgggcgcg accatctgcg ctgcggcgcc ggcgactcag cgctgcctca
      781 gtctgcggtg ggcagcggag gagtcgtgtc gtgcctgaga gcgcagtcga gaaggtaccg
      841 gatcctctag agtcgacatg gtgagcaagg gcgaggagga taacatggcc atcatcaagg
      901 agttcatgcg cttcaaggtg cacatggagg gctccgtgaa cggccacgag ttcgagatcg
      961 agggcgaggg cgagggccgc ccctacgagg gcacccagac cgccaagctg aaggtgacca
     1021 agggtggccc cctgcccttc gcctgggaca tcctgtcccc tcagttcatg tacggctcca
     1081 aggcctacgt gaagcacccc gccgacatcc ccgactactt gaagctgtcc ttccccgagg
     1141 gcttcaagtg ggagcgcgtg atgaacttcg aggacggcgg cgtggtgacc gtgacccagg
     1201 actcctccct gcaggacggc gagttcatct acaaggtgaa gctgcgcggc accaacttcc
     1261 cctccgacgg ccccgtaatg cagaagaaga ccatgggctg ggaggcctcc tccgagcgga
     1321 tgtaccccga ggacggcgcc ctgaagggcg agatcaagca gaggctgaag ctgaaggacg
     1381 gcggccacta cgacgctgag gtcaagacca cctacaaggc caagaagccc gtgcagctgc
     1441 ccggcgccta caacgtcaac atcaagttgg acatcacctc ccacaacgag gactacacca
     1501 tcgtggaaca gtacgaacgc gccgagggcc gccactccac cggcggcatg gacgagctgt
     1561 acaagtagtc cggactcaga tctcgagagg aggaggagga gacagacagc aggatgcccc
     1621 acctcgacag ccccggcagc tcccagccga gacgctcctt cctctcaagg gtgatcaggg
     1681 cagcgctacc gttgcagctg cttctgctgc tgctgctgct cctggcctgc ctgctacctg
     1741 cctctgaaga tgactacagc tgcacccagg ccaacaactt tgcccgatcc ttctacccca
     1801 tgctgcggta caccaacggg ccacctccca cctaggaatt cgatatcaag cttatcgata
     1861 ccgagcgctg ctcgagagat ctacgggtgg catccctgtg acccctcccc agtgcctctc
     1921 ctggccctgg aagttgccac tccagtgccc accagccttg tcctaataaa attaagttgc
     1981 atcattttgt ctgactaggt gtccttctat aatattatgg ggtggagggg ggtggtatgg
     2041 agcaaggggc aagttgggaa gacaacctgt agggcctgcg gggtctattg ggaaccaagc
     2101 tggagtgcag tggcacaatc ttggctcact gcaatctccg cctcctgggt tcaagcgatt
     2161 ctcctgcctc agcctcccga gttgttggga ttccaggcat gcatgaccag gctcagctaa
     2221 tttttgtttt tttggtagag acggggtttc accatattgg ccaggctggt ctccaactcc
     2281 taatctcagg tgatctaccc accttggcct cccaaattgc tgggattaca ggcgtgaacc
     2341 actgctccct tccctgtcct tctgattttg taggtaacca cgtgcggacc gagcggccgc
     2401 aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg
     2461 ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc
     2521 gagcgcgcag ctgcctgcag gggcgcctga tgcggtattt tctccttacg catctgtgcg
     2581 gtatttcaca ccgcatacgt caaagcaacc atagtacgcg ccctgtagcg gcgcattaag
     2641 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccttagcgcc
     2701 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
     2761 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
     2821 aaaacttgat ttgggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg
     2881 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac
     2941 actcaactct atctcgggct attcttttga tttataaggg attttgccga tttcggtcta
     3001 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac
     3061 gtttacaatt ttatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca
     3121 gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc
     3181 cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc
     3241 atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt
     3301 catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac
     3361 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc
     3421 ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt
     3481 cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct
     3541 ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga
     3601 tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag
     3661 cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca
     3721 actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga
     3781 aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag
     3841 tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc
     3901 ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa
     3961 tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt
     4021 gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg
     4081 gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt
     4141 tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg
     4201 gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat
     4261 ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact
     4321 gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa
     4381 aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt
     4441 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     4501 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     4561 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     4621 gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     4681 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     4741 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     4801 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     4861 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
     4921 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     4981 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     5041 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
     5101 acggttcctg gccttttgct ggccttttgc tcacatgtcc tgcaggcagc tgcgcgctcg
     5161 ctcgctcact gaggccgccc gggcgtcggg cgacctttgg tcgcccggcc tcagtgagcg
     5221 agcgagcgcg cagagaggga gtggccaact ccatcactag gggttcctgc ggccgcacgc
     5281 gt
//