LOCUS       sequence-174844-        5280 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  sgRNA expressing AAV construct with a mCherry reporter driven by 
            hSyn promoter. (replaced the GFP in pX552 from Zhang lab with 
            mCherry).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5280)
  AUTHORS   Hung SS, Chrysostomou V, Li F, Lim JK, Wang JH, Powell JE, Tu L, 
            Daniszewski M, Lo C, Wong RC, Crowston JG, Pebay A, King AE, Bui BV,
            Liu GS, Hewitt AW
  TITLE     AAV-Mediated CRISPR/Cas Gene Editing of Retinal Cells In Vivo.
  JOURNAL   Invest Ophthalmol Vis Sci. 2016 Jun 1;57(7):3470-6. doi: 
            10.1167/iovs.16-19316.
  PUBMED    27367513
REFERENCE   2  (bases 1 to 5280)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     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"
COMMENT     Sequence Label: AAV-U6-sgRNA-hSyn-mCherry
FEATURES             Location/Qualifiers
     source          1..5280
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..249
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1..21
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     172..191
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        268..343
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        377..824
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     CDS             856..1566
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(1001..1019)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1269..1288
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    1883..2359
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(2082..2101)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   2399..2539
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2399..2528
                     /label=AAV2 ITR
     rep_origin      2614..3069
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2701..2720)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2911..2932
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3086..3105)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3205..3227
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3265..3283)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3351..3455
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3456..3524,3525..4316)
                     /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     complement(3674..3693)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4487..5075
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4976..4995
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   5137..5266
                     /label=AAV2 ITR (alternate)
                     /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 cgaaacaccg gaagagcgag ctcttctgtt ttagagctag aaatagcaag ttaaaataag
      301 gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttt ctagactgca
      361 gagggccctg cgtatgagtg caagtgggtt ttaggaccag gatgaggcgg ggtgggggtg
      421 cctacctgac gaccgacccc gacccactgg acaagcaccc aacccccatt ccccaaattg
      481 cgcatcccct atcagagagg gggaggggaa acaggatgcg gcgaggcgcg tgcgcactgc
      541 cagcttcagc accgcggaca gtgccttcgc ccccgcctgg cggcgcgcgc caccgccgcc
      601 tcagcactga aggcgcgctg acgtcactcg ccggtccccc gcaaactccc cttcccggcc
      661 accttggtcg cgtccgcgcc gccgccggcc cagccggacc gcaccacgcg aggcgcgaga
      721 taggggggca cgggcgcgac catctgcgct gcggcgccgg cgactcagcg ctgcctcagt
      781 ctgcggtggg cagcggagga gtcgtgtcgt gcctgagagc gcagtcgaga aggtaccgga
      841 tcctctagag tcgacatggt gagcaagggc gaggaggata acatggccat catcaaggag
      901 ttcatgcgct tcaaggtgca catggagggc tccgtgaacg gccacgagtt cgagatcgag
      961 ggcgagggcg agggccgccc ctacgagggc acccagaccg ccaagctgaa ggtgaccaag
     1021 ggtggccccc tgcccttcgc ctgggacatc ctgtcccctc agttcatgta cggctccaag
     1081 gcctacgtga agcaccccgc cgacatcccc gactacttga agctgtcctt ccccgagggc
     1141 ttcaagtggg agcgcgtgat gaacttcgag gacggcggcg tggtgaccgt gacccaggac
     1201 tcctccctgc aggacggcga gttcatctac aaggtgaagc tgcgcggcac caacttcccc
     1261 tccgacggcc ccgtaatgca gaagaagacc atgggctggg aggcctcctc cgagcggatg
     1321 taccccgagg acggcgccct gaagggcgag atcaagcaga ggctgaagct gaaggacggc
     1381 ggccactacg acgctgaggt caagaccacc tacaaggcca agaagcccgt gcagctgccc
     1441 ggcgcctaca acgtcaacat caagttggac atcacctccc acaacgagga ctacaccatc
     1501 gtggaacagt acgaacgcgc cgagggccgc cactccaccg gcggcatgga cgagctgtac
     1561 aagtagtccg gactcagatc tcgagaggag gaggaggaga cagacagcag gatgccccac
     1621 ctcgacagcc ccggcagctc ccagccgaga cgctccttcc tctcaagggt gatcagggca
     1681 gcgctaccgt tgcagctgct tctgctgctg ctgctgctcc tggcctgcct gctacctgcc
     1741 tctgaagatg actacagctg cacccaggcc aacaactttg cccgatcctt ctaccccatg
     1801 ctgcggtaca ccaacgggcc acctcccacc taggaattcg atatcaagct tatcgatacc
     1861 gagcgctgct cgagagatct acgggtggca tccctgtgac ccctccccag tgcctctcct
     1921 ggccctggaa gttgccactc cagtgcccac cagccttgtc ctaataaaat taagttgcat
     1981 cattttgtct gactaggtgt ccttctataa tattatgggg tggagggggg tggtatggag
     2041 caaggggcaa gttgggaaga caacctgtag ggcctgcggg gtctattggg aaccaagctg
     2101 gagtgcagtg gcacaatctt ggctcactgc aatctccgcc tcctgggttc aagcgattct
     2161 cctgcctcag cctcccgagt tgttgggatt ccaggcatgc atgaccaggc tcagctaatt
     2221 tttgtttttt tggtagagac ggggtttcac catattggcc aggctggtct ccaactccta
     2281 atctcaggtg atctacccac cttggcctcc caaattgctg ggattacagg cgtgaaccac
     2341 tgctcccttc cctgtccttc tgattttgta ggtaaccacg tgcggaccga gcggccgcag
     2401 gaacccctag tgatggagtt ggccactccc tctctgcgcg ctcgctcgct cactgaggcc
     2461 gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg cggcctcagt gagcgagcga
     2521 gcgcgcagct gcctgcaggg gcgcctgatg cggtattttc tccttacgca tctgtgcggt
     2581 atttcacacc gcatacgtca aagcaaccat agtacgcgcc ctgtagcggc gcattaagcg
     2641 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ttagcgcccg
     2701 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc
     2761 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
     2821 aacttgattt gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc
     2881 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac
     2941 tcaactctat ctcgggctat tcttttgatt tataagggat tttgccgatt tcggtctatt
     3001 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt
     3061 ttacaatttt atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagc
     3121 cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg
     3181 cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat
     3241 caccgaaacg cgcgagacga aagggcctcg tgatacgcct atttttatag gttaatgtca
     3301 tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc
     3361 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct
     3421 gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg
     3481 cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg
     3541 tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc
     3601 tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca
     3661 cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac
     3721 tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa
     3781 agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg
     3841 ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt
     3901 ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg
     3961 aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc
     4021 gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga
     4081 tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta
     4141 ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc
     4201 cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg
     4261 atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt
     4321 cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa
     4381 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt
     4441 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     4501 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     4561 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     4621 taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     4681 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     4741 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     4801 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     4861 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     4921 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     4981 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     5041 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     5101 ggttcctggc cttttgctgg ccttttgctc acatgtcctg caggcagctg cgcgctcgct
     5161 cgctcactga ggccgcccgg gcgtcgggcg acctttggtc gcccggcctc agtgagcgag
     5221 cgagcgcgca gagagggagt ggccaactcc atcactaggg gttcctgcgg ccgcacgcgt
//
