LOCUS       sequence-331048-        7177 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express N-terminal part of split dCas9-VPR and 3 sgRNAs targeting 
            murine Opn1mw promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7177)
  AUTHORS   Bohm S, Splith V, Riedmayr LM, Rotzer RD, Gasparoni G, Nordstrom 
            KJV, Wagner JE, Hinrichsmeyer KS, Walter J, Wahl-Schott C, Fenske S,
            Biel M, Michalakis S, Becirovic E
  TITLE     A gene therapy for inherited blindness using dCas9-VPR-mediated 
            transcriptional activation.
  JOURNAL   Sci Adv. 2020 Aug 19;6(34):eaba5614. doi: 10.1126/sciadv.aba5614. 
            eCollection 2020 Aug.
  PUBMED    32875106
REFERENCE   2  (bases 1 to 7177)
  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"; journalName: "Sci Adv."; 
            date: "2020-08-19"; volume: "6(34):eaba5614. doi"; pages: " 
            10.1126/sciadv.aba5614. eCollection 2020 Aug"
COMMENT     Sequence Label: pAAV-3xsgRNA_Opn1mw-RHO-Cas9N-IntN-polyA
FEATURES             Location/Qualifiers
     source          1..7177
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    107..128
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(143..148,149..166,167..173)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    181..197
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     186..208
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     205..221
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     205..221
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   259..388
                     /label=AAV2 ITR
     promoter        480..728
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        749..824
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        838..1086
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        1108..1183
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        1197..1445
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        1467..1542
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             1787..1807
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(4287..4306)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     polyA_signal    4316..4397
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     4341..4360
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   4485..4614
                     /label=AAV2 ITR
     primer_bind     complement(4633..4650)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4633..4649)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4642..4664)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4791..5246
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4878..4897)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5088..5109
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5272..5376
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5377..5445,5446..6237)
                     /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(5595..5614)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6408..6996
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6897..6916
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
       61 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc
      121 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa
      181 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccagattta
      241 attaaggcct taattaggct gcgcgctcgc tcgctcactg aggccgcccg ggcaaagccc
      301 gggcgtcggg cgacctttgg tcgcccggcc tcagtgagcg agcgagcgcg cagagaggga
      361 gtggccaact ccatcactag gggttccttg tagttaatga ttaacccgcc atgctactta
      421 tctacgtagc catgctctag gaagatcgga attcgccctt aagggcgcgc cgtttaaacg
      481 agggcctatt tcccatgatt ccttcatatt tgcatatacg atacaaggct gttagagaga
      541 taattggaat taatttgact gtaaacacaa agatattagt acaaaatacg tgacgtagaa
      601 agtaataatt tcttgggtag tttgcagttt taaaattatg ttttaaaatg gactatcata
      661 tgcttaccgt aacttgaaag tatttcgatt tcttggcttt atatatcttg tggaaaggac
      721 gaaacaccgt ttgggggcct ttaaggtagt tttagagcta gaaatagcaa gttaaaataa
      781 ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt tgtatacgag
      841 ggcctatttc ccatgattcc ttcatatttg catatacgat acaaggctgt tagagagata
      901 attggaatta atttgactgt aaacacaaag atattagtac aaaatacgtg acgtagaaag
      961 taataatttc ttgggtagtt tgcagtttta aaattatgtt ttaaaatgga ctatcatatg
     1021 cttaccgtaa cttgaaagta tttcgatttc ttggctttat atatcttgtg gaaaggacga
     1081 aacaccgcct gagccacccc tgtggatgtt ttagagctag aaatagcaag ttaaaataag
     1141 gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttt gttaacgagg
     1201 gcctatttcc catgattcct tcatatttgc atatacgata caaggctgtt agagagataa
     1261 ttggaattaa tttgactgta aacacaaaga tattagtaca aaatacgtga cgtagaaagt
     1321 aataatttct tgggtagttt gcagttttaa aattatgttt taaaatggac tatcatatgc
     1381 ttaccgtaac ttgaaagtat ttcgatttct tggctttata tatcttgtgg aaaggacgaa
     1441 acaccgtagc tcttgcttgt ttacaagttt tagagctaga aatagcaagt taaaataagg
     1501 ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gcttttttta tttaaatagc
     1561 tagccctctc ctccctgacc tcaggcttcc tcctagtgtc accttggccc ctcttagaag
     1621 ccaattaggc cctcagtttc tgcagcgggg attaatatga ttatgaacac ccccaatctc
     1681 ccagatgctg attcagccag gagcttagga gggggaggtc actttataag ggtctggggg
     1741 ggtcagaacc cagagtcatc accggtcgac tagaggatcc atggccccaa agaagaagcg
     1801 gaaggtcggt atccacggag tcccagcagc cgacaagaag tactccattg ggctcgctat
     1861 cggcacaaac agcgtcggct gggccgtcat tacggacgag tacaaggtgc cgagcaaaaa
     1921 attcaaagtt ctgggcaata ccgatcgcca cagcataaag aagaacctca ttggcgccct
     1981 cctgttcgac tccggggaaa cggccgaagc cacgcggctc aaaagaacag cacggcgcag
     2041 atatacccgc agaaagaatc ggatctgcta cctgcaggag atctttagta atgagatggc
     2101 taaggtggat gactctttct tccataggct ggaggagtcc tttttggtgg aggaggataa
     2161 aaagcacgag cgccacccaa tctttggcaa tatcgtggac gaggtggcgt accatgaaaa
     2221 gtacccaacc atatatcatc tgaggaagaa gcttgtagac agtactgata aggctgactt
     2281 gcggttgatc tatctcgcgc tggcgcatat gatcaaattt cggggacact tcctcatcga
     2341 gggggacctg aacccagaca acagcgatgt cgacaaactc tttatccaac tggttcagac
     2401 ttacaatcag cttttcgaag agaacccgat caacgcatcc ggagttgacg ccaaagcaat
     2461 cctgagcgct aggctgtcca aatcccggcg gctcgaaaac ctcatcgcac agctccctgg
     2521 ggagaagaag aacggcctgt ttggtaatct tatcgccctg tcactcgggc tgacccccaa
     2581 ctttaaatct aacttcgacc tggccgaaga tgccaagctt caactgagca aagacaccta
     2641 cgatgatgat ctcgacaatc tgctggccca gatcggcgac cagtacgcag accttttttt
     2701 ggcggcaaag aacctgtcag acgccattct gctgagtgat attctgcgag tgaacacgga
     2761 gatcaccaaa gctccgctga gcgctagtat gatcaagcgc tatgatgagc accaccaaga
     2821 cttgactttg ctgaaggccc ttgtcagaca gcaactgcct gagaagtaca aggaaatttt
     2881 cttcgatcag tctaaaaatg gctacgccgg atacattgac ggcggagcaa gccaggagga
     2941 attttacaaa tttattaagc ccatcttgga aaaaatggac ggcaccgagg agctgctggt
     3001 aaagcttaac agagaagatc tgttgcgcaa acagcgcact ttcgacaatg gaagcatccc
     3061 ccaccagatt cacctgggcg aactgcacgc tatcctcagg cggcaagagg atttctaccc
     3121 ctttttgaaa gataacaggg aaaagattga gaaaatcctc acatttcgga taccctacta
     3181 tgtaggcccc ctcgcccggg gaaattccag attcgcgtgg atgactcgca aatcagaaga
     3241 gaccatcact ccctggaact tcgaggaagt cgtggataag ggggcctctg cccagtcctt
     3301 catcgaaagg atgactaact ttgataaaaa tctgcctaac gaaaaggtgc ttcctaaaca
     3361 ctctctgctg tacgagtact tcacagttta taacgagctc accaaggtca aatacgtcac
     3421 agaagggatg agaaagccag cattcctgtc tggagagcag aagaaagcta tcgtggacct
     3481 cctcttcaag acgaaccgga aagttaccgt gaaacagctc aaagaagact atttcaaaaa
     3541 gattgaatgt ttcgactctg ttgaaatcag cggagtggag gatcgcttca acgcatccct
     3601 gggaacgtat cacgatctcc tgaaaatcat taaagacaag gacttcctgg acaatgagga
     3661 gaacgaggac attcttgagg acattgtcct cacccttacg ttgtttgaag atagggagat
     3721 gattgaagaa cgcttgaaaa cttacgctca tctcttcgac gacaaagtca tgaaacagct
     3781 caagaggcgc cgatatacag gatgggggcg gctgtcaaga aaactgatca atgggatccg
     3841 agacaagcag agtggaaaga caatcctgga ttttcttaag tccgatggat ttgccaaccg
     3901 gaacttcatg cagttgatcc atgatgactc tctcaccttt aaggaggaca tccagaaagc
     3961 acaagtttgt ctggctggcg atactctcat taccctggcc gatggacgac gagtgcctat
     4021 tagagaactg gtgtcacagc agaatttttc cgtgtgggct ctgaatcctc agacttaccg
     4081 cctggagagg gctagagtga gtagagcttt ctgtaccggc atcaaacctg tgtaccgcct
     4141 caccactaga ctggggagat ccattagggc cactgccaac caccgatttc tcacacctca
     4201 gggctggaaa cgagtcgatg aactccagcc tggagattac ctggctctgc ctaggagaat
     4261 ccctactgcc tcctgatctc gagctcgatg agtttggaca aaccacaact agaatgcagt
     4321 gaaaaaaatg ctttatttgt gaaatttgtg atgctattgc tttatttgta accattataa
     4381 gctgcaataa acaagttctc gagttaaggg cgaattcccg ataaggatct tcctagagca
     4441 tggctacgta gataagtagc atggcgggtt aatcattaac tacaaggaac ccctagtgat
     4501 ggagttggcc actccctctc tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt
     4561 cgcccgacgc ccgggctttg cccgggcggc ctcagtgagc gagcgagcgc gcagccttaa
     4621 ttaacctaat tcactggccg tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac
     4681 ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata gcgaagaggc
     4741 ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatggg acgcgccctg
     4801 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc
     4861 cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg
     4921 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg
     4981 gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg
     5041 atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt
     5101 ccaaactgga acaacactca accctatctc ggtctattct tttgatttat aagggatttt
     5161 gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt
     5221 taacaaaata ttaacgctta caatttaggt ggcacttttc ggggaaatgt gcgcggaacc
     5281 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc
     5341 tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc
     5401 gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg
     5461 gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat
     5521 ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc
     5581 acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa
     5641 ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa
     5701 aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt
     5761 gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct
     5821 tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat
     5881 gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg
     5941 cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg
     6001 atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt
     6061 attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg
     6121 ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg
     6181 gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg
     6241 tcagaccaag tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa
     6301 aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt
     6361 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     6421 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     6481 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     6541 ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     6601 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     6661 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     6721 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     6781 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     6841 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     6901 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     6961 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     7021 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
     7081 tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg
     7141 accgagcgca gcgagtcagt gagcgaggaa gcggaag
//
