LOCUS       sequence-324866-        7167 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 7167)
  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 7167)
  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..7167
                     /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   248..377
                     /label=AAV2 ITR
     promoter        469..717
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        738..813
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        827..1075
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        1097..1172
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        1186..1434
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        1456..1531
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             1776..1796
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(4276..4295)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     polyA_signal    4305..4386
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     4330..4349
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   4474..4603
                     /label=AAV2 ITR
     primer_bind     complement(4622..4639)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4622..4638)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4631..4653)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4780..5235
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4867..4886)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5077..5098
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5262..5366
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5367..5435,5436..6227)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVVMVTTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(5585..5604)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6398..6986
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6887..6906
                     /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 attaaggctg cgcgctcgct cgctcactga ggccgcccgg gcaaagcccg ggcgtcgggc
      301 gacctttggt cgcccggcct cagtgagcga gcgagcgcgc agagagggag tggccaactc
      361 catcactagg ggttccttgt agttaatgat taacccgcca tgctacttat ctacgtagcc
      421 atgctctagg aagatcggaa ttcgccctta agggcgcgcc gtttaaacga gggcctattt
      481 cccatgattc cttcatattt gcatatacga tacaaggctg ttagagagat aattggaatt
      541 aatttgactg taaacacaaa gatattagta caaaatacgt gacgtagaaa gtaataattt
      601 cttgggtagt ttgcagtttt aaaattatgt tttaaaatgg actatcatat gcttaccgta
      661 acttgaaagt atttcgattt cttggcttta tatatcttgt ggaaaggacg aaacaccgtt
      721 tgggggcctt taaggtagtt ttagagctag aaatagcaag ttaaaataag gctagtccgt
      781 tatcaacttg aaaaagtggc accgagtcgg tgcttttttt gtatacgagg gcctatttcc
      841 catgattcct tcatatttgc atatacgata caaggctgtt agagagataa ttggaattaa
      901 tttgactgta aacacaaaga tattagtaca aaatacgtga cgtagaaagt aataatttct
      961 tgggtagttt gcagttttaa aattatgttt taaaatggac tatcatatgc ttaccgtaac
     1021 ttgaaagtat ttcgatttct tggctttata tatcttgtgg aaaggacgaa acaccgcctg
     1081 agccacccct gtggatgttt tagagctaga aatagcaagt taaaataagg ctagtccgtt
     1141 atcaacttga aaaagtggca ccgagtcggt gctttttttg ttaacgaggg cctatttccc
     1201 atgattcctt catatttgca tatacgatac aaggctgtta gagagataat tggaattaat
     1261 ttgactgtaa acacaaagat attagtacaa aatacgtgac gtagaaagta ataatttctt
     1321 gggtagtttg cagttttaaa attatgtttt aaaatggact atcatatgct taccgtaact
     1381 tgaaagtatt tcgatttctt ggctttatat atcttgtgga aaggacgaaa caccgtagct
     1441 cttgcttgtt tacaagtttt agagctagaa atagcaagtt aaaataaggc tagtccgtta
     1501 tcaacttgaa aaagtggcac cgagtcggtg ctttttttat ttaaatagct agccctctcc
     1561 tccctgacct caggcttcct cctagtgtca ccttggcccc tcttagaagc caattaggcc
     1621 ctcagtttct gcagcgggga ttaatatgat tatgaacacc cccaatctcc cagatgctga
     1681 ttcagccagg agcttaggag ggggaggtca ctttataagg gtctgggggg gtcagaaccc
     1741 agagtcatca ccggtcgact agaggatcca tggccccaaa gaagaagcgg aaggtcggta
     1801 tccacggagt cccagcagcc gacaagaagt actccattgg gctcgctatc ggcacaaaca
     1861 gcgtcggctg ggccgtcatt acggacgagt acaaggtgcc gagcaaaaaa ttcaaagttc
     1921 tgggcaatac cgatcgccac agcataaaga agaacctcat tggcgccctc ctgttcgact
     1981 ccggggaaac ggccgaagcc acgcggctca aaagaacagc acggcgcaga tatacccgca
     2041 gaaagaatcg gatctgctac ctgcaggaga tctttagtaa tgagatggct aaggtggatg
     2101 actctttctt ccataggctg gaggagtcct ttttggtgga ggaggataaa aagcacgagc
     2161 gccacccaat ctttggcaat atcgtggacg aggtggcgta ccatgaaaag tacccaacca
     2221 tatatcatct gaggaagaag cttgtagaca gtactgataa ggctgacttg cggttgatct
     2281 atctcgcgct ggcgcatatg atcaaatttc ggggacactt cctcatcgag ggggacctga
     2341 acccagacaa cagcgatgtc gacaaactct ttatccaact ggttcagact tacaatcagc
     2401 ttttcgaaga gaacccgatc aacgcatccg gagttgacgc caaagcaatc ctgagcgcta
     2461 ggctgtccaa atcccggcgg ctcgaaaacc tcatcgcaca gctccctggg gagaagaaga
     2521 acggcctgtt tggtaatctt atcgccctgt cactcgggct gacccccaac tttaaatcta
     2581 acttcgacct ggccgaagat gccaagcttc aactgagcaa agacacctac gatgatgatc
     2641 tcgacaatct gctggcccag atcggcgacc agtacgcaga cctttttttg gcggcaaaga
     2701 acctgtcaga cgccattctg ctgagtgata ttctgcgagt gaacacggag atcaccaaag
     2761 ctccgctgag cgctagtatg atcaagcgct atgatgagca ccaccaagac ttgactttgc
     2821 tgaaggccct tgtcagacag caactgcctg agaagtacaa ggaaattttc ttcgatcagt
     2881 ctaaaaatgg ctacgccgga tacattgacg gcggagcaag ccaggaggaa ttttacaaat
     2941 ttattaagcc catcttggaa aaaatggacg gcaccgagga gctgctggta aagcttaaca
     3001 gagaagatct gttgcgcaaa cagcgcactt tcgacaatgg aagcatcccc caccagattc
     3061 acctgggcga actgcacgct atcctcaggc ggcaagagga tttctacccc tttttgaaag
     3121 ataacaggga aaagattgag aaaatcctca catttcggat accctactat gtaggccccc
     3181 tcgcccgggg aaattccaga ttcgcgtgga tgactcgcaa atcagaagag accatcactc
     3241 cctggaactt cgaggaagtc gtggataagg gggcctctgc ccagtccttc atcgaaagga
     3301 tgactaactt tgataaaaat ctgcctaacg aaaaggtgct tcctaaacac tctctgctgt
     3361 acgagtactt cacagtttat aacgagctca ccaaggtcaa atacgtcaca gaagggatga
     3421 gaaagccagc attcctgtct ggagagcaga agaaagctat cgtggacctc ctcttcaaga
     3481 cgaaccggaa agttaccgtg aaacagctca aagaagacta tttcaaaaag attgaatgtt
     3541 tcgactctgt tgaaatcagc ggagtggagg atcgcttcaa cgcatccctg ggaacgtatc
     3601 acgatctcct gaaaatcatt aaagacaagg acttcctgga caatgaggag aacgaggaca
     3661 ttcttgagga cattgtcctc acccttacgt tgtttgaaga tagggagatg attgaagaac
     3721 gcttgaaaac ttacgctcat ctcttcgacg acaaagtcat gaaacagctc aagaggcgcc
     3781 gatatacagg atgggggcgg ctgtcaagaa aactgatcaa tgggatccga gacaagcaga
     3841 gtggaaagac aatcctggat tttcttaagt ccgatggatt tgccaaccgg aacttcatgc
     3901 agttgatcca tgatgactct ctcaccttta aggaggacat ccagaaagca caagtttgtc
     3961 tggctggcga tactctcatt accctggccg atggacgacg agtgcctatt agagaactgg
     4021 tgtcacagca gaatttttcc gtgtgggctc tgaatcctca gacttaccgc ctggagaggg
     4081 ctagagtgag tagagctttc tgtaccggca tcaaacctgt gtaccgcctc accactagac
     4141 tggggagatc cattagggcc actgccaacc accgatttct cacacctcag ggctggaaac
     4201 gagtcgatga actccagcct ggagattacc tggctctgcc taggagaatc cctactgcct
     4261 cctgatctcg agctcgatga gtttggacaa accacaacta gaatgcagtg aaaaaaatgc
     4321 tttatttgtg aaatttgtga tgctattgct ttatttgtaa ccattataag ctgcaataaa
     4381 caagttctcg agttaagggc gaattcccga ttaggatctt cctagagcat ggctacgtag
     4441 ataagtagca tggcgggtta atcattaact acaaggaacc cctagtgatg gagttggcca
     4501 ctccctctct gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc
     4561 cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg cagccttaat taacctaatt
     4621 cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc caacttaatc
     4681 gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc
     4741 gcccttccca acagttgcgc agcctgaatg gcgaatggga cgcgccctgt agcggcgcat
     4801 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag
     4861 cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc
     4921 aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc
     4981 ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccccga tagacggttt
     5041 ttcgcccttt gacgctggag ttcacgttcc tcaatagtgg actcttgttc caaactggaa
     5101 caacactcaa ccctatctcg gtctattctt ttgatttata agggattttt ccgatttcgg
     5161 cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat
     5221 taacgtttat aatttcaggt ggcatctttc ggggaaatgt gcgcggaacc cctatttgtt
     5281 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
     5341 ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc
     5401 ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa
     5461 aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaatagtg
     5521 gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag
     5581 ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc
     5641 gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta
     5701 cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg
     5761 cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca
     5821 acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac
     5881 caaacgacga gcgtgacacc acgatgcctg tagtaatggt aacaacgttg cgcaaactat
     5941 taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg
     6001 ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata
     6061 aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta
     6121 agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa
     6181 atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag
     6241 tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg
     6301 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     6361 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     6421 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     6481 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     6541 ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     6601 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     6661 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     6721 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     6781 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     6841 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     6901 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     6961 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     7021 ccttttgctg cggttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
     7081 accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca
     7141 gcgagtcagt gagcgaggaa gcggaag
//
