LOCUS       sequence-331105-        7353 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express C-terminal part of split dCas9-VPR.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7353)
  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 7353)
  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-RHO-IntC-dCas9C-VPR-polyA
FEATURES             Location/Qualifiers
     source          1..7353
                     /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
     regulatory      707..716
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1313..1324
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             2842..2862
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     protein_bind    2899..2923
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             2953..3102
                     /codon_start=1
                     /product="tetrameric repeat of the minimal activation 
                     domain of herpes simplex virus VP16 (Beerli et al., 1998)"
                     /label=VP64
                     /translation="DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDF
                     DLDML"
     CDS             3127..3147
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3577..3933
                     /codon_start=1
                     /product="transcriptional activation domain of human RelA, 
                     also known as p65 (O'Shea and Perkins, 2008)"
                     /label=RelA (p65) AD
                     /translation="PTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNS
                     EFQQLLNQGIPVAPHTTEPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGD
                     EDFSSIADMDFSALL"
     CDS             3952..4521
                     /codon_start=1
                     /product="transcriptional activation domain from the human 
                     herpesvirus 4 (Epstein-Barr virus) replication and 
                     transcription activator Rta/BRLF1 (Hardwick et al., 1992; 
                     Chavez et al., 2015)"
                     /label=Rta AD
                     /translation="RDSREGMFLPKPEAGSAISDVFEGREVCQPKRIRPFHPPGSPWAN
                     RPLPASLAPTPTGPVHEPVGSLTPAPVPQPLDPAPAVTPEASHLLEDPDEETSQAVKAL
                     REMADTVIPQKEEAAICGQMDLSHPPPRGHLDELTTTLESMTEDLNLDSPLTPELNEIL
                     DTFLNDECLLHAMHISTGLSIFDTSLF"
     repeat_region   4661..4790
                     /label=AAV2 ITR
     primer_bind     complement(4809..4826)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4809..4825)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4818..4840)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4967..5422
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5054..5073)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5264..5285
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5448..5552
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5553..5621,5622..6413)
                     /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(5771..5790)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6584..7172
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7073..7092
                     /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 cgtttaaata
      481 gctagccctc tcctccctga cctcaggctt cctcctagtg tcaccttggc ccctcttaga
      541 agccaattag gccctcagtt tctgcagcgg ggattaatat gattatgaac acccccaatc
      601 tcccagatgc tgattcagcc aggagcttag gagggggagg tcactttata agggtctggg
      661 ggggtcagaa cccagagtca tcggtaccac cggtcgacta gaggatccat ggcggcggcg
      721 tgcccggaac tgcgtcagct ggcgcagagc gatgtgtatt gggatccgat tgtgagcatt
      781 gaaccggatg gcgtggaaga agtgtttgat ctgaccgtgc cgggcccgca taactttgtg
      841 gcgaacgata ttattgcgca taactctggc cagggggaca gtcttcacga gcacatcgct
      901 aatcttgcag gtagcccagc tatcaaaaag ggaatactgc agaccgttaa ggtcgtggat
      961 gaactcgtca aagtaatggg aaggcataag cccgagaata tcgttatcga gatggcccga
     1021 gagaaccaaa ctacccagaa gggacagaag aacagtaggg aaaggatgaa gaggattgaa
     1081 gagggtataa aagaactggg gtcccaaatc cttaaggaac acccagttga aaacacccag
     1141 cttcagaatg agaagctcta cctgtactac ctgcagaacg gcagggacat gtacgtggat
     1201 caggaactgg acatcaatcg gctctccgac tacgacgtgg atgctatcgt gccccagtct
     1261 tttctcaaag atgattctat tgataataaa gtgttgacaa gatccgataa aaatagaggg
     1321 aagagtgata acgtcccctc agaagaagtt gtcaagaaaa tgaaaaatta ttggcggcag
     1381 ctgctgaacg ccaaactgat cacacaacgg aagttcgata atctgactaa ggctgaacga
     1441 ggtggcctgt ctgagttgga taaagccggc ttcatcaaaa ggcagcttgt tgagacacgc
     1501 cagatcacca agcacgtggc ccaaattctc gattcacgca tgaacaccaa gtacgatgaa
     1561 aatgacaaac tgattcgaga ggtgaaagtt attactctga agtctaagct ggtctcagat
     1621 ttcagaaagg actttcagtt ttataaggtg agagagatca acaattacca ccatgcgcat
     1681 gatgcctacc tgaatgcagt ggtaggcact gcacttatca aaaaatatcc caagcttgaa
     1741 tctgaatttg tttacggaga ctataaagtg tacgatgtta ggaaaatgat cgcaaagtct
     1801 gagcaggaaa taggcaaggc caccgctaag tacttctttt acagcaatat tatgaatttt
     1861 ttcaagaccg agattacact ggccaatgga gagattcgga agcgaccact tatcgaaaca
     1921 aacggagaaa caggagaaat cgtgtgggac aagggtaggg atttcgcgac agtccggaag
     1981 gtcctgtcca tgccgcaggt gaacatcgtt aaaaagaccg aagtacagac cggaggcttc
     2041 tccaaggaaa gtatcctccc gaaaaggaac agcgacaagc tgatcgcacg caaaaaagat
     2101 tgggacccca agaaatacgg cggattcgat tctcctacag tcgcttacag tgtactggtt
     2161 gtggccaaag tggagaaagg gaagtctaaa aaactcaaaa gcgtcaagga actgctgggc
     2221 atcacaatca tggagcgatc aagcttcgaa aaaaacccca tcgactttct cgaggcgaaa
     2281 ggatataaag aggtcaaaaa agacctcatc attaagcttc ccaagtactc tctctttgag
     2341 cttgaaaacg gccggaaacg aatgctcgct agtgcgggcg agctgcagaa aggtaacgag
     2401 ctggcactgc cctctaaata cgttaatttc ttgtatctgg ccagccacta tgaaaagctc
     2461 aaagggtctc ccgaagataa tgagcagaag cagctgttcg tggaacaaca caaacactac
     2521 cttgatgaga tcatcgagca aataagcgaa ttctccaaaa gagtgatcct cgccgacgct
     2581 aacctcgata aggtgctttc tgcttacaat aagcacaggg ataagcccat cagggagcag
     2641 gcagaaaaca ttatccactt gtttactctg accaacttgg gcgcgcctgc agccttcaag
     2701 tacttcgaca ccaccataga cagaaagcgg tacacctcta caaaggaggt cctggacgcc
     2761 acactgattc atcagtcaat tacggggctc tatgaaacaa gaatcgacct ctctcagctc
     2821 ggtggagaca gcagggctga ccccaagaag aagaggaagg tgtcgccagg gatccgtcga
     2881 cttgacgcgt tgatatcaac aagtttgtac aaaaaagcag gctacaaaga ggccagcggt
     2941 tccggacggg ctgacgcatt ggacgatttt gatctggata tgctgggaag tgacgccctc
     3001 gatgattttg accttgacat gcttggttcg gatgcccttg atgactttga cctcgacatg
     3061 ctcggcagtg acgcccttga tgatttcgac ctggacatgc tgattaactc tagaagttcc
     3121 ggatctccga aaaagaaacg caaagttggt agccagtacc tgcccgacac cgacgaccgg
     3181 caccggatcg aggaaaagcg gaagcggacc tacgagacat tcaagagcat catgaagaag
     3241 tcccccttca gcggccccac cgaccctaga cctccaccta gaagaatcgc cgtgcccagc
     3301 agatccagcg ccagcgtgcc aaaacctgcc ccccagcctt accccttcac cagcagcctg
     3361 agcaccatca actacgacga gttccctacc atggtgttcc ccagcggcca gatctctcag
     3421 gcctctgctc tggctccagc ccctcctcag gtgctgcctc aggctcctgc tcctgcacca
     3481 gctccagcca tggtgtctgc actggctcag gcaccagcac ccgtgcctgt gctggctcct
     3541 ggacctccac aggctgtggc tccaccagcc cctaaaccta cacaggccgg cgagggcaca
     3601 ctgtctgaag ctctgctgca gctgcagttc gacgacgagg atctgggagc cctgctggga
     3661 aacagcaccg atcctgccgt gttcaccgac ctggccagcg tggacaacag cgagttccag
     3721 cagctgctga accagggcat ccctgtggcc cctcacacca ccgagcccat gctgatggaa
     3781 taccccgagg ccatcacccg gctcgtgaca ggcgctcaga ggcctcctga tccagctcct
     3841 gcccctctgg gagcaccagg cctgcctaat ggactgctgt ctggcgacga ggacttcagc
     3901 tctatcgccg atatggattt ctcagccttg ctgggctctg gcagcggcag ccgggattcc
     3961 agggaaggga tgtttttgcc gaagcctgag gccggctccg ctattagtga cgtgtttgag
     4021 ggccgcgagg tgtgccagcc aaaacgaatc cggccatttc atcctccagg aagtccatgg
     4081 gccaaccgcc cactccccgc cagcctcgca ccaacaccaa ccggtccagt acatgagcca
     4141 gtcgggtcac tgaccccggc accagtccct cagccactgg atccagcgcc cgcagtgact
     4201 cccgaggcca gtcacctgtt ggaggatccc gatgaagaga cgagccaggc tgtcaaagcc
     4261 cttcgggaga tggccgatac tgtgattccc cagaaggaag aggctgcaat ctgtggccaa
     4321 atggaccttt cccatccgcc cccaaggggc catctggatg agctgacaac cacacttgag
     4381 tccatgaccg aggatctgaa cctggactca cccctgaccc cggaattgaa cgagattctg
     4441 gataccttcc tgaacgacga gtgcctcttg catgccatgc atatcagcac aggactgtcc
     4501 atcttcgaca catctctgtt ttgacaataa aatatcttta ttttcattac atctgtgtgt
     4561 tggttttttg tgtctcgagt taagggcgaa ttcccgataa ggatcttcct agagcatggc
     4621 tacgtagata agtagcatgg cgggttaatc attaactaca aggaacccct agtgatggag
     4681 ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc
     4741 cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag ccttaattaa
     4801 cctaattcac tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg cgttacccaa
     4861 cttaatcgcc ttgcagcaca tccccctttc gccagctggc gtaatagcga agaggcccgc
     4921 accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg aatgggacgc gccctgtagc
     4981 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc
     5041 gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt
     5101 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac
     5161 ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag
     5221 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa
     5281 actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg
     5341 atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac
     5401 aaaatattaa cgcttacaat ttaggtggca cttttcgggg aaatgtgcgc ggaaccccta
     5461 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
     5521 aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc
     5581 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga
     5641 aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca
     5701 acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt
     5761 ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg
     5821 gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc
     5881 atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata
     5941 acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt
     6001 tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag
     6061 ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca
     6121 aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg
     6181 aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg
     6241 ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag
     6301 atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg
     6361 aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag
     6421 accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga
     6481 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
     6541 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     6601 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     6661 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     6721 caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     6781 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     6841 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     6901 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     6961 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     7021 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     7081 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     7141 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     7201 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg
     7261 tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg
     7321 agcgcagcga gtcagtgagc gaggaagcgg aag
//
