LOCUS       sequence-408439-        7777 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  In vitro transcription plasmid for production of dSaCas9-VPR mRNA 
            for transcriptional activation (VPR = tripartite transactivator 
            VP64, p65, and Rta). Insert is derived from Addgene plasmid #68495.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7777)
  AUTHORS   Jensen TI, Mikkelsen NS, Gao Z, Fosselteder J, Pabst G, Axelgaard E,
            Laustsen A, Konig S, Reinisch A, Bak RO
  TITLE     Targeted regulation of transcription in primary cells using CRISPRa 
            and CRISPRi.
  JOURNAL   Genome Res. 2021 Nov;31(11):2120-2130. doi: 10.1101/gr.275607.121. 
            Epub 2021 Aug 18.
  PUBMED    34407984
REFERENCE   2  (bases 1 to 7777)
  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: 
            "10.1101/gr.275607.121"; journalName: "Genome Res"; date: "2021-11";
            volume: "31"; issue: "11"; pages: "2120-2130"
COMMENT     Sequence Label: dSaCas9-VPR
FEATURES             Location/Qualifiers
     source          1..7777
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     364..386
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     378..395
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     379..395
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     regulatory      475..484
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             487..507
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3688..3735
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             3766..3915
                     /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             3940..3960
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4390..4746
                     /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             4765..5334
                     /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"
     primer_bind     complement(5556..5572)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5556..5572)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5569..5591)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5580..5596
                     /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)."
     promoter        complement(join(5604..5610,5611..5628,5629..5634))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5649..5670
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5787..5804)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5958..6546)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6038..6057)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6717..7508,7509..7577))
                     /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     7340..7359
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7578..7682)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7750..7768
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgagctcggt acctaatacg
      421 actcactata aggaaataag agagaaaaga agagtaagaa gaaatatagc tagcgccacc
      481 atggccccaa agaagaagcg gaaggtcggt atccacggag tcccagcagc caagcggaac
      541 tacatcctgg gcctggccat cggcatcacc agcgtgggct acggcatcat cgactacgag
      601 acacgggacg tgatcgatgc cggcgtgcgg ctgttcaaag aggccaacgt ggaaaacaac
      661 gagggcaggc ggagcaagag aggcgccaga aggctgaagc ggcggaggcg gcatagaatc
      721 cagagagtga agaagctgct gttcgactac aacctgctga ccgaccacag cgagctgagc
      781 ggcatcaacc cctacgaggc cagagtgaag ggcctgagcc agaagctgag cgaggaagag
      841 ttctctgccg ccctgctgca cctggccaag agaagaggcg tgcacaacgt gaacgaggtg
      901 gaagaggaca ccggcaacga gctgtccacc aaagagcaga tcagccggaa cagcaaggcc
      961 ctggaagaga aatacgtggc cgaactgcag ctggaacggc tgaagaaaga cggcgaagtg
     1021 cggggcagca tcaacagatt caagaccagc gactacgtga aagaagccaa acagctgctg
     1081 aaggtgcaga aggcctacca ccagctggac cagagcttca tcgacaccta catcgacctg
     1141 ctggaaaccc ggcggaccta ctatgaggga cctggcgagg gcagcccctt cggctggaag
     1201 gacatcaaag aatggtacga gatgctgatg ggccactgca cctacttccc cgaggaactg
     1261 cggagcgtga agtacgccta caacgccgac ctgtacaacg ccctgaacga cctgaacaat
     1321 ctcgtgatca ccagggacga gaacgagaag ctggaatatt acgagaagtt ccagatcatc
     1381 gagaacgtgt tcaagcagaa gaagaagccc accctgaagc agatcgccaa agaaatcctc
     1441 gtgaacgaag aggatattaa gggctacaga gtgaccagca ccggcaagcc cgagttcacc
     1501 aacctgaagg tgtaccacga catcaaggac attaccgccc ggaaagagat tattgagaac
     1561 gccgagctgc tggatcagat tgccaagatc ctgaccatct accagagcag cgaggacatc
     1621 caggaagaac tgaccaatct gaactccgag ctgacccagg aagagatcga gcagatctct
     1681 aatctgaagg gctataccgg cacccacaac ctgagcctga aggccatcaa cctgatcctg
     1741 gacgagctgt ggcacaccaa cgacaaccag atcgctatct tcaaccggct gaagctggtg
     1801 cccaagaagg tggacctgtc ccagcagaaa gagatcccca ccaccctggt ggacgacttc
     1861 atcctgagcc ccgtcgtgaa gagaagcttc atccagagca tcaaagtgat caacgccatc
     1921 atcaagaagt acggcctgcc caacgacatc attatcgagc tggcccgcga gaagaactcc
     1981 aaggacgccc agaaaatgat caacgagatg cagaagcgga accggcagac caacgagcgg
     2041 atcgaggaaa tcatccggac caccggcaaa gagaacgcca agtacctgat cgagaagatc
     2101 aagctgcacg acatgcagga aggcaagtgc ctgtacagcc tggaagccat ccctctggaa
     2161 gatctgctga acaacccctt caactatgag gtggaccaca tcatccccag aagcgtgtcc
     2221 ttcgacaaca gcttcaacaa caaggtgctc gtgaagcagg aagaagccag caagaagggc
     2281 aaccggaccc cattccagta cctgagcagc agcgacagca agatcagcta cgaaaccttc
     2341 aagaagcaca tcctgaatct ggccaagggc aagggcagaa tcagcaagac caagaaagag
     2401 tatctgctgg aagaacggga catcaacagg ttctccgtgc agaaagactt catcaaccgg
     2461 aacctggtgg ataccagata cgccaccaga ggcctgatga acctgctgcg gagctacttc
     2521 agagtgaaca acctggacgt gaaagtgaag tccatcaatg gcggcttcac cagctttctg
     2581 cggcggaagt ggaagtttaa gaaagagcgg aacaaggggt acaagcacca cgccgaggac
     2641 gccctgatca ttgccaacgc cgatttcatc ttcaaagagt ggaagaaact ggacaaggcc
     2701 aaaaaagtga tggaaaacca gatgttcgag gaaaagcagg ccgagagcat gcccgagatc
     2761 gaaaccgagc aggagtacaa agagatcttc atcacccccc accagatcaa gcacattaag
     2821 gacttcaagg actacaagta cagccaccgg gtggacaaga agcctaatag agagctgatt
     2881 aacgacaccc tgtactccac ccggaaggac gacaagggca acaccctgat cgtgaacaat
     2941 ctgaacggcc tgtacgacaa ggacaatgac aagctgaaaa agctgatcaa caagagcccc
     3001 gaaaagctgc tgatgtacca ccacgacccc cagacctacc agaaactgaa gctgattatg
     3061 gaacagtacg gcgacgagaa gaatcccctg tacaagtact acgaggaaac cgggaactac
     3121 ctgaccaagt actccaaaaa ggacaacggc cccgtgatca agaagattaa gtattacggc
     3181 aacaaactga acgcccatct ggacatcacc gacgactacc ccaacagcag aaacaaggtc
     3241 gtgaagctgt ccctgaagcc ctacagattc gacgtgtacc tggacaatgg cgtgtacaag
     3301 ttcgtgaccg tgaagaatct ggatgtgatc aaaaaagaaa actactacga agtgaatagc
     3361 aagtgctatg aggaagctaa gaagctgaag aagatcagca accaggccga gtttatcgcc
     3421 tccttctaca acaacgatct gatcaagatc aacggcgagc tgtatagagt gatcggcgtg
     3481 aacaacgacc tgctgaaccg gatcgaagtg aacatgatcg acatcaccta ccgcgagtac
     3541 ctggaaaaca tgaacgacaa gaggcccccc aggatcatta agacaatcgc ctccaagacc
     3601 cagagcatta agaagtacag cacagacatt ctgggcaacc tgtatgaagt gaaatctaag
     3661 aagcaccctc agatcatcaa aaagggcaaa aggccggcgg ccacgaaaaa ggccggccag
     3721 gcaaaaaaga aaaagggatc cgaggccagc ggttccggac gggctgacgc attggacgat
     3781 tttgatctgg atatgctggg aagtgacgcc ctcgatgatt ttgaccttga catgcttggt
     3841 tcggatgccc ttgatgactt tgacctcgac atgctcggca gtgacgccct tgatgatttc
     3901 gacctggaca tgctgattaa ctctagaagt tccggatctc cgaaaaagaa acgcaaagtt
     3961 ggtagccagt acctgcccga caccgacgac cggcaccgga tcgaggaaaa gcggaagcgg
     4021 acctacgaga cattcaagag catcatgaag aagtccccct tcagcggccc caccgaccct
     4081 agacctccac ctagaagaat cgccgtgccc agcagatcca gcgccagcgt gccaaaacct
     4141 gccccccagc cttacccctt caccagcagc ctgagcacca tcaactacga cgagttccct
     4201 accatggtgt tccccagcgg ccagatctct caggcctctg ctctggctcc agcccctcct
     4261 caggtgctgc ctcaggctcc tgctcctgca ccagctccag ccatggtgtc tgcactggct
     4321 caggcaccag cacccgtgcc tgtgctggct cctggacctc cacaggctgt ggctccacca
     4381 gcccctaaac ctacacaggc cggcgagggc acactgtctg aagctctgct gcagctgcag
     4441 ttcgacgacg aggatctggg agccctgctg ggaaacagca ccgatcctgc cgtgttcacc
     4501 gacctggcca gcgtggacaa cagcgagttc cagcagctgc tgaaccaggg catccctgtg
     4561 gcccctcaca ccaccgagcc catgctgatg gaataccccg aggccatcac ccggctcgtg
     4621 acaggcgctc agaggcctcc tgatccagct cctgcccctc tgggagcacc aggcctgcct
     4681 aatggactgc tgtctggcga cgaggacttc agctctatcg ccgatatgga tttctcagcc
     4741 ttgctgggct ctggcagcgg cagccgggat tccagggaag ggatgttttt gccgaagcct
     4801 gaggccggct ccgctattag tgacgtgttt gagggccgcg aggtgtgcca gccaaaacga
     4861 atccggccat ttcatcctcc aggaagtcca tgggccaacc gcccactccc cgccagcctc
     4921 gcaccaacac caaccggtcc agtacatgag ccagtcgggt cactgacccc ggcaccagtc
     4981 cctcagccac tggatccagc gcccgcagtg actcccgagg ccagtcacct gttggaggat
     5041 cccgatgaag agacgagcca ggctgtcaaa gcccttcggg agatggccga tactgtgatt
     5101 ccccagaagg aagaggctgc aatctgtggc caaatggacc tttcccatcc gcccccaagg
     5161 ggccatctgg atgagctgac aaccacactt gagtccatga ccgaggatct gaacctggac
     5221 tcacccctga ccccggaatt gaacgagatt ctggatacct tcctgaacga cgagtgcctc
     5281 ttgcatgcca tgcatatcag cacaggactg tccatcttcg acacatctct gttttgaggc
     5341 ggccgcttaa ttaagctgcc ttctgcgggg cttgccttct ggccatgccc ttcttctctc
     5401 ccttgcacct gtacctcttg gtctttgaat aaagcctgag taggaagtct agaaaaaaaa
     5461 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaagggtctt catcatcgcc
     5521 tgcagaggcc tgcatgcaag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat
     5581 tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg
     5641 ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag
     5701 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     5761 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     5821 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     5881 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     5941 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     6001 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     6061 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     6121 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     6181 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     6241 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     6301 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     6361 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct
     6421 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     6481 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     6541 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     6601 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     6661 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     6721 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     6781 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
     6841 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag
     6901 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     6961 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     7021 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     7081 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     7141 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     7201 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     7261 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     7321 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     7381 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     7441 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     7501 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     7561 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat
     7621 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     7681 cgcacatttc cccgaaaagt gccacctgac gtctaagaaa ccattattat catgacatta
     7741 acctataaaa ataggcgtat cacgaggccc tttcgtc
//
