LOCUS       sequence-375253-        6158 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV-CRISPRa base vector .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6158)
  AUTHORS   Wang G, Chow RD, Bai Z, Zhu L, Errami Y, Dai X, Dong MB, Ye L, Zhang
            X, Renauer PA, Park JJ, Shen L, Ye H, Fuchs CS, Chen S
  TITLE     Multiplexed activation of endogenous genes by CRISPRa elicits potent
            antitumor immunity.
  JOURNAL   Nat Immunol. 2019 Nov;20(11):1494-1505. doi: 
            10.1038/s41590-019-0500-4. Epub 2019 Oct 14.
  PUBMED    31611701
REFERENCE   2  (bases 1 to 6158)
  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: 
            "10.1038/s41590-019-0500-4"; journalName: "Nat Immunol"; date: 
            "2019-11"; volume: "20"; issue: "11"; pages: "1494-1505"
COMMENT     Sequence Label: pGW05 AAV-CRISPRa base vector
            pAAV-OE_U6-Lib(M...
FEATURES             Location/Qualifiers
     source          1..6158
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        152..400
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     152..172
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     323..342
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        436..454
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        506..524
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     promoter        714..1892
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          945..1883
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1840..1860
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      1899..1908
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1908..2294
                     /codon_start=1
                     /product="bacteriophage MS2 coat protein"
                     /label=MS2-N55K
                     /note="binds with high affinity to a specific stem-loop 
                     structure in the viral RNA (Lim et al., 1994)"
                     /translation="ASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVT
                     CSVRQSSAQKRKYTIKVEVPKVATQTVGGVELPVAAWRSYLNMELTIPIFATNSDCELI
                     VKAMQGLLKDGNPIPSAIAANSGIY"
     CDS             2349..2369
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2385..2927
                     /codon_start=1
                     /product="C-terminal portion of the p65 subunit of mouse 
                     NF-kappa-B"
                     /label=p65
                     /translation="PSGQISNQALALAPSSAPVLAQTMVPSSAMVPLAQPPAPAPVLTP
                     GPPQSLSAPVPKSTQAGEGTLSEALLHLQFDADEDLGALLGNSTDPGVFTDLASVDNSE
                     FQQLLNQGVSMSHSTAEPMLMEYPEAITRLVTGSQRPPDPAPTPLGTSGLPNGLSGDED
                     FSSIADMDFSALLSQISS"
     CDS             2952..3323
                     /codon_start=1
                     /product="C-terminal activation domain from the human heat 
                     shock transcription factor HSF1"
                     /label=HSF1
                     /translation="GFSVDTSALLDLFSPSVTVPDMSLPDLDSSLASIQELLSPQEPPR
                     PPEAENSSPDSGKQLVHYTAQPLFLLDPGSVDTGSNDLPVLFELGEGSYFSEGDGFAED
                     PTISLLTGSEPPKAKDPTVS"
     polyA_signal    3345..3393
                     /note="synthetic polyadenylation signal"
     repeat_region   3421..3561
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3421..3550
                     /label=AAV2 ITR
     rep_origin      3636..4091
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3723..3742)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3933..3954
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4108..4127)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4227..4249
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4287..4305)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4373..4477
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4478..4546,4547..5338)
                     /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(4696..4715)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5509..6097
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5998..6017
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gcggccgcac gcgttctaga agagggccta tttcccatga ttccttcata
      181 tttgcatata cgatacaagg ctgttagaga gataattgga attaatttga ctgtaaacac
      241 aaagatatta gtacaaaata cgtgacgtag aaagtaataa tttcttgggt agtttgcagt
      301 tttaaaatta tgttttaaaa tggactatca tatgcttacc gtaacttgaa agtatttcga
      361 tttcttggct ttatatatct tgtggaaagg acgaaacacc ggaagagcga gctcttctgt
      421 tttagagcta ggccaacatg aggatcaccc atgtctgcag ggcctagcaa gttaaaataa
      481 ggctagtccg ttatcaactt ggccaacatg aggatcaccc atgtctgcag ggccaagtgg
      541 caccgagtcg gtgctttttt ggatccaagc ttggcgtaac tagatcttga gacaaatggg
      601 acagcagaga tccagtttgg ttaattagct agctgcaaag atggataaag ttttaaacag
      661 agaggaatct ttgcagctaa tggaccttct aggtcttgaa aggagtggga attggctccg
      721 gtgcccgtca gtgggcagag cgcacatcgc ccacagtccc cgagaagttg gggggagggg
      781 tcggcaattg aaccggtgcc tagagaaggt ggcgcggggt aaactgggaa agtgatgtcg
      841 tgtactggct ccgccttttt cccgagggtg ggggagaacc gtatataagt gcagtagtcg
      901 ccgtgaacgt tctttttcgc aacgggtttg ccgccagaac acaggtaagt gccgtgtgtg
      961 gttcccgcgg gcctggcctc tttacgggtt atggcccttg cgtgccttga attacttcca
     1021 cctggctgca gtacgtgatt cttgatcccg agcttcgggt tggaagtggg tgggagagtt
     1081 cgaggccttg cgcttaagga gccccttcgc ctcgtgcttg agttgaggcc tggcctgggc
     1141 gctggggccg ccgcgtgcga atctggtggc accttcgcgc ctgtctcgct gctttcgata
     1201 agtctctagc catttaaaat ttttgatgac ctgctgcgac gctttttttc tggcaagata
     1261 gtcttgtaaa tgcgggccaa gatctgcaca ctggtatttc ggtttttggg gccgcgggcg
     1321 gcgacggggc ccgtgcgtcc cagcgcacat gttcggcgag gcggggcctg cgagcgcggc
     1381 caccgagaat cggacggggg tagtctcaag ctggccggcc tgctctggtg cctggcctcg
     1441 cgccgccgtg tatcgccccg ccctgggcgg caaggctggc ccggtcggca ccagttgcgt
     1501 gagcggaaag atggccgctt cccggccctg ctgcagggag ctcaaaatga aggacgcggc
     1561 gctcgggaga gcgggcgggt gagtcaccca cacaaaggaa aagggccttt ccgtcctcag
     1621 ccgtcgcttc atgtgactcc acggagtacc gggcgccgtc caggcacctc gattagttct
     1681 cgagcttttg gagtacgtcg tctttaggtt ggggggaggg gttttatgcg atggagtttc
     1741 cccacactga gtgggtggag actgaagtta ggccagcttg gcacttgatg taattctcct
     1801 tggaatttgc cctttttgag tttggatctt ggttcattct caagcctcag acagtggttc
     1861 aaagtttttt tcttccattt caggtgtcgt gacgtacggc caccatggct tcaaacttta
     1921 ctcagttcgt gctcgtggac aatggtggga caggggatgt gacagtggct ccttctaatt
     1981 tcgctaatgg ggtggcagag tggatcagct ccaactcacg gagccaggcc tacaaggtga
     2041 catgcagcgt caggcagtct agtgcccaga agagaaagta taccatcaag gtggaggtcc
     2101 ccaaagtggc tacccagaca gtgggcggag tcgaactgcc tgtcgccgct tggaggtcct
     2161 acctgaacat ggagctcact atcccaattt tcgctaccaa ttctgactgt gaactcatcg
     2221 tgaaggcaat gcaggggctc ctcaaagacg gtaatcctat cccttccgcc atcgccgcta
     2281 actcaggtat ctacagcgct ggaggaggtg gaagcggagg aggaggaagc ggaggaggag
     2341 gtagcggacc taagaaaaag aggaaggtgg cggccgctgg atccccttca gggcagatca
     2401 gcaaccaggc cctggctctg gcccctagct ccgctccagt gctggcccag actatggtgc
     2461 cctctagtgc tatggtgcct ctggcccagc cacctgctcc agcccctgtg ctgaccccag
     2521 gaccacccca gtcactgagc gctccagtgc ccaagtctac acaggccggc gaggggactc
     2581 tgagtgaagc tctgctgcac ctgcagttcg acgctgatga ggacctggga gctctgctgg
     2641 ggaacagcac cgatcccgga gtgttcacag atctggcctc cgtggacaac tctgagtttc
     2701 agcagctgct gaatcagggc gtgtccatgt ctcatagtac agccgaacca atgctgatgg
     2761 agtaccccga agccattacc cggctggtga ccggcagcca gcggcccccc gaccccgctc
     2821 caactcccct gggaaccagc ggcctgccta atgggctgtc cggagatgaa gacttctcaa
     2881 gcatcgctga tatggacttt agtgccctgc tgtcacagat ttcctctagt gggcagggag
     2941 gaggtggaag cggcttcagc gtggacacca gtgccctgct ggacctgttc agcccctcgg
     3001 tgaccgtgcc cgacatgagc ctgcctgacc ttgacagcag cctggccagt atccaagagc
     3061 tcctgtctcc ccaggagccc cccaggcctc ccgaggcaga gaacagcagc ccggattcag
     3121 ggaagcagct ggtgcactac acagcgcagc cgctgttcct gctggacccc ggctccgtgg
     3181 acaccgggag caacgacctg ccggtgctgt ttgagctggg agagggctcc tacttctccg
     3241 aaggggacgg cttcgccgag gaccccacca tctccctgct gacaggctcg gagcctccca
     3301 aagccaagga ccccactgtc tcctaagaat tcgatatcaa gcttaataaa agatctttat
     3361 tttcattaga tctgtgtgtt ggttttttgt gtggtaacca cgtgcggacc gagcggccgc
     3421 aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg
     3481 ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc
     3541 gagcgcgcag ctgcctgcag gggcgcctga tgcggtattt tctccttacg catctgtgcg
     3601 gtatttcaca ccgcatacgt caaagcaacc atagtacgcg ccctgtagcg gcgcattaag
     3661 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccttagcgcc
     3721 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
     3781 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
     3841 aaaacttgat ttgggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg
     3901 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac
     3961 actcaactct atctcgggct attcttttga tttataaggg attttgccga tttcggtcta
     4021 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac
     4081 gtttacaatt ttatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca
     4141 gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc
     4201 cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc
     4261 atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt
     4321 catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac
     4381 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc
     4441 ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt
     4501 cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct
     4561 ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga
     4621 tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag
     4681 cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca
     4741 actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga
     4801 aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag
     4861 tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc
     4921 ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa
     4981 tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt
     5041 gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg
     5101 gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt
     5161 tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg
     5221 gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat
     5281 ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact
     5341 gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa
     5401 aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt
     5461 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     5521 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     5581 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     5641 gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     5701 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     5761 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     5821 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     5881 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
     5941 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     6001 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     6061 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
     6121 acggttcctg gccttttgct ggccttttgc tcacatgt
//
