LOCUS       sequence-375163-        6717 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 6717)
  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 6717)
  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: pGW045 AAV-CRISPRa base vector
            pAAV_OE-U6sgbbS...
FEATURES             Location/Qualifiers
     source          1..6717
                     /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"
     misc_feature    3348..3936
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3401..3421)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3819..3830)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     repeat_region   3980..4120
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3980..4109
                     /label=AAV2 ITR
     rep_origin      4195..4650
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4282..4301)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4492..4513
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4667..4686)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4786..4808
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4846..4864)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4932..5036
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5037..5105,5106..5897)
                     /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(5255..5274)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6068..6656
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6557..6576
                     /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 tcacgcgtta agtcgacaat caacctctgg
     3361 attacaaaat ttgtgaaaga ttgactggta ttcttaacta tgttgctcct tttacgctat
     3421 gtggatacgc tgctttaatg cctttgtatc atgctattgc ttcccgtatg gctttcattt
     3481 tctcctcctt gtataaatcc tggttgctgt ctctttatga ggagttgtgg cccgttgtca
     3541 ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac ccccactggt tggggcattg
     3601 ccaccacctg tcagctcctt tccgggactt tcgctttccc cctccctatt gccacggcgg
     3661 aactcatcgc cgcctgcctt gcccgctgct ggacaggggc tcggctgttg ggcactgaca
     3721 attccgtggt gttgtcgggg aaatcatcgt cctttccttg gctgctcgcc tgtgttgcca
     3781 cctggattct gcgcgggacg tccttctgct acgtcccttc ggccctcaat ccagcggacc
     3841 ttccttcccg cggcctgctg ccggctctgc ggcctcttcc gcgtcttcgc cttcgccctc
     3901 agacgagtcg gatctccctt tgggccgcct ccccgcgtcg actttaagac caatgaccac
     3961 gtgcggaccg agcggccgca ggaaccccta gtgatggagt tggccactcc ctctctgcgc
     4021 gctcgctcgc tcactgaggc cgggcgacca aaggtcgccc gacgcccggg ctttgcccgg
     4081 gcggcctcag tgagcgagcg agcgcgcagc tgcctgcagg ggcgcctgat gcggtatttt
     4141 ctccttacgc atctgtgcgg tatttcacac cgcatacgtc aaagcaacca tagtacgcgc
     4201 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
     4261 ttgccagcgc cttagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
     4321 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
     4381 tacggcacct cgaccccaaa aaacttgatt tgggtgatgg ttcacgtagt gggccatcgc
     4441 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct
     4501 tgttccaaac tggaacaaca ctcaactcta tctcgggcta ttcttttgat ttataaggga
     4561 ttttgccgat ttcggtctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
     4621 attttaacaa aatattaacg tttacaattt tatggtgcac tctcagtaca atctgctctg
     4681 atgccgcata gttaagccag ccccgacacc cgccaacacc cgctgacgcg ccctgacggg
     4741 cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt
     4801 gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc
     4861 tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc
     4921 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc
     4981 cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga
     5041 gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt
     5101 ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag
     5161 tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag
     5221 aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta
     5281 ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg
     5341 agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca
     5401 gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag
     5461 gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc
     5521 gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg
     5581 tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc
     5641 ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg
     5701 cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg
     5761 gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga
     5821 cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac
     5881 tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa
     5941 aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca
     6001 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     6061 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     6121 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     6181 ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc
     6241 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     6301 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     6361 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     6421 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
     6481 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
     6541 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     6601 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
     6661 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgt
//
