LOCUS       sequence-181403-        7790 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  Modified CAG promoter-containing vector for ubiquitous expression of
            catalytically inactive Cas9 fused to VP64 transcriptional activation
            domain. For targeted gene activation in chicken embryos..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 7790)
  AUTHORS   Williams RM, Senanayake U, Artibani M, Taylor G, Wells D, Ahmed AA, 
            Sauka-Spengler T
  TITLE     Genome and epigenome engineering CRISPR toolkit for in vivo 
            modulation of cis-regulatory interactions and gene expression in the
            chicken embryo.
  JOURNAL   Development. 2018 Feb 23;145(4). pii: dev.160333. doi: 
            10.1242/dev.160333.
  PUBMED    29386245
REFERENCE   2  (bases 1 to 7790)
  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: 
            "Development."; date: "2018-02-23"; volume: "145(4). pii: 
            dev.160333. doi"; pages: " 10.1242/dev.160333"
COMMENT     Sequence Label: pX330a dCas9-VP64
FEATURES             Location/Qualifiers
     source          1..7790
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     CDS             13..33
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(43..69,70..72,73..2556,2557..2562,2563..2628,
                     2629..2631,2632..4146)
                     /codon_start=1
                     /product="catalytically dead mutant of the Cas9 
                     endonuclease from the Streptococcus pyogenes Type II 
                     CRISPR/Cas system"
                     /label=Cas9m4
                     /note="RNA-guided DNA-binding protein that lacks 
                     endonuclease activity due to the mutations D10A, D839A, 
                     H840A, and N863A (Mali et al., 2013)"
                     /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVAAIVPQSFLKDDSIDNKVLTRSDKARGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             4159..4179
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4204..4353
                     /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"
     primer_bind     complement(4396..4413)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4402..4609
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   4618..4758
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4618..4747
                     /label=AAV2 ITR
     rep_origin      4833..5288
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4920..4939)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5130..5151
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5305..5324)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5424..5446
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5484..5502)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5570..5674
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5675..5743,5744..6535)
                     /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(5893..5912)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6706..7294
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7195..7214
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        7356..7604
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     7356..7376
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     7527..7546
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        7623..7698
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
ORIGIN
        1 ggtgccacca tgcccaagaa gaagaggaag gtgggaaggg ggatggacaa gaagtactcc
       61 attgggctcg ctatcggcac aaacagcgtc ggctgggccg tcattacgga cgagtacaag
      121 gtgccgagca aaaaattcaa agttctgggc aataccgatc gccacagcat aaagaagaac
      181 ctcattggcg ccctcctgtt cgactccggg gagacggccg aagccacgcg gctcaaaaga
      241 acagcacggc gcagatatac ccgcagaaag aatcggatct gctacctgca ggagatcttt
      301 agtaatgaga tggctaaggt ggatgactct ttcttccata ggctggagga gtcctttttg
      361 gtggaggagg ataaaaagca cgagcgccac ccaatctttg gcaatatcgt ggacgaggtg
      421 gcgtaccatg aaaagtaccc aaccatatat catctgagga agaagcttgt agacagtact
      481 gataaggctg acttgcggtt gatctatctc gcgctggcgc atatgatcaa atttcgggga
      541 cacttcctca tcgaggggga cctgaaccca gacaacagcg atgtcgacaa actctttatc
      601 caactggttc agacttacaa tcagcttttc gaagagaacc cgatcaatgc atccggagtt
      661 gacgccaaag caatcctgag cgctaggctg tccaaatccc ggcggctcga aaacctcatc
      721 gcacagctcc ctggggagaa gaagaacggc ctgtttggta atcttatcgc cctgtcactc
      781 gggctgaccc ccaactttaa atctaacttc gacctggccg aagatgccaa gcttcaactg
      841 agcaaagaca cctacgatga tgatctcgac aatctgctgg cccagatcgg cgaccagtac
      901 gcagaccttt ttttggcggc aaagaacctg tcagacgcca ttctgctgag tgatattctg
      961 cgagtgaaca cggagatcac caaagctccg ctgagcgcta gtatgatcaa gcgctatgat
     1021 gagcaccacc aagacttgac tttgctgaag gcccttgtca gacagcaact gcctgagaag
     1081 tacaaggaaa ttttcttcga tcagtctaaa aatggctacg ccggatacat tgacggcgga
     1141 gcaagccagg aggaatttta caaatttatt aagcccatct tggaaaaaat ggacggcacc
     1201 gaggagctgc tggtaaagct taacagagaa gatctgttgc gcaaacagcg cactttcgac
     1261 aatggaagca tcccccacca gattcacctg ggcgaactgc acgctatcct caggcggcaa
     1321 gaggatttct accccttttt gaaagataac agggaaaaga ttgagaaaat cctcacattt
     1381 cggataccct actatgtagg ccccctcgcc cggggaaatt ccagattcgc gtggatgact
     1441 cgcaaatcag aagagaccat cactccctgg aacttcgagg aagtcgtgga taagggggcc
     1501 tctgcccagt ccttcatcga aaggatgact aactttgata aaaatctgcc taacgaaaag
     1561 gtgcttccta aacactctct gctgtacgag tacttcacag tttataacga gctcaccaag
     1621 gtcaaatacg tcacagaagg gatgagaaag ccagcattcc tgtctggaga gcagaagaaa
     1681 gctatcgtgg acctcctctt caagacgaac cggaaagtta ccgtgaaaca gctcaaagag
     1741 gactatttca aaaagattga atgtttcgac tctgttgaaa tcagcggagt ggaggatcgc
     1801 ttcaacgcat ccctgggaac gtatcacgat ctcctgaaaa tcattaaaga caaggacttc
     1861 ctggacaatg aggagaacga ggacattctt gaggacattg tcctcaccct tacgttgttt
     1921 gaagataggg agatgattga agaacgcttg aaaacttacg ctcatctctt cgacgacaaa
     1981 gtcatgaaac agctcaagag gcgccgatat acaggatggg ggcggctgtc aagaaaactg
     2041 atcaatggga tccgagacaa gcagagtgga aagacaatcc tggattttct taagtccgat
     2101 ggatttgcca accggaactt catgcagttg atccatgatg actctctcac ctttaaggag
     2161 gacatccaga aagcacaagt ttctggccag ggggacagtc tccacgagca catcgctaat
     2221 cttgcaggta gcccagctat caaaaaggga atactgcaga ccgttaaggt cgtggatgaa
     2281 ctcgtcaaag taatgggaag gcataagccc gagaatatcg ttatcgagat ggcccgagag
     2341 aaccaaacta cccagaaggg acagaagaac agtagggaaa ggatgaagag gattgaagag
     2401 ggtataaaag aactggggtc ccaaatcctt aaggaacacc cagttgaaaa cacccagctt
     2461 cagaatgaga agctctacct gtactacctg cagaacggca gggacatgta cgtggatcag
     2521 gaactggaca tcaatcggct ctccgactac gacgtggctg ctatcgtgcc ccagtctttt
     2581 ctcaaagatg attctattga taataaagtg ttgacaagat ccgataaagc tagagggaag
     2641 agtgataacg tcccctcaga agaagttgtc aagaaaatga aaaattattg gcggcagctg
     2701 ctgaacgcca aactgatcac acaacggaag ttcgataatc tgactaaggc tgaacgaggt
     2761 ggcctgtctg agttggataa agccggcttc atcaaaaggc agcttgttga gacacgccag
     2821 atcaccaagc acgtggccca aattctcgat tcacgcatga acaccaagta cgatgaaaat
     2881 gacaaactga ttcgagaggt gaaagttatt actctgaagt ctaagctggt ctcagatttc
     2941 agaaaggact ttcagtttta taaggtgaga gagatcaaca attaccacca tgcgcatgat
     3001 gcctacctga atgcagtggt aggcactgca cttatcaaaa aatatcccaa gcttgaatct
     3061 gaatttgttt acggagacta taaagtgtac gatgttagga aaatgatcgc aaagtctgag
     3121 caggaaatag gcaaggccac cgctaagtac ttcttttaca gcaatattat gaattttttc
     3181 aagaccgaga ttacactggc caatggagag attcggaagc gaccacttat cgaaacaaac
     3241 ggagaaacag gagaaatcgt gtgggacaag ggtagggatt tcgcgacagt ccggaaggtc
     3301 ctgtccatgc cgcaggtgaa catcgttaaa aagaccgaag tacagaccgg aggcttctcc
     3361 aaggaaagta tcctcccgaa aaggaacagc gacaagctga tcgcacgcaa aaaagattgg
     3421 gaccccaaga aatacggcgg attcgattct cctacagtcg cttacagtgt actggttgtg
     3481 gccaaagtgg agaaagggaa gtctaaaaaa ctcaaaagcg tcaaggaact gctgggcatc
     3541 acaatcatgg agcgatcaag cttcgaaaaa aaccccatcg actttctcga ggcgaaagga
     3601 tataaagagg tcaaaaaaga cctcatcatt aagcttccca agtactctct ctttgagctt
     3661 gaaaacggcc ggaaacgaat gctcgctagt gcgggcgagc tgcagaaagg taacgagctg
     3721 gcactgccct ctaaatacgt taatttcttg tatctggcca gccactatga aaagctcaaa
     3781 gggtctcccg aagataatga gcagaagcag ctgttcgtgg aacaacacaa acactacctt
     3841 gatgagatca tcgagcaaat aagcgaattc tccaaaagag tgatcctcgc cgacgctaac
     3901 ctcgataagg tgctttctgc ttacaataag cacagggata agcccatcag ggagcaggca
     3961 gaaaacatta tccacttgtt tactctgacc aacttgggcg cgcctgcagc cttcaagtac
     4021 ttcgacacca ccatagacag aaagcggtac acctctacaa aggaggtcct ggacgccaca
     4081 ctgattcatc agtcaattac ggggctctat gaaacaagaa tcgacctctc tcagctcggt
     4141 ggagacagca gggctgaccc caagaagaag aggaaggtgg aggccagcgg ttccggacgg
     4201 gctgacgcat tggacgattt tgatctggat atgctgggaa gtgacgccct cgatgatttt
     4261 gaccttgaca tgcttggttc ggatgccctt gatgactttg acctcgacat gctcggcagt
     4321 gacgcccttg atgatttcga cctggacatg ctgattaact ctagatgata agaattccta
     4381 gagctcgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct
     4441 cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg
     4501 aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc
     4561 aggacagcaa gggggaggat tgggaagaga atagcaggca tgctggggag cggccgcagg
     4621 aacccctagt gatggagttg gccactccct ctctgcgcgc tcgctcgctc actgaggccg
     4681 ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc ggcctcagtg agcgagcgag
     4741 cgcgcagctg cctgcagggg cgcctgatgc ggtattttct ccttacgcat ctgtgcggta
     4801 tttcacaccg catacgtcaa agcaaccata gtacgcgccc tgtagcggcg cattaagcgc
     4861 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgcct tagcgcccgc
     4921 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
     4981 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
     5041 acttgatttg ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
     5101 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
     5161 caactctatc tcgggctatt cttttgattt ataagggatt ttgccgattt cggtctattg
     5221 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt
     5281 tacaatttta tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagcc
     5341 ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc
     5401 ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc
     5461 accgaaacgc gcgagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat
     5521 gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc
     5581 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg
     5641 ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc
     5701 ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt
     5761 gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct
     5821 caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac
     5881 ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact
     5941 cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa
     6001 gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga
     6061 taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt
     6121 tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga
     6181 agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg
     6241 caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat
     6301 ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat
     6361 tgctgataaa tctggagccg gtgagcgtgg aagccgcggt atcattgcag cactggggcc
     6421 agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga
     6481 tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc
     6541 agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag
     6601 gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc
     6661 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     6721 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     6781 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     6841 accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     6901 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     6961 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     7021 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     7081 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     7141 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     7201 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     7261 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     7321 gttcctggcc ttttgctggc cttttgctca catgtgaggg cctatttccc atgattcctt
     7381 catatttgca tatacgatac aaggctgtta gagagataat tggaattaat ttgactgtaa
     7441 acacaaagat attagtacaa aatacgtgac gtagaaagta ataatttctt gggtagtttg
     7501 cagttttaaa attatgtttt aaaatggact atcatatgct taccgtaact tgaaagtatt
     7561 tcgatttctt ggctttatat atcttgtgga aaggacgaaa caccgggtct tcgagaagac
     7621 ctgttttaga gctagaaata gcaagttaaa ataaggctag tccgttatca acttgaaaaa
     7681 gtggcaccga gtcggtgctt ttttgtttta gagctagaaa tagcaagtta aaataaggct
     7741 agtccgtttt tagcgcgtgc gccaattctg cagacaaatg gctctagagg
//
