LOCUS       sequence-181400-        7935 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  Modified CAG promoter-containing vector for ubiquitous expression of
            catalytically inactive Cas9 fused to KRAB repressor domain. For 
            targeted enhancer inactivation in chicken embryos..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 7935)
  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 7935)
  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-KRAB
FEATURES             Location/Qualifiers
     source          1..7935
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     CDS             18..38
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(48..74,75..77,78..2561,2562..2567,2568..2633,
                     2634..2636,2637..4151)
                     /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             4164..4184
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4218..4412
                     /codon_start=1
                     /product="Kruppel-associated box (KRAB) transcriptional 
                     repression domain from the human zinc finger protein ZNF10 
                     (Margolin et al., 1994)"
                     /label=KRAB
                     /translation="RTLVTFKDVFVDFTREEWKLLDTAQQILYRNVMLENYKNLVSLGY
                     QLTKPDVILRLEKGEEPWLV"
     CDS             4479..4499
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(4536..4553)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4542..4749
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   4758..4898
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4758..4887
                     /label=AAV2 ITR
     rep_origin      4973..5428
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5060..5079)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5270..5291
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5445..5464)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5564..5586
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5624..5642)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5710..5814
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5815..5883,5884..6675)
                     /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(6033..6052)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6846..7434
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7335..7354
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        7496..7744
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     7496..7516
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     7667..7686
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        7763..7838
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
ORIGIN
        1 ggaccggtgc caccatgccc aagaagaaga ggaaggtggg aagggggatg gacaagaagt
       61 actccattgg gctcgctatc ggcacaaaca gcgtcggctg ggccgtcatt acggacgagt
      121 acaaggtgcc gagcaaaaaa ttcaaagttc tgggcaatac cgatcgccac agcataaaga
      181 agaacctcat tggcgccctc ctgttcgact ccggggagac ggccgaagcc acgcggctca
      241 aaagaacagc acggcgcaga tatacccgca gaaagaatcg gatctgctac ctgcaggaga
      301 tctttagtaa tgagatggct aaggtggatg actctttctt ccataggctg gaggagtcct
      361 ttttggtgga ggaggataaa aagcacgagc gccacccaat ctttggcaat atcgtggacg
      421 aggtggcgta ccatgaaaag tacccaacca tatatcatct gaggaagaag cttgtagaca
      481 gtactgataa ggctgacttg cggttgatct atctcgcgct ggcgcatatg atcaaatttc
      541 ggggacactt cctcatcgag ggggacctga acccagacaa cagcgatgtc gacaaactct
      601 ttatccaact ggttcagact tacaatcagc ttttcgaaga gaacccgatc aatgcatccg
      661 gagttgacgc caaagcaatc ctgagcgcta ggctgtccaa atcccggcgg ctcgaaaacc
      721 tcatcgcaca gctccctggg gagaagaaga acggcctgtt tggtaatctt atcgccctgt
      781 cactcgggct gacccccaac tttaaatcta acttcgacct ggccgaagat gccaagcttc
      841 aactgagcaa agacacctac gatgatgatc tcgacaatct gctggcccag atcggcgacc
      901 agtacgcaga cctttttttg gcggcaaaga acctgtcaga cgccattctg ctgagtgata
      961 ttctgcgagt gaacacggag atcaccaaag ctccgctgag cgctagtatg atcaagcgct
     1021 atgatgagca ccaccaagac ttgactttgc tgaaggccct tgtcagacag caactgcctg
     1081 agaagtacaa ggaaattttc ttcgatcagt ctaaaaatgg ctacgccgga tacattgacg
     1141 gcggagcaag ccaggaggaa ttttacaaat ttattaagcc catcttggaa aaaatggacg
     1201 gcaccgagga gctgctggta aagcttaaca gagaagatct gttgcgcaaa cagcgcactt
     1261 tcgacaatgg aagcatcccc caccagattc acctgggcga actgcacgct atcctcaggc
     1321 ggcaagagga tttctacccc tttttgaaag ataacaggga aaagattgag aaaatcctca
     1381 catttcggat accctactat gtaggccccc tcgcccgggg aaattccaga ttcgcgtgga
     1441 tgactcgcaa atcagaagag accatcactc cctggaactt cgaggaagtc gtggataagg
     1501 gggcctctgc ccagtccttc atcgaaagga tgactaactt tgataaaaat ctgcctaacg
     1561 aaaaggtgct tcctaaacac tctctgctgt acgagtactt cacagtttat aacgagctca
     1621 ccaaggtcaa atacgtcaca gaagggatga gaaagccagc attcctgtct ggagagcaga
     1681 agaaagctat cgtggacctc ctcttcaaga cgaaccggaa agttaccgtg aaacagctca
     1741 aagaggacta tttcaaaaag attgaatgtt tcgactctgt tgaaatcagc ggagtggagg
     1801 atcgcttcaa cgcatccctg ggaacgtatc acgatctcct gaaaatcatt aaagacaagg
     1861 acttcctgga caatgaggag aacgaggaca ttcttgagga cattgtcctc acccttacgt
     1921 tgtttgaaga tagggagatg attgaagaac gcttgaaaac ttacgctcat ctcttcgacg
     1981 acaaagtcat gaaacagctc aagaggcgcc gatatacagg atgggggcgg ctgtcaagaa
     2041 aactgatcaa tgggatccga gacaagcaga gtggaaagac aatcctggat tttcttaagt
     2101 ccgatggatt tgccaaccgg aacttcatgc agttgatcca tgatgactct ctcaccttta
     2161 aggaggacat ccagaaagca caagtttctg gccaggggga cagtctccac gagcacatcg
     2221 ctaatcttgc aggtagccca gctatcaaaa agggaatact gcagaccgtt aaggtcgtgg
     2281 atgaactcgt caaagtaatg ggaaggcata agcccgagaa tatcgttatc gagatggccc
     2341 gagagaacca aactacccag aagggacaga agaacagtag ggaaaggatg aagaggattg
     2401 aagagggtat aaaagaactg gggtcccaaa tccttaagga acacccagtt gaaaacaccc
     2461 agcttcagaa tgagaagctc tacctgtact acctgcagaa cggcagggac atgtacgtgg
     2521 atcaggaact ggacatcaat cggctctccg actacgacgt ggctgctatc gtgccccagt
     2581 cttttctcaa agatgattct attgataata aagtgttgac aagatccgat aaagctagag
     2641 ggaagagtga taacgtcccc tcagaagaag ttgtcaagaa aatgaaaaat tattggcggc
     2701 agctgctgaa cgccaaactg atcacacaac ggaagttcga taatctgact aaggctgaac
     2761 gaggtggcct gtctgagttg gataaagccg gcttcatcaa aaggcagctt gttgagacac
     2821 gccagatcac caagcacgtg gcccaaattc tcgattcacg catgaacacc aagtacgatg
     2881 aaaatgacaa actgattcga gaggtgaaag ttattactct gaagtctaag ctggtctcag
     2941 atttcagaaa ggactttcag ttttataagg tgagagagat caacaattac caccatgcgc
     3001 atgatgccta cctgaatgca gtggtaggca ctgcacttat caaaaaatat cccaagcttg
     3061 aatctgaatt tgtttacgga gactataaag tgtacgatgt taggaaaatg atcgcaaagt
     3121 ctgagcagga aataggcaag gccaccgcta agtacttctt ttacagcaat attatgaatt
     3181 ttttcaagac cgagattaca ctggccaatg gagagattcg gaagcgacca cttatcgaaa
     3241 caaacggaga aacaggagaa atcgtgtggg acaagggtag ggatttcgcg acagtccgga
     3301 aggtcctgtc catgccgcag gtgaacatcg ttaaaaagac cgaagtacag accggaggct
     3361 tctccaagga aagtatcctc ccgaaaagga acagcgacaa gctgatcgca cgcaaaaaag
     3421 attgggaccc caagaaatac ggcggattcg attctcctac agtcgcttac agtgtactgg
     3481 ttgtggccaa agtggagaaa gggaagtcta aaaaactcaa aagcgtcaag gaactgctgg
     3541 gcatcacaat catggagcga tcaagcttcg aaaaaaaccc catcgacttt ctcgaggcga
     3601 aaggatataa agaggtcaaa aaagacctca tcattaagct tcccaagtac tctctctttg
     3661 agcttgaaaa cggccggaaa cgaatgctcg ctagtgcggg cgagctgcag aaaggtaacg
     3721 agctggcact gccctctaaa tacgttaatt tcttgtatct ggccagccac tatgaaaagc
     3781 tcaaagggtc tcccgaagat aatgagcaga agcagctgtt cgtggaacaa cacaaacact
     3841 accttgatga gatcatcgag caaataagcg aattctccaa aagagtgatc ctcgccgacg
     3901 ctaacctcga taaggtgctt tctgcttaca ataagcacag ggataagccc atcagggagc
     3961 aggcagaaaa cattatccac ttgtttactc tgaccaactt gggcgcgcct gcagccttca
     4021 agtacttcga caccaccata gacagaaagc ggtacacctc tacaaaggag gtcctggacg
     4081 ccacactgat tcatcagtca attacggggc tctatgaaac aagaatcgac ctctctcagc
     4141 tcggtggaga cagcagggct gaccccaaga agaagaggaa ggtggctagc gatgctaagt
     4201 cactgactgc ctggtcccgg acactggtga ccttcaagga tgtgtttgtg gacttcacca
     4261 gggaggagtg gaagctgctg gacactgctc agcagatcct gtacagaaat gtgatgctgg
     4321 agaactataa gaacctggtt tccttgggtt atcagcttac taagccagat gtgatcctcc
     4381 ggttggagaa gggagaagag ccctggctgg tggagagaga aattcaccaa gagacccatc
     4441 ctgattcaga gactgcattt gaaatcaaat catcagttcc gaaaaagaaa cgcaaagttg
     4501 ctagctgata agaattccta gagctcgctg atcagcctcg actgtgcctt ctagttgcca
     4561 gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac
     4621 tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat
     4681 tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaga atagcaggca
     4741 tgctggggag cggccgcagg aacccctagt gatggagttg gccactccct ctctgcgcgc
     4801 tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc
     4861 ggcctcagtg agcgagcgag cgcgcagctg cctgcagggg cgcctgatgc ggtattttct
     4921 ccttacgcat ctgtgcggta tttcacaccg catacgtcaa agcaaccata gtacgcgccc
     4981 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt
     5041 gccagcgcct tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc
     5101 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta
     5161 cggcacctcg accccaaaaa acttgatttg ggtgatggtt cacgtagtgg gccatcgccc
     5221 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg
     5281 ttccaaactg gaacaacact caactctatc tcgggctatt cttttgattt ataagggatt
     5341 ttgccgattt cggtctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat
     5401 tttaacaaaa tattaacgtt tacaatttta tggtgcactc tcagtacaat ctgctctgat
     5461 gccgcatagt taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct
     5521 tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt
     5581 cagaggtttt caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta
     5641 tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg
     5701 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     5761 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
     5821 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
     5881 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
     5941 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
     6001 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt
     6061 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
     6121 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
     6181 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
     6241 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
     6301 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta
     6361 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
     6421 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
     6481 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg aagccgcggt
     6541 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
     6601 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
     6661 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
     6721 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     6781 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     6841 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     6901 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     6961 ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac
     7021 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     7081 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     7141 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     7201 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     7261 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     7321 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     7381 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     7441 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgtgaggg
     7501 cctatttccc atgattcctt catatttgca tatacgatac aaggctgtta gagagataat
     7561 tggaattaat ttgactgtaa acacaaagat attagtacaa aatacgtgac gtagaaagta
     7621 ataatttctt gggtagtttg cagttttaaa attatgtttt aaaatggact atcatatgct
     7681 taccgtaact tgaaagtatt tcgatttctt ggctttatat atcttgtgga aaggacgaaa
     7741 caccgggtct tcgagaagac ctgttttaga gctagaaata gcaagttaaa ataaggctag
     7801 tccgttatca acttgaaaaa gtggcaccga gtcggtgctt ttttgtttta gagctagaaa
     7861 tagcaagtta aaataaggct agtccgtttt tagcgcgtgc gccaattctg cagacaaatg
     7921 gctctagagg taccc
//
