LOCUS       40924_46983        8596 bp DNA     circular SYN 13-MAY-2021
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      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8596)
  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 8596)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 8596)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Development."; date: "2018-02-23"; pages: " 10.1242/dev.160333"
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..8596
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      24..612
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     promoter        674..914
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     misc_RNA        941..1016
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        1113..1398
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer;
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        1400..1677
                     /label=chicken beta-actin promoter
     intron          1678..1906
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute
                     virus of mice (MMV) introns (Gray et al., 2011)"
     CDS             1927..1947
                     /codon_start=1
                     /label=SV40 NLS
                     /note="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /translation="PKKKRKV"
     CDS             1957..6060
                     /codon_start=1
                     /label=Cas9m4
                     /note="catalytically dead mutant of the Cas9 endonuclease
                     from the Streptococcus pyogenes Type II CRISPR/Cas system"
                     /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             6073..6093
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6118..6267
                     /codon_start=1
                     /label=VP64
                     /note="tetrameric repeat of the minimal activation domain
                     of herpes simplex virus VP16 (Beerli et al., 1998)"
                     /translation="DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDF
                     DLDML"
     polyA_signal    6316..6523
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   6532..6672
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     rep_origin      6747..7202
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7219..7238)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable
                     marker"
     primer_bind     7338..7360
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(7398..7416)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward
                     primer"
     promoter        7484..7588
                     /label=AmpR promoter
     CDS             7589..8446
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
ORIGIN
        1 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
       61 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
      121 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg
      181 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
      241 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
      301 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
      361 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
      421 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
      481 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
      541 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
      601 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
      661 tttgctcaca tgtgagggcc tatttcccat gattccttca tatttgcata tacgatacaa
      721 ggctgttaga gagataattg gaattaattt gactgtaaac acaaagatat tagtacaaaa
      781 tacgtgacgt agaaagtaat aatttcttgg gtagtttgca gttttaaaat tatgttttaa
      841 aatggactat catatgctta ccgtaacttg aaagtatttc gatttcttgg ctttatatat
      901 cttgtggaaa ggacgaaaca ccgggtcttc gagaagacct gttttagagc tagaaatagc
      961 aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt
     1021 ttgttttaga gctagaaata gcaagttaaa ataaggctag tccgttttta gcgcgtgcgc
     1081 caattctgca gacaaatggc tctagaggta cccgttacat aacttacggt aaatggcccg
     1141 cctggctgac cgcccaacga cccccgccca ttgacgtcaa tagtaacgcc aatagggact
     1201 ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa
     1261 gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg
     1321 cattgtgccc agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta
     1381 gtcatcgcta ttaccatggt cgaggtgagc cccacgttct gcttcactct ccccatctcc
     1441 cccccctccc cacccccaat tttgtattta tttatttttt aattattttg tgcagcgatg
     1501 ggggcggggg gggggggggg gcgcgcgcca ggcggggcgg ggcggggcga ggggcggggc
     1561 ggggcgaggc ggagaggtgc ggcggcagcc aatcagagcg gcgcgctccg aaagtttcct
     1621 tttatggcga ggcggcggcg gcggcggccc tataaaaagc gaagcgcgcg gcgggcggga
     1681 gtcgctgcga cgctgccttc gccccgtgcc ccgctccgcc gccgcctcgc gccgcccgcc
     1741 ccggctctga ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc cttctcctcc
     1801 gggctgtaat tagctgagca agaggtaagg gtttaaggga tggttggttg gtggggtatt
     1861 aatgtttaat tacctggagc acctgcctga aatcactttt tttcaggttg gaccggtgcc
     1921 accatgccca agaagaagag gaaggtggga agggggatgg acaagaagta ctccattggg
     1981 ctcgctatcg gcacaaacag cgtcggctgg gccgtcatta cggacgagta caaggtgccg
     2041 agcaaaaaat tcaaagttct gggcaatacc gatcgccaca gcataaagaa gaacctcatt
     2101 ggcgccctcc tgttcgactc cggggagacg gccgaagcca cgcggctcaa aagaacagca
     2161 cggcgcagat atacccgcag aaagaatcgg atctgctacc tgcaggagat ctttagtaat
     2221 gagatggcta aggtggatga ctctttcttc cataggctgg aggagtcctt tttggtggag
     2281 gaggataaaa agcacgagcg ccacccaatc tttggcaata tcgtggacga ggtggcgtac
     2341 catgaaaagt acccaaccat atatcatctg aggaagaagc ttgtagacag tactgataag
     2401 gctgacttgc ggttgatcta tctcgcgctg gcgcatatga tcaaatttcg gggacacttc
     2461 ctcatcgagg gggacctgaa cccagacaac agcgatgtcg acaaactctt tatccaactg
     2521 gttcagactt acaatcagct tttcgaagag aacccgatca acgcatccgg agttgacgcc
     2581 aaagcaatcc tgagcgctag gctgtccaaa tcccggcggc tcgaaaacct catcgcacag
     2641 ctccctgggg agaagaagaa cggcctgttt ggtaatctta tcgccctgtc actcgggctg
     2701 acccccaact ttaaatctaa cttcgacctg gccgaagatg ccaagcttca actgagcaaa
     2761 gacacctacg atgatgatct cgacaatctg ctggcccaga tcggcgacca gtacgcagac
     2821 ctttttttgg cggcaaagaa cctgtcagac gccattctgc tgagtgatat tctgcgagtg
     2881 aacacggaga tcaccaaagc tccgctgagc gctagtatga tcaagcgcta tgatgagcac
     2941 caccaagact tgactttgct gaaggccctt gtcagacagc aactgcctga gaagtacaag
     3001 gaaattttct tcgatcagtc taaaaatggc tacgccggat acattgacgg cggagcaagc
     3061 caggaggaat tttacaaatt tattaagccc atcttggaaa aaatggacgg caccgaggag
     3121 ctgctggtaa agcttaacag agaagatctg ttgcgcaaac agcgcacttt cgacaatgga
     3181 agcatccccc accagattca cctgggcgaa ctgcacgcta tcctcaggcg gcaagaggat
     3241 ttctacccct ttttgaaaga taacagggaa aagattgaga aaatcctcac atttcggata
     3301 ccctactatg taggccccct cgcccgggga aattccagat tcgcgtggat gactcgcaaa
     3361 tcagaagaga ccatcactcc ctggaacttc gaggaagtcg tggataaggg ggcctctgcc
     3421 cagtccttca tcgaaaggat gactaacttt gataaaaatc tgcctaacga aaaggtgctt
     3481 cctaaacact ctctgctgta cgagtacttc acagtttata acgagctcac caaggtcaaa
     3541 tacgtcacag aagggatgag aaagccagca ttcctgtctg gagagcagaa gaaagctatc
     3601 gtggacctcc tcttcaagac gaaccggaaa gttaccgtga aacagctcaa agaagactat
     3661 ttcaaaaaga ttgaatgttt cgactctgtt gaaatcagcg gagtggagga tcgcttcaac
     3721 gcatccctgg gaacgtatca cgatctcctg aaaatcatta aagacaagga cttcctggac
     3781 aatgaggaga acgaggacat tcttgaggac attgtcctca cccttacgtt gtttgaagat
     3841 agggagatga ttgaagaacg cttgaaaact tacgctcatc tcttcgacga caaagtcatg
     3901 aaacagctca agaggcgccg atatacagga tgggggcggc tgtcaagaaa actgatcaat
     3961 gggatccgag acaagcagag tggaaagaca atcctggatt ttcttaagtc cgatggattt
     4021 gccaaccgga acttcatgca gttgatccat gatgactctc tcacctttaa ggaggacatc
     4081 cagaaagcac aagtttctgg ccagggggac agtcttcacg agcacatcgc taatcttgca
     4141 ggtagcccag ctatcaaaaa gggaatactg cagaccgtta aggtcgtgga tgaactcgtc
     4201 aaagtaatgg gaaggcataa gcccgagaat atcgttatcg agatggcccg agagaaccaa
     4261 actacccaga agggacagaa gaacagtagg gaaaggatga agaggattga agagggtata
     4321 aaagaactgg ggtcccaaat ccttaaggaa cacccagttg aaaacaccca gcttcagaat
     4381 gagaagctct acctgtacta cctgcagaac ggcagggaca tgtacgtgga tcaggaactg
     4441 gacatcaatc ggctctccga ctacgacgtg gctgctatcg tgccccagtc ttttctcaaa
     4501 gatgattcta ttgataataa agtgttgaca agatccgata aagctagagg gaagagtgat
     4561 aacgtcccct cagaagaagt tgtcaagaaa atgaaaaatt attggcggca gctgctgaac
     4621 gccaaactga tcacacaacg gaagttcgat aatctgacta aggctgaacg aggtggcctg
     4681 tctgagttgg ataaagccgg cttcatcaaa aggcagcttg ttgagacacg ccagatcacc
     4741 aagcacgtgg cccaaattct cgattcacgc atgaacacca agtacgatga aaatgacaaa
     4801 ctgattcgag aggtgaaagt tattactctg aagtctaagc tggtctcaga tttcagaaag
     4861 gactttcagt tttataaggt gagagagatc aacaattacc accatgcgca tgatgcctac
     4921 ctgaatgcag tggtaggcac tgcacttatc aaaaaatatc ccaagcttga atctgaattt
     4981 gtttacggag actataaagt gtacgatgtt aggaaaatga tcgcaaagtc tgagcaggaa
     5041 ataggcaagg ccaccgctaa gtacttcttt tacagcaata ttatgaattt tttcaagacc
     5101 gagattacac tggccaatgg agagattcgg aagcgaccac ttatcgaaac aaacggagaa
     5161 acaggagaaa tcgtgtggga caagggtagg gatttcgcga cagtccggaa ggtcctgtcc
     5221 atgccgcagg tgaacatcgt taaaaagacc gaagtacaga ccggaggctt ctccaaggaa
     5281 agtatcctcc cgaaaaggaa cagcgacaag ctgatcgcac gcaaaaaaga ttgggacccc
     5341 aagaaatacg gcggattcga ttctcctaca gtcgcttaca gtgtactggt tgtggccaaa
     5401 gtggagaaag ggaagtctaa aaaactcaaa agcgtcaagg aactgctggg catcacaatc
     5461 atggagcgat caagcttcga aaaaaacccc atcgactttc tcgaggcgaa aggatataaa
     5521 gaggtcaaaa aagacctcat cattaagctt cccaagtact ctctctttga gcttgaaaac
     5581 ggccggaaac gaatgctcgc tagtgcgggc gagctgcaga aaggtaacga gctggcactg
     5641 ccctctaaat acgttaattt cttgtatctg gccagccact atgaaaagct caaagggtct
     5701 cccgaagata atgagcagaa gcagctgttc gtggaacaac acaaacacta ccttgatgag
     5761 atcatcgagc aaataagcga attctccaaa agagtgatcc tcgccgacgc taacctcgat
     5821 aaggtgcttt ctgcttacaa taagcacagg gataagccca tcagggagca ggcagaaaac
     5881 attatccact tgtttactct gaccaacttg ggcgcgcctg cagccttcaa gtacttcgac
     5941 accaccatag acagaaagcg gtacacctct acaaaggagg tcctggacgc cacactgatt
     6001 catcagtcaa ttacggggct ctatgaaaca agaatcgacc tctctcagct cggtggagac
     6061 agcagggctg accccaagaa gaagaggaag gtggaggcca gcggttccgg acgggctgac
     6121 gcattggacg attttgatct ggatatgctg ggaagtgacg ccctcgatga ttttgacctt
     6181 gacatgcttg gttcggatgc ccttgatgac tttgacctcg acatgctcgg cagtgacgcc
     6241 cttgatgatt tcgacctgga catgctgatt aactctagat gataagaatt cctagagctc
     6301 gctgatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg
     6361 tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa
     6421 ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca
     6481 gcaaggggga ggattgggaa gagaatagca ggcatgctgg ggagcggccg caggaacccc
     6541 tagtgatgga gttggccact ccctctctgc gcgctcgctc gctcactgag gccgggcgac
     6601 caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc agtgagcgag cgagcgcgca
     6661 gctgcctgca ggggcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac
     6721 accgcatacg tcaaagcaac catagtacgc gccctgtagc ggcgcattaa gcgcggcggg
     6781 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt
     6841 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
     6901 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
     6961 tttgggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
     7021 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc
     7081 tatctcgggc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa
     7141 aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa cgtttacaat
     7201 tttatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agccccgaca
     7261 cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag
     7321 acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa
     7381 acgcgcgaga cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat
     7441 aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
     7501 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
     7561 gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat
     7621 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt
     7681 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag
     7741 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa
     7801 agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg
     7861 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct
     7921 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac
     7981 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca
     8041 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
     8101 accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact
     8161 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc
     8221 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga
     8281 taaatctgga gccggtgagc gtggaagccg cggtatcatt gcagcactgg ggccagatgg
     8341 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
     8401 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca
     8461 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
     8521 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     8581 ctgagcgtca gacccc
//
