LOCUS       40924_46973        8731 bp DNA     circular SYN 13-MAY-2021
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      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8731)
  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 8731)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 8731)
  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..8731
                     /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             6127..6321
                     /codon_start=1
                     /label=KRAB
                     /note="Kruppel-associated box (KRAB) transcriptional
                     repression domain from the human zinc finger protein ZNF10 
                     (Margolin et al., 1994)"
                     /translation="RTLVTFKDVFVDFTREEWKLLDTAQQILYRNVMLENYKNLVSLGY
                     QLTKPDVILRLEKGEEPWLV"
     CDS             6388..6408
                     /codon_start=1
                     /label=SV40 NLS
                     /note="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /translation="PKKKRKV"
     polyA_signal    6451..6658
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   6667..6807
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     rep_origin      6882..7337
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7354..7373)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable
                     marker"
     primer_bind     7473..7495
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(7533..7551)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward
                     primer"
     promoter        7619..7723
                     /label=AmpR promoter
     CDS             7724..8581
                     /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 agaggactat
     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 agtctccacg 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 gtggctagcg atgctaagtc actgactgcc
     6121 tggtcccgga cactggtgac cttcaaggat gtgtttgtgg acttcaccag ggaggagtgg
     6181 aagctgctgg acactgctca gcagatcctg tacagaaatg tgatgctgga gaactataag
     6241 aacctggttt ccttgggtta tcagcttact aagccagatg tgatcctccg gttggagaag
     6301 ggagaagagc cctggctggt ggagagagaa attcaccaag agacccatcc tgattcagag
     6361 actgcatttg aaatcaaatc atcagttccg aaaaagaaac gcaaagttgc tagctgataa
     6421 gaattcctag agctcgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg
     6481 tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct
     6541 aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg
     6601 gggtggggca ggacagcaag ggggaggatt gggaagagaa tagcaggcat gctggggagc
     6661 ggccgcagga acccctagtg atggagttgg ccactccctc tctgcgcgct cgctcgctca
     6721 ctgaggccgg gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga
     6781 gcgagcgagc gcgcagctgc ctgcaggggc gcctgatgcg gtattttctc cttacgcatc
     6841 tgtgcggtat ttcacaccgc atacgtcaaa gcaaccatag tacgcgccct gtagcggcgc
     6901 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct
     6961 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
     7021 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
     7081 ccccaaaaaa cttgatttgg gtgatggttc acgtagtggg ccatcgccct gatagacggt
     7141 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg
     7201 aacaacactc aaccctatct cgggctattc ttttgattta taagggattt tgccgatttc
     7261 ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat
     7321 attaacgttt acaattttat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt
     7381 aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc
     7441 ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc
     7501 accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat ttttataggt
     7561 taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg
     7621 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca
     7681 ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt
     7741 ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga
     7801 aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga
     7861 actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat
     7921 gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca
     7981 agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt
     8041 cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac
     8101 catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct
     8161 aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga
     8221 gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac
     8281 aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat
     8341 agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg
     8401 ctggtttatt gctgataaat ctggagccgg tgagcgtgga agccgcggta tcattgcagc
     8461 actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc
     8521 aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg
     8581 gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta
     8641 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg
     8701 tgagttttcg ttccactgag cgtcagaccc c
//
