BB3cK_pGAP_23*_pLAT1_Cas9

LOCUS       BB3cK_pGAP_23*_pLAT1_Cas9                9104 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  hCas9 under control of LAT1 for direct cloning of HH-sgRNA-HDV PCR 
            products for episomal expression in P. pastoris and selection on 
            G418.
ACCESSION   .
VERSION     .
KEYWORDS    BB3cK_pGAP_23*_pLAT1_Cas9
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9104)
  AUTHORS   Gassler T, Heistinger L, Mattanovich D, Gasser B, Prielhofer R.
  TITLE     CRISPR/Cas9-mediated homology directed genome editing in Pichia 
            pastoris
  JOURNAL   (2018) In Press
REFERENCE   2  (bases 1 to 9104)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..9104
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     rep_origin      complement(70..658)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(150..169)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        745..1221
                     /label=GAP promoter
                     /note="promoter for Pichia pastoris 
                     glyceraldehyde-3-phosphate dehydrogenase"
     CDS             2765..6868
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             6881..6901
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     terminator      6973..7220
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(6989..7007)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     gene            7240..8596
                     /label=kanMX
                     /note="yeast selectable marker conferring kanamycin 
                     resistance (Wach et al., 1994)"
     promoter        7240..7583
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             7584..8393
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MGKEKTHVSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(7651..7670)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      8399..8596
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     misc_feature    8608..7
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
ORIGIN
        1 agcgatcgcg ctgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
       61 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
      121 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
      181 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
      241 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
      301 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
      361 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
      421 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
      481 gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa
      541 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
      601 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
      661 agatcctttg atcttttcta cggggtggag cgcaaaaaac cccgcccctg acagggcggg
      721 gttttttcgc gatcggagga tccttttttg tagaaatgtc ttggtgtcct cgtccaatca
      781 ggtagccatc tctgaaatat ctggctccgt tgcaactccg aacgacctgc tggcaacgta
      841 aaattctccg gggtaaaact taaatgtgga gtaatggaac cagaaacgtc tcttcccttc
      901 tctctccttc caccgcccgt taccgtccct aggaaatttt actctgctgg agagcttctt
      961 ctacggcccc cttgcagcaa tgctcttccc agcattacgt tgcgggtaaa acggaggtcg
     1021 tgtacccgac ctagcagccc agggatggaa aagtcccggc cgtcgctggc aataatagcg
     1081 ggcggacgca tgtcatgaga ttattggaaa ccaccagaat cgaatataaa aggcgaacac
     1141 ctttcccaat tttggtttct cctgacccaa agactttaaa tttaatttat ttgtccctat
     1201 ttcaatcaat tgaacaacta tcaaaacacc atggcgtctt cggatccctc gaggaagacg
     1261 cgcttttagt gtacatctga taatatagta ctaccacgta tgataatgta gagaataggc
     1321 ttccttgtcg agtgtgtttg cagttttctt gagtttcaag gtttaaatgc tggtatatta
     1381 gttcatcgaa ggtttcagcc aatagcacct taaatcaatc aaactaattc gactcttacg
     1441 aaagagccta ctgtgtttag tatcgaagtc gtttaccttt catgttgaat agcttcctct
     1501 ctgaccctaa catttcaaga tcctcctaaa gttacccgga ttgtgaaatt ctaatgatcc
     1561 acctgcccaa tgcatttttt ctttattcag tttacctttt ttacctaata tacgagcttg
     1621 ttaaagtaag tggcactgca atactaggct tattgttgat attatgatga atcgttttca
     1681 caaacttgat ttcctgtgaa ctcaccatgt actaaggaaa aaaacatgca tcaccatctg
     1741 aatatttgac cgctccgggg agggaagaat atgaaccacg attgaaagac tttgggtttg
     1801 ccgattctaa aacacttact gaacagaaaa gaagtgaatt attagagagt ttatgcaacg
     1861 gagatcatga gcttagaaag aacgtaggct gggcaacaac atccatttct gcctgcgata
     1921 tttccagtgc aatgctaaag ccacataata taggcaatta caacttgaat gagcaggctc
     1981 atgatgtcac catggaactg atagataaag tcatccaaaa aggtgtcaat ttacggcacg
     2041 tctacgtaga tactgtcgga cctccagcat cataccaaaa gaaactgcaa caaaggtttc
     2101 ccgcactgct gatcaccgta tctaaaaaag cagatgccat ctatccatgt gtttcaagtg
     2161 cttcagtttg cgccaaggtc actagagacg tctgtttgag aacctcaaat ggggaggaga
     2221 acctattaga ttgtgggtct ggatatccct cggacccaaa tactaagaaa tggctagccc
     2281 agtctataca tccattgttt ggctggagtc gtgatgtcag gttttcgtgg cagacggcca
     2341 aagattgtct tgagagacac aactcaatca aagttgtgtt tggcgaagag gagcctaccc
     2401 cggttaattt cggtgatctg gggttgagca aacagaagcc cgactttaag gatttagaaa
     2461 tcagtaccag ttggtgggga cattaattgg tggaggagag ccaatattct caactgttcg
     2521 taggaccata accccccact cgcaccagaa atactcattg gcgctgacaa ctagtttgag
     2581 ccgcttcaat tgcggcaagt ccccagtttt gtttggtact cccctctcgc ctcgcgtgtt
     2641 tttgttcctc cgaattgtgc atcaccctgt cacatccgta caccatcgga acactctgct
     2701 cctcctcctt tctcctctta attgaacgta tatctaatta ccctaataac aagtatcttc
     2761 aaccatggac aagaagtact ccattgggct cgatatcggc acaaacagcg tcggttgggc
     2821 cgtcattacg gacgagtaca aggtgccgag caaaaaattc aaagttctgg gcaataccga
     2881 tcgccacagc ataaagaaga acctcattgg cgccctcctg ttcgactccg gggagacggc
     2941 cgaagccacg cggctcaaaa gaacagcacg gcgcagatat acccgcagaa agaatcggat
     3001 ctgctacctg caggagatct ttagtaatga gatggctaag gtggatgact ctttcttcca
     3061 taggctggag gagtcctttt tggtggagga ggataaaaag cacgagcgcc acccaatctt
     3121 tggcaatatc gtggacgagg tggcgtacca tgaaaagtac ccaaccatat atcatctgag
     3181 gaagaagctt gtagacagta ctgataaggc tgacttgcgg ttgatctatc tcgcgctggc
     3241 gcatatgatc aaatttcggg gacacttcct catcgagggg gacctgaacc cagacaacag
     3301 cgatgtcgac aaactcttta tccaactggt tcagacttac aatcagcttt tcgaagagaa
     3361 cccgatcaac gcatccggag ttgacgccaa agcaatcctg agcgctaggc tgtccaaatc
     3421 ccggcggctc gaaaacctca tcgcacagct ccctggggag aagaagaacg gcctgtttgg
     3481 taatcttatc gccctgtcac tcgggctgac ccccaacttt aaatctaact tcgacctggc
     3541 cgaagatgcc aagcttcaac tgagcaaaga cacctacgat gatgatctcg acaatctgct
     3601 ggcccagatc ggcgaccagt acgcagacct ttttttggcg gcaaagaacc tgtcagacgc
     3661 cattctgctg agtgatattc tgcgagtgaa cacggagatc accaaagctc cgctgagcgc
     3721 tagtatgatc aagcgctatg atgagcacca ccaagacttg actttgctga aggcccttgt
     3781 cagacagcaa ctgcctgaga agtacaagga aattttcttc gatcagtcta aaaatggcta
     3841 cgccggatac attgacggcg gagcaagcca ggaggaattt tacaaattta ttaagcccat
     3901 cttggaaaaa atggacggca ccgaggagct gctggtaaag cttaacagag aagatctgtt
     3961 gcgcaaacag cgcactttcg acaatggaag catcccccac cagattcacc tgggcgaact
     4021 gcacgctatc ctcaggcggc aagaggattt ctaccccttt ttgaaagata acagggaaaa
     4081 gattgagaaa atcctcacat ttcggatacc ctactatgta ggccccctcg cccggggaaa
     4141 ttccagattc gcgtggatga ctcgcaaatc agaagagact atcactccct ggaacttcga
     4201 ggaagtcgtg gataaggggg cctctgccca gtccttcatc gaaaggatga ctaactttga
     4261 taaaaatctg cctaacgaaa aggtgcttcc taaacactct ctgctgtacg agtacttcac
     4321 agtttataac gagctcacca aggtcaaata cgtcacagaa gggatgagaa agccagcatt
     4381 cctgtctgga gagcagaaga aagctatcgt ggacctcctc ttcaagacga accggaaagt
     4441 taccgtgaaa cagctcaaag aggactattt caaaaagatt gaatgtttcg actctgttga
     4501 aatcagcgga gtggaggatc gcttcaacgc atccctggga acgtatcacg atctcctgaa
     4561 aatcattaaa gacaaggact tcctggacaa tgaggagaac gaggacattc ttgaggacat
     4621 tgtcctcacc cttacgttgt ttgaagatag ggagatgatt gaagaacgct tgaaaactta
     4681 cgctcatctc ttcgacgaca aagtcatgaa acagctcaag aggcgccgat atacaggatg
     4741 ggggcggctg tcaagaaaac tgatcaatgg gatccgagac aagcagagtg gaaagacaat
     4801 cctggatttt cttaagtccg atggatttgc caaccggaac ttcatgcagt tgatccatga
     4861 tgactctctc acctttaagg aggacatcca gaaagcacaa gtttctggcc agggggacag
     4921 ccttcacgag cacatcgcta atcttgcagg tagcccagct atcaaaaagg gaatactgca
     4981 gaccgttaag gtcgtggatg aactcgtcaa agtaatggga aggcataagc ccgagaatat
     5041 cgttatcgag atggcccgag agaaccaaac tacccagaag ggacagaaga acagtaggga
     5101 aaggatgaag aggattgaag agggtataaa agaactgggg tcccaaatcc ttaaggaaca
     5161 cccagttgaa aacacccagc ttcagaatga gaagctctac ctgtactacc tgcagaacgg
     5221 cagggacatg tacgtggatc aggaactgga catcaatcgg ctctccgact acgacgtgga
     5281 tcatatcgtg ccccagtctt ttctcaaaga tgattctatt gataataaag tgttgacaag
     5341 atccgataaa aatagaggga agagtgataa cgtcccctca gaagaagttg tcaagaaaat
     5401 gaaaaattat tggcggcagc tgctgaacgc caaactgatc acacaacgga agttcgataa
     5461 tctgactaag gctgaacgag gtggcctgtc tgagttggat aaagccggct tcatcaaaag
     5521 gcagcttgtt gagacacgcc agatcaccaa gcacgtggcc caaattctcg attcacgcat
     5581 gaacaccaag tacgatgaaa atgacaaact gattcgagag gtgaaagtta ttactctgaa
     5641 gtctaagctc gtctcagatt tcagaaagga ctttcagttt tataaggtga gagagatcaa
     5701 caattaccac catgcgcatg atgcctacct gaatgcagtg gtaggcactg cacttatcaa
     5761 aaaatatccc aagcttgaat ctgaatttgt ttacggagac tataaagtgt acgatgttag
     5821 gaaaatgatc gcaaagtctg agcaggaaat aggcaaggcc accgctaagt acttctttta
     5881 cagcaatatt atgaattttt tcaagaccga gattacactg gccaatggag agattcggaa
     5941 gcgaccactt atcgaaacaa acggagaaac aggagaaatc gtgtgggaca agggtaggga
     6001 tttcgcgaca gtccggaagg tcctgtccat gccgcaggtg aacatcgtta aaaagaccga
     6061 agtacagacc ggaggcttct ccaaggaaag tatcctcccg aaaaggaaca gcgacaagct
     6121 gatcgcacgc aaaaaagatt gggaccccaa gaaatacggc ggattcgatt ctcctacagt
     6181 cgcttacagt gtactggttg tggccaaagt ggagaaaggg aagtctaaaa aactcaaaag
     6241 cgtcaaggaa ctgctgggca tcacaatcat ggagcgatca agcttcgaaa aaaaccccat
     6301 cgactttctc gaggcgaaag gatataaaga ggtcaaaaaa gacctcatca ttaagcttcc
     6361 caagtactct ctctttgagc ttgaaaacgg ccggaaacga atgctcgcta gtgcgggcga
     6421 gctgcagaaa ggtaacgagc tggcactgcc ctctaaatac gttaatttct tgtatctggc
     6481 cagccactat gaaaagctca aagggtcccc cgaagataat gagcagaagc agctgttcgt
     6541 ggaacaacac aaacactacc ttgatgagat catcgagcaa ataagcgaat tctccaaaag
     6601 agtgatcctc gccgacgcta acctcgataa ggtgctttct gcttacaata agcacaggga
     6661 taagcccatc agggagcagg cagaaaacat tatccacttg tttactctga ccaacttggg
     6721 cgcgcctgca gccttcaagt acttcgacac caccatagac agaaagcggt acacctctac
     6781 aaaggaggtc ctggacgcca cactgattca tcagtcaatt acggggctct atgaaacaag
     6841 aatcgacctc tctcagctcg gtggagacag cagggctgac cccaagaaga agaggaaggt
     6901 gtgagcttcg tccgacggcg gcccgacggg tccgaggcct cggagatccg tccccctttt
     6961 cctttgtcga tatcatgtaa ttagttatgt cacgcttaca ttcacgccct ccccccacat
     7021 ccgctctaac cgaaaaggaa ggagttagac aacctgaagt ctaggtccct atttattttt
     7081 ttatagttat gttagtatta agaacgttat ttatatttca aatttttctt ttttttctgt
     7141 acagacgcgt gtacgcatgt aacattatac tgaaaacctt gcttgagaag gttttgggac
     7201 gctcgaaggc tttaatttgc aagctggcgc taattcatgg acatggaggc ccagaatacc
     7261 ctccttgaca gtcttgacgt gcgcagctca ggggcatgat gtgactgtcg cccgtacatt
     7321 tagcccatac atccccatgt ataatcattt gcatccatac attttgatgg ccgcacggcg
     7381 cgaagcaaaa attacggctc ctcgctgcag acgtgcgagc agggaaacgc tcccctcaca
     7441 gacgcgttga attgtcccca cgccgcgccc ctgtagagaa atataaaagg ttaggatttg
     7501 ccactgaggt tcttctttca tatacttcct tttaaaatct tgctaggata cagttctcac
     7561 atcacatccg aacataaaca accatgggta aggaaaagac tcacgtttcg aggccgcgat
     7621 taaattccaa catggatgct gatttatatg ggtataaatg ggctcgcgat aatgtcgggc
     7681 aatcaggtgc gacaatctat cgattgtatg ggaagcccga tgcgccagag ttgtttctga
     7741 aacatggcaa aggtagcgtt gccaatgatg ttacagatga gatggtcaga ctaaactggc
     7801 tgacggaatt tatgcctctt ccgaccatca agcattttat ccgtactcct gatgatgcat
     7861 ggttactcac cactgcgatc cccggcaaaa cagcattcca ggtattagaa gaatatcctg
     7921 attcaggtga aaatattgtt gatgcgctgg cagtgttcct gcgccggttg cattcgattc
     7981 ctgtttgtaa ttgtcctttt aacagcgatc gcgtatttcg tctcgctcag gcgcaatcac
     8041 gaatgaataa cggtttggtt gatgcgagtg attttgatga cgagcgtaat ggctggcctg
     8101 ttgaacaagt ctggaaagaa atgcataagc ttttgccatt ctcaccggat tcagtcgtca
     8161 ctcatggtga tttctcactt gataacctta tttttgacga ggggaaatta ataggttgta
     8221 ttgatgttgg acgagtcgga atcgcagacc gataccagga tcttgccatc ctatggaact
     8281 gcctcggtga gttttctcct tcattacaga aacggctttt tcaaaaatat ggtattgata
     8341 atcctgatat gaataaattg cagtttcatt tgatgctcga tgagtttttc taatcagtac
     8401 tgacaataaa aagattcttg ttttcaagaa cttgtcattt gtatagtttt tttatattgt
     8461 agttgttcta ttttaatcaa atgttagcgt gatttatatt ttttttcgcc tcgacatcat
     8521 ctgcccagat gcgaagttaa gtgcgcagaa agtaatatca tgcgtcaatc gtatgtgaat
     8581 gctggtcgct atactggctt ccttttcatc acgtgctata aaaataatta taatttaaat
     8641 tttttaatat aaatatataa attaaaaata gaaagtaaaa aaagaaatta aagaaaaaat
     8701 agtttttgtt ttccgaagat gtaaaagact ctagggggat cgccaacaaa tactaccttt
     8761 tatcttgctc ttcctgctct caggtattaa tgccgaattg tttcatcttg tctgtgtaga
     8821 acaccacaca cgaaaatcct gtgattttac attttactta tcgttaatcg aatgtatatc
     8881 tatttaatct gcttttcttg tctaataaat atatatgtaa agtacgcttt ttgttgaaat
     8941 tttttaaacc tttgtttatt tttttttctt cattccgtaa ctcttctacc ttctttattt
     9001 actttctaaa atccaaatac aaaacataaa aataaataaa cacagagtaa attcccaaat
     9061 tattccatca ttaaaagata cgaggcgcgt gtaagttaca ggca
//