BB3cH_pGAP_23*_pLAT1_Cas9

LOCUS       BB3cH_pGAP_23*_pLAT1_Cas9                9323 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 
            Hyg.
ACCESSION   .
VERSION     .
KEYWORDS    BB3cH_pGAP_23*_pLAT1_Cas9
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9323)
  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 9323)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.biofeng.com
FEATURES             Location/Qualifiers
     source          1..9323
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     rep_origin      complement(62..650)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(142..161)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        737..1213
                     /label=GAP promoter
                     /note="promoter for Pichia pastoris 
                     glyceraldehyde-3-phosphate dehydrogenase"
     CDS             2757..6860
                     /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             6873..6893
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     terminator      6965..7212
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(6981..6999)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     gene            7232..8807
                     /label=hphMX6
                     /note="yeast selectable marker conferring hygromycin 
                     resistance"
     promoter        7232..7575
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             7576..8604
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     terminator      8610..8807
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     misc_feature    8819..9322
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
ORIGIN
        1 cgctgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
       61 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
      121 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
      181 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
      241 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
      301 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
      361 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
      421 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
      481 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
      541 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
      601 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
      661 tgatcttttc tacggggtgg agcgcaaaaa accccgcccc tgacagggcg gggttttttc
      721 gcgatcggag gatccttttt tgtagaaatg tcttggtgtc ctcgtccaat caggtagcca
      781 tctctgaaat atctggctcc gttgcaactc cgaacgacct gctggcaacg taaaattctc
      841 cggggtaaaa cttaaatgtg gagtaatgga accagaaacg tctcttccct tctctctcct
      901 tccaccgccc gttaccgtcc ctaggaaatt ttactctgct ggagagcttc ttctacggcc
      961 cccttgcagc aatgctcttc ccagcattac gttgcgggta aaacggaggt cgtgtacccg
     1021 acctagcagc ccagggatgg aaaagtcccg gccgtcgctg gcaataatag cgggcggacg
     1081 catgtcatga gattattgga aaccaccaga atcgaatata aaaggcgaac acctttccca
     1141 attttggttt ctcctgaccc aaagacttta aatttaattt atttgtccct atttcaatca
     1201 attgaacaac tatcaaaaca ccatggcgtc ttcggatccc tcgaggaaga cgcgctttta
     1261 gtgtacatct gataatatag tactaccacg tatgataatg tagagaatag gcttccttgt
     1321 cgagtgtgtt tgcagttttc ttgagtttca aggtttaaat gctggtatat tagttcatcg
     1381 aaggtttcag ccaatagcac cttaaatcaa tcaaactaat tcgactctta cgaaagagcc
     1441 tactgtgttt agtatcgaag tcgtttacct ttcatgttga atagcttcct ctctgaccct
     1501 aacatttcaa gatcctccta aagttacccg gattgtgaaa ttctaatgat ccacctgccc
     1561 aatgcatttt ttctttattc agtttacctt ttttacctaa tatacgagct tgttaaagta
     1621 agtggcactg caatactagg cttattgttg atattatgat gaatcgtttt cacaaacttg
     1681 atttcctgtg aactcaccat gtactaagga aaaaaacatg catcaccatc tgaatatttg
     1741 accgctccgg ggagggaaga atatgaacca cgattgaaag actttgggtt tgccgattct
     1801 aaaacactta ctgaacagaa aagaagtgaa ttattagaga gtttatgcaa cggagatcat
     1861 gagcttagaa agaacgtagg ctgggcaaca acatccattt ctgcctgcga tatttccagt
     1921 gcaatgctaa agccacataa tataggcaat tacaacttga atgagcaggc tcatgatgtc
     1981 accatggaac tgatagataa agtcatccaa aaaggtgtca atttacggca cgtctacgta
     2041 gatactgtcg gacctccagc atcataccaa aagaaactgc aacaaaggtt tcccgcactg
     2101 ctgatcaccg tatctaaaaa agcagatgcc atctatccat gtgtttcaag tgcttcagtt
     2161 tgcgccaagg tcactagaga cgtctgtttg agaacctcaa atggggagga gaacctatta
     2221 gattgtgggt ctggatatcc ctcggaccca aatactaaga aatggctagc ccagtctata
     2281 catccattgt ttggctggag tcgtgatgtc aggttttcgt ggcagacggc caaagattgt
     2341 cttgagagac acaactcaat caaagttgtg tttggcgaag aggagcctac cccggttaat
     2401 ttcggtgatc tggggttgag caaacagaag cccgacttta aggatttaga aatcagtacc
     2461 agttggtggg gacattaatt ggtggaggag agccaatatt ctcaactgtt cgtaggacca
     2521 taacccccca ctcgcaccag aaatactcat tggcgctgac aactagtttg agccgcttca
     2581 attgcggcaa gtccccagtt ttgtttggta ctcccctctc gcctcgcgtg tttttgttcc
     2641 tccgaattgt gcatcaccct gtcacatccg tacaccatcg gaacactctg ctcctcctcc
     2701 tttctcctct taattgaacg tatatctaat taccctaata acaagtatct tcaaccatgg
     2761 acaagaagta ctccattggg ctcgatatcg gcacaaacag cgtcggttgg gccgtcatta
     2821 cggacgagta caaggtgccg agcaaaaaat tcaaagttct gggcaatacc gatcgccaca
     2881 gcataaagaa gaacctcatt ggcgccctcc tgttcgactc cggggagacg gccgaagcca
     2941 cgcggctcaa aagaacagca cggcgcagat atacccgcag aaagaatcgg atctgctacc
     3001 tgcaggagat ctttagtaat gagatggcta aggtggatga ctctttcttc cataggctgg
     3061 aggagtcctt tttggtggag gaggataaaa agcacgagcg ccacccaatc tttggcaata
     3121 tcgtggacga ggtggcgtac catgaaaagt acccaaccat atatcatctg aggaagaagc
     3181 ttgtagacag tactgataag gctgacttgc ggttgatcta tctcgcgctg gcgcatatga
     3241 tcaaatttcg gggacacttc ctcatcgagg gggacctgaa cccagacaac agcgatgtcg
     3301 acaaactctt tatccaactg gttcagactt acaatcagct tttcgaagag aacccgatca
     3361 acgcatccgg agttgacgcc aaagcaatcc tgagcgctag gctgtccaaa tcccggcggc
     3421 tcgaaaacct catcgcacag ctccctgggg agaagaagaa cggcctgttt ggtaatctta
     3481 tcgccctgtc actcgggctg acccccaact ttaaatctaa cttcgacctg gccgaagatg
     3541 ccaagcttca actgagcaaa gacacctacg atgatgatct cgacaatctg ctggcccaga
     3601 tcggcgacca gtacgcagac ctttttttgg cggcaaagaa cctgtcagac gccattctgc
     3661 tgagtgatat tctgcgagtg aacacggaga tcaccaaagc tccgctgagc gctagtatga
     3721 tcaagcgcta tgatgagcac caccaagact tgactttgct gaaggccctt gtcagacagc
     3781 aactgcctga gaagtacaag gaaattttct tcgatcagtc taaaaatggc tacgccggat
     3841 acattgacgg cggagcaagc caggaggaat tttacaaatt tattaagccc atcttggaaa
     3901 aaatggacgg caccgaggag ctgctggtaa agcttaacag agaagatctg ttgcgcaaac
     3961 agcgcacttt cgacaatgga agcatccccc accagattca cctgggcgaa ctgcacgcta
     4021 tcctcaggcg gcaagaggat ttctacccct ttttgaaaga taacagggaa aagattgaga
     4081 aaatcctcac atttcggata ccctactatg taggccccct cgcccgggga aattccagat
     4141 tcgcgtggat gactcgcaaa tcagaagaga ctatcactcc ctggaacttc gaggaagtcg
     4201 tggataaggg ggcctctgcc cagtccttca tcgaaaggat gactaacttt gataaaaatc
     4261 tgcctaacga aaaggtgctt cctaaacact ctctgctgta cgagtacttc acagtttata
     4321 acgagctcac caaggtcaaa tacgtcacag aagggatgag aaagccagca ttcctgtctg
     4381 gagagcagaa gaaagctatc gtggacctcc tcttcaagac gaaccggaaa gttaccgtga
     4441 aacagctcaa agaggactat ttcaaaaaga ttgaatgttt cgactctgtt gaaatcagcg
     4501 gagtggagga tcgcttcaac gcatccctgg gaacgtatca cgatctcctg aaaatcatta
     4561 aagacaagga cttcctggac aatgaggaga acgaggacat tcttgaggac attgtcctca
     4621 cccttacgtt gtttgaagat agggagatga ttgaagaacg cttgaaaact tacgctcatc
     4681 tcttcgacga caaagtcatg aaacagctca agaggcgccg atatacagga tgggggcggc
     4741 tgtcaagaaa actgatcaat gggatccgag acaagcagag tggaaagaca atcctggatt
     4801 ttcttaagtc cgatggattt gccaaccgga acttcatgca gttgatccat gatgactctc
     4861 tcacctttaa ggaggacatc cagaaagcac aagtttctgg ccagggggac agccttcacg
     4921 agcacatcgc taatcttgca ggtagcccag ctatcaaaaa gggaatactg cagaccgtta
     4981 aggtcgtgga tgaactcgtc aaagtaatgg gaaggcataa gcccgagaat atcgttatcg
     5041 agatggcccg agagaaccaa actacccaga agggacagaa gaacagtagg gaaaggatga
     5101 agaggattga agagggtata aaagaactgg ggtcccaaat ccttaaggaa cacccagttg
     5161 aaaacaccca gcttcagaat gagaagctct acctgtacta cctgcagaac ggcagggaca
     5221 tgtacgtgga tcaggaactg gacatcaatc ggctctccga ctacgacgtg gatcatatcg
     5281 tgccccagtc ttttctcaaa gatgattcta ttgataataa agtgttgaca agatccgata
     5341 aaaatagagg gaagagtgat aacgtcccct cagaagaagt tgtcaagaaa atgaaaaatt
     5401 attggcggca gctgctgaac gccaaactga tcacacaacg gaagttcgat aatctgacta
     5461 aggctgaacg aggtggcctg tctgagttgg ataaagccgg cttcatcaaa aggcagcttg
     5521 ttgagacacg ccagatcacc aagcacgtgg cccaaattct cgattcacgc atgaacacca
     5581 agtacgatga aaatgacaaa ctgattcgag aggtgaaagt tattactctg aagtctaagc
     5641 tcgtctcaga tttcagaaag gactttcagt tttataaggt gagagagatc aacaattacc
     5701 accatgcgca tgatgcctac ctgaatgcag tggtaggcac tgcacttatc aaaaaatatc
     5761 ccaagcttga atctgaattt gtttacggag actataaagt gtacgatgtt aggaaaatga
     5821 tcgcaaagtc tgagcaggaa ataggcaagg ccaccgctaa gtacttcttt tacagcaata
     5881 ttatgaattt tttcaagacc gagattacac tggccaatgg agagattcgg aagcgaccac
     5941 ttatcgaaac aaacggagaa acaggagaaa tcgtgtggga caagggtagg gatttcgcga
     6001 cagtccggaa ggtcctgtcc atgccgcagg tgaacatcgt taaaaagacc gaagtacaga
     6061 ccggaggctt ctccaaggaa agtatcctcc cgaaaaggaa cagcgacaag ctgatcgcac
     6121 gcaaaaaaga ttgggacccc aagaaatacg gcggattcga ttctcctaca gtcgcttaca
     6181 gtgtactggt tgtggccaaa gtggagaaag ggaagtctaa aaaactcaaa agcgtcaagg
     6241 aactgctggg catcacaatc atggagcgat caagcttcga aaaaaacccc atcgactttc
     6301 tcgaggcgaa aggatataaa gaggtcaaaa aagacctcat cattaagctt cccaagtact
     6361 ctctctttga gcttgaaaac ggccggaaac gaatgctcgc tagtgcgggc gagctgcaga
     6421 aaggtaacga gctggcactg ccctctaaat acgttaattt cttgtatctg gccagccact
     6481 atgaaaagct caaagggtcc cccgaagata atgagcagaa gcagctgttc gtggaacaac
     6541 acaaacacta ccttgatgag atcatcgagc aaataagcga attctccaaa agagtgatcc
     6601 tcgccgacgc taacctcgat aaggtgcttt ctgcttacaa taagcacagg gataagccca
     6661 tcagggagca ggcagaaaac attatccact tgtttactct gaccaacttg ggcgcgcctg
     6721 cagccttcaa gtacttcgac accaccatag acagaaagcg gtacacctct acaaaggagg
     6781 tcctggacgc cacactgatt catcagtcaa ttacggggct ctatgaaaca agaatcgacc
     6841 tctctcagct cggtggagac agcagggctg accccaagaa gaagaggaag gtgtgagctt
     6901 cgtccgacgg cggcccgacg ggtccgaggc ctcggagatc cgtccccctt ttcctttgtc
     6961 gatatcatgt aattagttat gtcacgctta cattcacgcc ctccccccac atccgctcta
     7021 accgaaaagg aaggagttag acaacctgaa gtctaggtcc ctatttattt ttttatagtt
     7081 atgttagtat taagaacgtt atttatattt caaatttttc ttttttttct gtacagacgc
     7141 gtgtacgcat gtaacattat actgaaaacc ttgcttgaga aggttttggg acgctcgaag
     7201 gctttaattt gcaagctggc gctaattcat ggacatggag gcccagaata ccctccttga
     7261 cagtcttgac gtgcgcagct caggggcatg atgtgactgt cgcccgtaca tttagcccat
     7321 acatccccat gtataatcat ttgcatccat acattttgat ggccgcacgg cgcgaagcaa
     7381 aaattacggc tcctcgctgc agacgtgcga gcagggaaac gctcccctca cagacgcgtt
     7441 gaattgtccc cacgccgcgc ccctgtagag aaatataaaa ggttaggatt tgccactgag
     7501 gttcttcttt catatacttc cttttaaaat cttgctagga tacagttctc acatcacatc
     7561 cgaacataaa caaccatggg taaaaagcct gaactcaccg cgacgtctgt cgagaagttt
     7621 ctgatcgaaa agttcgacag cgtctccgac ctgatgcagc tctcggaggg cgaagaatct
     7681 cgtgctttca gcttcgatgt aggagggcgt ggatatgtcc tgcgggtaaa tagctgcgcc
     7741 gatggtttct acaaagatcg ttatgtttat cggcactttg catcggccgc gctcccgatt
     7801 ccggaagtgc ttgacattgg ggaattcagc gagagcctga cctattgcat ctcccgccgt
     7861 gcacagggtg tcacgttgca agacctccct gaaaccgaac tgcccgctgt tctgcagccg
     7921 gtcgcggagg ccatggatgc gatcgctgcg gccgatctta gccagacgag cgggttcggc
     7981 ccattcggac cgcaaggaat cggtcaatac actacatggc gtgatttcat atgcgcgatt
     8041 gctgatcccc atgtgtatca ctggcaaact gtgatggacg acaccgtcag tgcgtccgtc
     8101 gcgcaggctc tcgatgagct gatgctttgg gccgaggact gccccgaagt ccggcacctc
     8161 gtgcacgcgg atttcggctc caacaatgtc ctgacggaca atggccgcat aacagcggtc
     8221 attgactgga gcgaggcgat gttcggggat tcccaatacg aggtcgccaa catcttcttc
     8281 tggaggccgt ggttggcttg tatggagcag cagacgcgct acttcgagcg gaggcatccg
     8341 gagcttgcag gatcgccgcg gctccgggcg tatatgctcc gcattggtct tgaccaactc
     8401 tatcagagct tggttgacgg caatttcgat gatgcagctt gggcgcaggg tcgatgcgac
     8461 gcaatcgtcc gatccggagc cgggactgtc gggcgtacac aaatcgcccg cagaagcgcg
     8521 gccgtctgga ccgatggctg tgtagaagta ctcgccgata gtggaaaccg acgccccagc
     8581 actcgtccga gggcaaagga ataatcagta ctgacaataa aaagattctt gttttcaaga
     8641 acttgtcatt tgtatagttt ttttatattg tagttgttct attttaatca aatgttagcg
     8701 tgatttatat tttttttcgc ctcgacatca tctgcccaga tgcgaagtta agtgcgcaga
     8761 aagtaatatc atgcgtcaat cgtatgtgaa tgctggtcgc tatactggct tccttttcat
     8821 cacgtgctat aaaaataatt ataatttaaa ttttttaata taaatatata aattaaaaat
     8881 agaaagtaaa aaaagaaatt aaagaaaaaa tagtttttgt tttccgaaga tgtaaaagac
     8941 tctaggggga tcgccaacaa atactacctt ttatcttgct cttcctgctc tcaggtatta
     9001 atgccgaatt gtttcatctt gtctgtgtag aacaccacac acgaaaatcc tgtgatttta
     9061 cattttactt atcgttaatc gaatgtatat ctatttaatc tgcttttctt gtctaataaa
     9121 tatatatgta aagtacgctt tttgttgaaa ttttttaaac ctttgtttat ttttttttct
     9181 tcattccgta actcttctac cttctttatt tactttctaa aatccaaata caaaacataa
     9241 aaataaataa acacagagta aattcccaaa ttattccatc attaaaagat acgaggcgcg
     9301 tgtaagttac aggcaagcga tcg
//