LOCUS       Exported                4569 bp ds-DNA     circular SYN 17-AUG-2018
DEFINITION  Construct for expressing BTL2 with the alpha-factor signal sequence 
            and pro region in P. pastoris.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4569)
  AUTHORS   Glick Lab
  TITLE     Direct Submission
  JOURNAL   Exported Sep 12, 2018 from SnapGene 4.2.2
            http://www.snapgene.com
COMMENT     Linearize with PmeI for integration at the AOX1 locus.
FEATURES             Location/Qualifiers
     source          1..4569
                     /organism="synthetic DNA construct"
                     /lab_host="Pichia pastoris"
                     /mol_type="other DNA"
     promoter        2..940
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 promoter
                     /note="inducible promoter, regulated by methanol"
     CDS             941..997
                     /codon_start=1
                     /product="signal sequence of S. cerevisiae alpha-factor"
                     /label=pre-alpha-factor
                     /translation="MRFPSIFTAVLFAASSALA"
     CDS             998..1207
                     /codon_start=1
                     /product="S. cerevisiae alpha-factor pro region"
                     /label=pro-alpha-factor
                     /translation="APVNTTTEDETAQIPAEAVIGYLDLEGDFDVAVLPFSNSTNNGLL
                     FINTTIASIAAKEEGVSLDKREAEA"
     CDS             1208..2371
                     /codon_start=1
                     /product="mature domain of the BTL2 lipase from Bacillus 
                     thermocatenulatus"
                     /label=BTL2
                     /note="codon-optimized for expression in Pichia pastoris"
                     /translation="ASPRANDAPIVLLHGFTGWGREEMLGFKYWGGVRGDIEQWLNDNG
                     YRTYTLAVGPLSSNWDRACEAYAQLVGGTVDYGAAHAAKHGHARFGRTYPGLLPELKRG
                     GRVHIIAHSQGGQTARMLVSLLENGSQEEREYAKAHNVSLSPLFEGGHHFVLSVTTIAT
                     PHDGTTLVNMVDFTDRFFDLQKAVLKAAAVASNVPYTSQVYDFKLDQWGLRRQPGESFD
                     HYFERLKRSPVWTSTDTARYDLSIPGAEKLNQWVQASPNTYYLSFSTERTHRGALTGNY
                     YPELGMNAFSAVVCAPFLGSYRNEALGIDDRWLENDGIVNTVSMNGPKRGSSDRIVPYD
                     GTLKKGVWNDMGTCNVDHLEVIGVDPNPSFDIRAFYLRLAEQLASLRP"
     CDS             2372..2389
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      2393..2639
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     promoter        2679..3065
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     promoter        3073..3120
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             3139..3513
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     terminator      3579..3826
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     rep_origin      complement(3901..4489)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 agatctaaca tccaaagacg aaaggttgaa tgaaaccttt ttgccatccg acatccacag
       61 gtccattctc acacataagt gccaaacgca acaggagggg atacactagc agcagaccgt
      121 tgcaaacgca ggacctccac tcctcttctc ctcaacaccc acttttgcca tcgaaaaacc
      181 agcccagtta ttgggcttga ttggagctcg ctcattccaa ttccttctat taggctacta
      241 acaccatgac tttattagcc tgtctatcct ggcccccctg gcgaggttca tgtttgttta
      301 tttccgaatg caacaagctc cgcattacac ccgaacatca ctccagatga gggctttctg
      361 agtgtggggt caaatagttt catgttcccc aaatggccca aaactgacag tttaaacgct
      421 gtcttggaac ctaatatgac aaaagcgtga tctcatccaa gatgaactaa gtttggttcg
      481 ttgaaatgct aacggccagt tggtcaaaaa gaaacttcca aaagtcggca taccgtttgt
      541 cttgtttggt attgattgac gaatgctcaa aaataatctc attaatgctt agcgcagtct
      601 ctctatcgct tctgaacccc ggtgcacctg tgccgaaacg caaatgggga aacacccgct
      661 ttttggatga ttatgcattg tctccacatt gtatgcttcc aagattctgg tgggaatact
      721 gctgatagcc taacgttcat gatcaaaatt taactgttct aacccctact tgacagcaat
      781 atataaacag aaggaagctg ccctgtctta aacctttttt tttatcatca ttattagctt
      841 actttcataa ttgcgactgg ttccaattga caagcttttg attttaacga cttttaacga
      901 caacttgaga agatcaaaaa acaactaatt attcgaaacg atgagatttc cttcaatttt
      961 tactgcagtt ttattcgcag catcctccgc attagctgct ccagtcaaca ctacaacaga
     1021 agatgaaacg gcacaaattc cggctgaagc tgtcatcggt tacttagatt tagaagggga
     1081 tttcgatgtt gctgttttgc cattttccaa cagcacaaat aacgggttat tgtttataaa
     1141 tactactatt gccagcattg ctgctaaaga agaaggggta tctttggata aaagagaggc
     1201 tgaagctgct tctccaagag ctaatgatgc tcctattgtt ttgttgcatg gttttactgg
     1261 ttggggtaga gaagagatgt tgggtttcaa atattggggt ggtgttagag gagatattga
     1321 acaatggttg aacgataatg gttacagaac ttatactttg gctgttggtc cattgtcttc
     1381 taactgggat agagcttgtg aggcttacgc tcaattggtt ggtggtactg ttgattatgg
     1441 tgctgctcac gctgctaagc atggtcacgc tagatttggt agaacttatc caggtttgtt
     1501 gcctgaattg aaaagaggtg gtagagttca tattattgct cactctcaag gtggtcaaac
     1561 tgctagaatg ttggtttctt tgttggaaaa tggttctcaa gaagagagag agtacgctaa
     1621 ggctcataac gtttctttgt ctccattgtt tgagggtggt catcacttcg ttttgtctgt
     1681 tactactatt gctactcctc acgatggtac tactttggtt aacatggttg atttcactga
     1741 tagatttttc gatttgcaaa aggctgtttt gaaagctgct gctgttgctt ctaacgttcc
     1801 ttacacttct caagtttacg atttcaagtt ggatcaatgg ggtttgagaa gacaacctgg
     1861 tgaatctttt gatcattact tcgagagatt gaaaagatcc cctgtttgga cttctactga
     1921 tactgctaga tatgatttgt ctattccagg tgctgaaaag ttgaatcaat gggttcaggc
     1981 ttctcctaac acttactatt tgtctttctc tactgaaaga actcacagag gtgctttgac
     2041 tggtaattac tatccagagt tgggtatgaa cgctttttct gctgttgttt gtgctccttt
     2101 cttgggttct tacagaaatg aggctttggg tattgatgat agatggttgg agaacgatgg
     2161 tattgttaat actgtttcta tgaacggtcc aaaaagaggt tcttctgata gaattgttcc
     2221 ttatgatggt actttgaaga aaggtgtttg gaatgatatg ggtacttgta acgttgatca
     2281 tttggaagtt attggtgttg atccaaaccc ttcttttgat attagagctt tctacttgag
     2341 attggctgag caattggctt ctttgagacc acatcaccat caccatcact aatcaagagg
     2401 atgtcagaat gccatttgcc tgagagatgc aggcttcatt tttgatactt ttttatttgt
     2461 aacctatata gtataggatt ttttttgtca ttttgtttct tctcgtacga gcttgctcct
     2521 gatcagccta tctcgcagct gatgaatatc ttgtggtagg ggtttgggaa aatcattcga
     2581 gtttgatgtt tttcttggta tttcccactc ctcttcagag tacagaagat taagtgagac
     2641 cttcgtttgt gcggatcccc cacacaccat agcttcaaaa tgtttctact ccttttttac
     2701 tcttccagat tttctcggac tccgcgcatc gccgtaccac ttcaaaacac ccaagcacag
     2761 catactaaat tttccctctt tcttcctcta gggtgtcgtt aattacccgt actaaaggtt
     2821 tggaaaagaa aaaagagacc gcctcgtttc tttttcttcg tcgaaaaagg caataaaaat
     2881 ttttatcacg tttctttttc ttgaaatttt tttttttagt ttttttctct ttcagtgacc
     2941 tccattgata tttaagttaa taaacggtct tcaatttctc aagtttcagt ttcatttttc
     3001 ttgttctatt acaacttttt ttacttcttg ttcattagaa agaaagcata gcaatctaat
     3061 ctaaggggcg gtgttgacaa ttaatcatcg gcatagtata tcggcatagt ataatacgac
     3121 aaggtgagga actaaaccat ggccaagttg accagtgccg ttccggtgct caccgcgcgc
     3181 gacgtcgccg gagcggtcga gttctggacc gaccggctcg ggttctcccg ggacttcgtg
     3241 gaggacgact tcgccggtgt ggtccgggac gacgtgaccc tgttcatcag cgcggtccag
     3301 gaccaggtgg tgccggacaa caccctggcc tgggtgtggg tgcgcggcct ggacgagctg
     3361 tacgccgagt ggtcggaggt cgtgtccacg aacttccggg acgcctccgg gccggccatg
     3421 accgagatcg gcgagcagcc gtgggggcgg gagttcgccc tgcgcgaccc ggccggcaac
     3481 tgcgtgcact tcgtggccga ggagcaggac tgacacgtcc gacggcggcc cacgggtccc
     3541 aggcctcgga gatccgtccc ccttttcctt tgtcgatatc atgtaattag ttatgtcacg
     3601 cttacattca cgccctcccc ccacatccgc tctaaccgaa aaggaaggag ttagacaacc
     3661 tgaagtctag gtccctattt atttttttat agttatgtta gtattaagaa cgttatttat
     3721 atttcaaatt tttctttttt ttctgtacag acgcgtgtac gcatgtaaca ttatactgaa
     3781 aaccttgctt gagaaggttt tgggacgctc gaaggcttta atttgcaagc tggagaccaa
     3841 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
     3901 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
     3961 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
     4021 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
     4081 cgtggcgctt tctcaatgct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     4141 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
     4201 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
     4261 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
     4321 taactacggc tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac
     4381 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
     4441 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     4501 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
     4561 catgagatc
//
