LOCUS       Exported                4578 bp ds-DNA     circular SYN 17-AUG-2018
DEFINITION  Construct for expressing BTL2 with the Ost1 signal sequence and the 
            Invitrogen variant of the alpha-factor pro region in P. pastoris.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4578)
  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..4578
                     /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..1006
                     /codon_start=1
                     /product="signal sequence of S. cerevisiae Ost1"
                     /label=pre-Ost1
                     /translation="MRQVWFSWIVGLFLCFFNVSSA"
     CDS             1007..1216
                     /codon_start=1
                     /product="S. cerevisiae alpha-factor pro region with the 
                     Leu42 to Ser and Asp83 to Glu mutations"
                     /label=pro-alpha-factor(I)
                     /translation="APVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNNGLL
                     FINTTIASIAAKEEGVSLEKREAEA"
     CDS             1217..2380
                     /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             2381..2398
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      2402..2648
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     promoter        2688..3074
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     promoter        3082..3129
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             3148..3522
                     /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      3588..3835
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     rep_origin      complement(3910..4498)
                     /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 atgaggcagg tttggttctc
      961 ttggattgtg ggattgttcc tatgtttttt caacgtgtct tctgctgctc cagtcaacac
     1021 tacaacagaa gatgaaacgg cacaaattcc ggctgaagct gtcatcggtt actcagattt
     1081 agaaggggat ttcgatgttg ctgttttgcc attttccaac agcacaaata acgggttatt
     1141 gtttataaat actactattg ccagcattgc tgctaaagaa gaaggggtat ctctcgagaa
     1201 aagagaggct gaagctgctt ctccaagagc taatgatgct cctattgttt tgttgcatgg
     1261 ttttactggt tggggtagag aagagatgtt gggtttcaaa tattggggtg gtgttagagg
     1321 agatattgaa caatggttga acgataatgg ttacagaact tatactttgg ctgttggtcc
     1381 attgtcttct aactgggata gagcttgtga ggcttacgct caattggttg gtggtactgt
     1441 tgattatggt gctgctcacg ctgctaagca tggtcacgct agatttggta gaacttatcc
     1501 aggtttgttg cctgaattga aaagaggtgg tagagttcat attattgctc actctcaagg
     1561 tggtcaaact gctagaatgt tggtttcttt gttggaaaat ggttctcaag aagagagaga
     1621 gtacgctaag gctcataacg tttctttgtc tccattgttt gagggtggtc atcacttcgt
     1681 tttgtctgtt actactattg ctactcctca cgatggtact actttggtta acatggttga
     1741 tttcactgat agatttttcg atttgcaaaa ggctgttttg aaagctgctg ctgttgcttc
     1801 taacgttcct tacacttctc aagtttacga tttcaagttg gatcaatggg gtttgagaag
     1861 acaacctggt gaatcttttg atcattactt cgagagattg aaaagatccc ctgtttggac
     1921 ttctactgat actgctagat atgatttgtc tattccaggt gctgaaaagt tgaatcaatg
     1981 ggttcaggct tctcctaaca cttactattt gtctttctct actgaaagaa ctcacagagg
     2041 tgctttgact ggtaattact atccagagtt gggtatgaac gctttttctg ctgttgtttg
     2101 tgctcctttc ttgggttctt acagaaatga ggctttgggt attgatgata gatggttgga
     2161 gaacgatggt attgttaata ctgtttctat gaacggtcca aaaagaggtt cttctgatag
     2221 aattgttcct tatgatggta ctttgaagaa aggtgtttgg aatgatatgg gtacttgtaa
     2281 cgttgatcat ttggaagtta ttggtgttga tccaaaccct tcttttgata ttagagcttt
     2341 ctacttgaga ttggctgagc aattggcttc tttgagacca catcaccatc accatcacta
     2401 atcaagagga tgtcagaatg ccatttgcct gagagatgca ggcttcattt ttgatacttt
     2461 tttatttgta acctatatag tataggattt tttttgtcat tttgtttctt ctcgtacgag
     2521 cttgctcctg atcagcctat ctcgcagctg atgaatatct tgtggtaggg gtttgggaaa
     2581 atcattcgag tttgatgttt ttcttggtat ttcccactcc tcttcagagt acagaagatt
     2641 aagtgagacc ttcgtttgtg cggatccccc acacaccata gcttcaaaat gtttctactc
     2701 cttttttact cttccagatt ttctcggact ccgcgcatcg ccgtaccact tcaaaacacc
     2761 caagcacagc atactaaatt ttccctcttt cttcctctag ggtgtcgtta attacccgta
     2821 ctaaaggttt ggaaaagaaa aaagagaccg cctcgtttct ttttcttcgt cgaaaaaggc
     2881 aataaaaatt tttatcacgt ttctttttct tgaaattttt ttttttagtt tttttctctt
     2941 tcagtgacct ccattgatat ttaagttaat aaacggtctt caatttctca agtttcagtt
     3001 tcatttttct tgttctatta caactttttt tacttcttgt tcattagaaa gaaagcatag
     3061 caatctaatc taaggggcgg tgttgacaat taatcatcgg catagtatat cggcatagta
     3121 taatacgaca aggtgaggaa ctaaaccatg gccaagttga ccagtgccgt tccggtgctc
     3181 accgcgcgcg acgtcgccgg agcggtcgag ttctggaccg accggctcgg gttctcccgg
     3241 gacttcgtgg aggacgactt cgccggtgtg gtccgggacg acgtgaccct gttcatcagc
     3301 gcggtccagg accaggtggt gccggacaac accctggcct gggtgtgggt gcgcggcctg
     3361 gacgagctgt acgccgagtg gtcggaggtc gtgtccacga acttccggga cgcctccggg
     3421 ccggccatga ccgagatcgg cgagcagccg tgggggcggg agttcgccct gcgcgacccg
     3481 gccggcaact gcgtgcactt cgtggccgag gagcaggact gacacgtccg acggcggccc
     3541 acgggtccca ggcctcggag atccgtcccc cttttccttt gtcgatatca tgtaattagt
     3601 tatgtcacgc ttacattcac gccctccccc cacatccgct ctaaccgaaa aggaaggagt
     3661 tagacaacct gaagtctagg tccctattta tttttttata gttatgttag tattaagaac
     3721 gttatttata tttcaaattt ttcttttttt tctgtacaga cgcgtgtacg catgtaacat
     3781 tatactgaaa accttgcttg agaaggtttt gggacgctcg aaggctttaa tttgcaagct
     3841 ggagaccaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
     3901 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
     3961 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
     4021 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
     4081 ttcgggaagc gtggcgcttt ctcaatgctc acgctgtagg tatctcagtt cggtgtaggt
     4141 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
     4201 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
     4261 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
     4321 gtggtggcct aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa
     4381 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
     4441 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
     4501 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg
     4561 gattttggtc atgagatc
//
