LOCUS       Exported                4068 bp ds-DNA     circular SYN 17-AUG-2018
DEFINITION  Construct for expressing E2-Crimson with the Ost1 signal sequence 
            and the MUT1 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 4068)
  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..4068
                     /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 mutation (MUT1)"
                     /label=pro-alpha-factor(MUT1)
                     /translation="APVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNNGLL
                     FINTTIASIAAKEEGVSLDKREAEA"
     CDS             1217..1891
                     /codon_start=1
                     /product="far-red noncytotoxic tetrameric variant of DsRed 
                     fluorescent protein"
                     /label=E2-Crimson
                     /note="mammalian codon-optimized"
                     /translation="DSTENVIKPFMRFKVHMEGSVNGHEFEIEGVGEGKPYEGTQTAKL
                     QVTKGGPLPFAWDILSPQFFYGSKAYIKHPADIPDYLKQSFPEGFKWERVMNFEDGGVV
                     TVTQDSSLQDGTLIYHVKFIGVNFPSDGPVMQKKTLGWEPSTERNYPRDGVLKGENHMA
                     LKLKGGGHYLCEFKSIYMAKKPVKLPGYHYVDYKLDITSHNEDYTVVEQYERAEARHHL
                     FQ"
     terminator      1892..2138
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     promoter        2178..2564
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     promoter        2572..2619
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             2638..3012
                     /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      3078..3325
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     rep_origin      complement(3400..3988)
                     /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 ctttggataa
     1201 aagagaggct gaagctgata gcactgagaa cgtcatcaag cccttcatgc gcttcaaggt
     1261 gcacatggag ggctccgtga acggccacga gttcgagatc gagggcgtgg gcgagggcaa
     1321 gccctacgag ggcacccaga ccgccaagct gcaagtgacc aagggcggcc ccctgccctt
     1381 cgcctgggac atcctgtccc cccagttctt ctacggctcc aaggcgtaca tcaagcaccc
     1441 cgccgacatc cccgactacc tcaagcagtc cttccccgag ggcttcaagt gggagcgcgt
     1501 gatgaacttc gaggacggcg gcgtggtgac cgtgacccag gactcctccc tgcaggacgg
     1561 caccctcatc taccacgtga agttcatcgg cgtgaacttc ccctccgacg gccccgtaat
     1621 gcagaagaag actctgggct gggagccctc cactgagcgc aactaccccc gcgacggcgt
     1681 gctgaagggc gagaaccaca tggcgctgaa gctgaagggc ggcggccact acctgtgtga
     1741 gttcaagtcc atctacatgg ccaagaagcc cgtgaagctg cccggctacc actacgtgga
     1801 ctacaagctc gacatcacct cccacaacga ggactacacc gtggtggagc agtacgagcg
     1861 cgccgaggcc cgccaccacc tgttccagta gtcaagagga tgtcagaatg ccatttgcct
     1921 gagagatgca ggcttcattt ttgatacttt tttatttgta acctatatag tataggattt
     1981 tttttgtcat tttgtttctt ctcgtacgag cttgctcctg atcagcctat ctcgcagctg
     2041 atgaatatct tgtggtaggg gtttgggaaa atcattcgag tttgatgttt ttcttggtat
     2101 ttcccactcc tcttcagagt acagaagatt aagtgagacc ttcgtttgtg cggatccccc
     2161 acacaccata gcttcaaaat gtttctactc cttttttact cttccagatt ttctcggact
     2221 ccgcgcatcg ccgtaccact tcaaaacacc caagcacagc atactaaatt ttccctcttt
     2281 cttcctctag ggtgtcgtta attacccgta ctaaaggttt ggaaaagaaa aaagagaccg
     2341 cctcgtttct ttttcttcgt cgaaaaaggc aataaaaatt tttatcacgt ttctttttct
     2401 tgaaattttt ttttttagtt tttttctctt tcagtgacct ccattgatat ttaagttaat
     2461 aaacggtctt caatttctca agtttcagtt tcatttttct tgttctatta caactttttt
     2521 tacttcttgt tcattagaaa gaaagcatag caatctaatc taaggggcgg tgttgacaat
     2581 taatcatcgg catagtatat cggcatagta taatacgaca aggtgaggaa ctaaaccatg
     2641 gccaagttga ccagtgccgt tccggtgctc accgcgcgcg acgtcgccgg agcggtcgag
     2701 ttctggaccg accggctcgg gttctcccgg gacttcgtgg aggacgactt cgccggtgtg
     2761 gtccgggacg acgtgaccct gttcatcagc gcggtccagg accaggtggt gccggacaac
     2821 accctggcct gggtgtgggt gcgcggcctg gacgagctgt acgccgagtg gtcggaggtc
     2881 gtgtccacga acttccggga cgcctccggg ccggccatga ccgagatcgg cgagcagccg
     2941 tgggggcggg agttcgccct gcgcgacccg gccggcaact gcgtgcactt cgtggccgag
     3001 gagcaggact gacacgtccg acggcggccc acgggtccca ggcctcggag atccgtcccc
     3061 cttttccttt gtcgatatca tgtaattagt tatgtcacgc ttacattcac gccctccccc
     3121 cacatccgct ctaaccgaaa aggaaggagt tagacaacct gaagtctagg tccctattta
     3181 tttttttata gttatgttag tattaagaac gttatttata tttcaaattt ttcttttttt
     3241 tctgtacaga cgcgtgtacg catgtaacat tatactgaaa accttgcttg agaaggtttt
     3301 gggacgctcg aaggctttaa tttgcaagct ggagaccaac atgtgagcaa aaggccagca
     3361 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
     3421 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
     3481 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
     3541 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcaatgctc
     3601 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
     3661 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
     3721 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
     3781 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
     3841 gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
     3901 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
     3961 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
     4021 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagatc
//
