LOCUS       Exported                4059 bp ds-DNA     circular SYN 17-AUG-2018
DEFINITION  Construct for expressing E2-Crimson 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 4059)
  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..4059
                     /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..1882
                     /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      1883..2129
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     promoter        2169..2555
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     promoter        2563..2610
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             2629..3003
                     /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      3069..3316
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     rep_origin      complement(3391..3979)
                     /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 tgaagctgat agcactgaga acgtcatcaa gcccttcatg cgcttcaagg tgcacatgga
     1261 gggctccgtg aacggccacg agttcgagat cgagggcgtg ggcgagggca agccctacga
     1321 gggcacccag accgccaagc tgcaagtgac caagggcggc cccctgccct tcgcctggga
     1381 catcctgtcc ccccagttct tctacggctc caaggcgtac atcaagcacc ccgccgacat
     1441 ccccgactac ctcaagcagt ccttccccga gggcttcaag tgggagcgcg tgatgaactt
     1501 cgaggacggc ggcgtggtga ccgtgaccca ggactcctcc ctgcaggacg gcaccctcat
     1561 ctaccacgtg aagttcatcg gcgtgaactt cccctccgac ggccccgtaa tgcagaagaa
     1621 gactctgggc tgggagccct ccactgagcg caactacccc cgcgacggcg tgctgaaggg
     1681 cgagaaccac atggcgctga agctgaaggg cggcggccac tacctgtgtg agttcaagtc
     1741 catctacatg gccaagaagc ccgtgaagct gcccggctac cactacgtgg actacaagct
     1801 cgacatcacc tcccacaacg aggactacac cgtggtggag cagtacgagc gcgccgaggc
     1861 ccgccaccac ctgttccagt agtcaagagg atgtcagaat gccatttgcc tgagagatgc
     1921 aggcttcatt tttgatactt ttttatttgt aacctatata gtataggatt ttttttgtca
     1981 ttttgtttct tctcgtacga gcttgctcct gatcagccta tctcgcagct gatgaatatc
     2041 ttgtggtagg ggtttgggaa aatcattcga gtttgatgtt tttcttggta tttcccactc
     2101 ctcttcagag tacagaagat taagtgagac cttcgtttgt gcggatcccc cacacaccat
     2161 agcttcaaaa tgtttctact ccttttttac tcttccagat tttctcggac tccgcgcatc
     2221 gccgtaccac ttcaaaacac ccaagcacag catactaaat tttccctctt tcttcctcta
     2281 gggtgtcgtt aattacccgt actaaaggtt tggaaaagaa aaaagagacc gcctcgtttc
     2341 tttttcttcg tcgaaaaagg caataaaaat ttttatcacg tttctttttc ttgaaatttt
     2401 tttttttagt ttttttctct ttcagtgacc tccattgata tttaagttaa taaacggtct
     2461 tcaatttctc aagtttcagt ttcatttttc ttgttctatt acaacttttt ttacttcttg
     2521 ttcattagaa agaaagcata gcaatctaat ctaaggggcg gtgttgacaa ttaatcatcg
     2581 gcatagtata tcggcatagt ataatacgac aaggtgagga actaaaccat ggccaagttg
     2641 accagtgccg ttccggtgct caccgcgcgc gacgtcgccg gagcggtcga gttctggacc
     2701 gaccggctcg ggttctcccg ggacttcgtg gaggacgact tcgccggtgt ggtccgggac
     2761 gacgtgaccc tgttcatcag cgcggtccag gaccaggtgg tgccggacaa caccctggcc
     2821 tgggtgtggg tgcgcggcct ggacgagctg tacgccgagt ggtcggaggt cgtgtccacg
     2881 aacttccggg acgcctccgg gccggccatg accgagatcg gcgagcagcc gtgggggcgg
     2941 gagttcgccc tgcgcgaccc ggccggcaac tgcgtgcact tcgtggccga ggagcaggac
     3001 tgacacgtcc gacggcggcc cacgggtccc aggcctcgga gatccgtccc ccttttcctt
     3061 tgtcgatatc atgtaattag ttatgtcacg cttacattca cgccctcccc ccacatccgc
     3121 tctaaccgaa aaggaaggag ttagacaacc tgaagtctag gtccctattt atttttttat
     3181 agttatgtta gtattaagaa cgttatttat atttcaaatt tttctttttt ttctgtacag
     3241 acgcgtgtac gcatgtaaca ttatactgaa aaccttgctt gagaaggttt tgggacgctc
     3301 gaaggcttta atttgcaagc tggagaccaa catgtgagca aaaggccagc aaaaggccag
     3361 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     3421 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     3481 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     3541 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcaatgct cacgctgtag
     3601 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     3661 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     3721 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     3781 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt
     3841 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     3901 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     3961 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     4021 gaacgaaaac tcacgttaag ggattttggt catgagatc
//
