LOCUS       sequence-231075-        4069 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Test intracellular localisation of E2-Crimson and study the 
            secretion efficiency.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4069)
  AUTHORS   Barrero JJ, Casler JC, Valero F, Ferrer P, Glick BS
  TITLE     An improved secretion signal enhances the secretion of model 
            proteins from Pichia pastoris.
  JOURNAL   Microb Cell Fact. 2018 Oct 12;17(1):161. doi: 
            10.1186/s12934-018-1009-5.
  PUBMED    30314480
REFERENCE   2  (bases 1 to 4069)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Microb Cell
            Fact."; date: "2018-10-12"; volume: "17(1):161. doi"; pages: " 
            10.1186/s12934-018-1009-5"
COMMENT     Sequence Label: pPICZ-pre-Ost1-alphaf(I)-E2-Crimson
FEATURES             Location/Qualifiers
     source          1..4069
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        2..940
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 promoter
                     /note="inducible promoter, regulated by methanol"
     CDS             1217..1891
                     /codon_start=1
                     /product="far-red noncytotoxic tetrameric variant of DsRed 
                     fluorescent protein (Strack et al., 2009)"
                     /label=E2-Crimson
                     /note="mammalian codon-optimized"
                     /translation="DSTENVIKPFMRFKVHMEGSVNGHEFEIEGVGEGKPYEGTQTAKL
                     QVTKGGPLPFAWDILSPQFFYGSKAYIKHPADIPDYLKQSFPEGFKWERVMNFEDGGVV
                     TVTQDSSLQDGTLIYHVKFIGVNFPSDGPVMQKKTLGWEPSTERNYPRDGVLKGENHMA
                     LKLKGGGHYLCEFKSIYMAKKPVKLPGYHYVDYKLDITSHNEDYTVVEQYERAEARHHL
                     FQ"
     primer_bind     1612..1631
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     terminator      1892..2138
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     promoter        2153..2566
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     promoter        2573..2620
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             2639..3013
                     /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      3079..3326
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(3095..3113)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     rep_origin      complement(3401..3989)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3481..3500)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
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 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 tcccctcttt
     2281 cttcctctag ggtgtcgtta attacccgta ctaaaggttt ggaaaagaaa aaagagaccg
     2341 cctcgtttct ttttcttcgt cgaaaaaggc aataaaaatt tttatcacgt ttctttttct
     2401 tgaaaatttt tttttttgat ttttttctct ttcgatgacc tcccattgat atttaagtta
     2461 ataaacggtc ttcaatttct caagtttcag tttcattttt cttgttctat tacaactttt
     2521 tttacttctt gctcattaga aagaaagcat agcaatctaa tctaagggcg gtgttgacaa
     2581 ttaatcatcg gcatagtata tcggcatagt ataatacgac aaggtgagga actaaaccat
     2641 ggccaagttg accagtgccg ttccggtgct caccgcgcgc gacgtcgccg gagcggtcga
     2701 gttctggacc gaccggctcg ggttctcccg ggacttcgtg gaggacgact tcgccggtgt
     2761 ggtccgggac gacgtgaccc tgttcatcag cgcggtccag gaccaggtgg tgccggacaa
     2821 caccctggcc tgggtgtggg tgcgcggcct ggacgagctg tacgccgagt ggtcggaggt
     2881 cgtgtccacg aacttccggg acgcctccgg gccggccatg accgagatcg gcgagcagcc
     2941 gtgggggcgg gagttcgccc tgcgcgaccc ggccggcaac tgcgtgcact tcgtggccga
     3001 ggagcaggac tgacacgtcc gacgcggccc gacgggtccg aggcctcgga gatccgtccc
     3061 ccttttcctt tgtcgatatc atgtaattag ttatgtcacg cttacattca cgccctcccc
     3121 ccacatccgc tctaaccgaa aaggaaggag ttagacaacc tgaagtctag gtccctattt
     3181 atttttttat agttatgtta gtattaagaa cgttatttat atttcaaatt tttctttttt
     3241 ttctgtacag acgcgtgtac gcatgtaaca ttatactgaa aaccttgctt gagaaggttt
     3301 tgggacgctc gaaggcttta atttgcaagc tggagaccaa catgtgagca aaaggccagc
     3361 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     3421 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     3481 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     3541 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     3601 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     3661 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     3721 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     3781 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     3841 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     3901 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     3961 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     4021 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatc
//
