LOCUS       sequence-231065-        4060 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 4060)
  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 4060)
  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-pro-alphaf-E2-Crimson
FEATURES             Location/Qualifiers
     source          1..4060
                     /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             join(941..997,998..1195,1196..1207)
                     /codon_start=1
                     /gene="MF-alpha-1"
                     /product="N-terminal secretion signal from S. cerevisiae 
                     alpha-factor"
                     /label=alpha-factor secretion signal
                     /note="Cleavage by the Kex2 protease occurs after the 
                     dibasic KR sequence. The EA dipeptides are then removed by 
                     dipeptidyl aminopeptidase A."
                     /translation="MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYLDLE
                     GDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLDKREAEA"
     CDS             1208..1882
                     /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     1603..1622
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     terminator      1883..2129
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     promoter        2144..2557
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     promoter        2564..2611
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             2630..3004
                     /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      3070..3317
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(3086..3104)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     rep_origin      complement(3392..3980)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3472..3491)
                     /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 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 atgtcaaaat 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 ttcccctctt tcttcctcta
     2281 gggtgtcgtt aattacccgt actaaaggtt tggaaaagaa aaaagagacc gcctcgtttc
     2341 tttttcttcg tcgaaaaagg caataaaaat ttttatcacg tttctttttc ttgaaaattt
     2401 ttttttttga tttttttctc tttcgatgac ctcccattga tatttaagtt aataaacggt
     2461 cttcaatttc tcaagtttca gtttcatttt tcttgttcta ttacaacttt ttttacttct
     2521 tgctcattag aaagaaagca tagcaatcta atctaagggc ggtgttgaca attaatcatc
     2581 ggcatagtat atcggcatag tataatacga caaggtgagg aactaaacca tggccaagtt
     2641 gaccagtgcc gttccggtgc tcaccgcgcg cgacgtcgcc ggagcggtcg agttctggac
     2701 cgaccggctc gggttctccc gggacttcgt ggaggacgac ttcgccggtg tggtccggga
     2761 cgacgtgacc ctgttcatca gcgcggtcca ggaccaggtg gtgccggaca acaccctggc
     2821 ctgggtgtgg gtgcgcggcc tggacgagct gtacgccgag tggtcggagg tcgtgtccac
     2881 gaacttccgg gacgcctccg ggccggccat gaccgagatc ggcgagcagc cgtgggggcg
     2941 ggagttcgcc ctgcgcgacc cggccggcaa ctgcgtgcac ttcgtggccg aggagcagga
     3001 ctgacacgtc cgacgcggcc cgacgggtcc gaggcctcgg agatccgtcc cccttttcct
     3061 ttgtcgatat catgtaatta gttatgtcac gcttacattc acgccctccc cccacatccg
     3121 ctctaaccga aaaggaagga gttagacaac ctgaagtcta ggtccctatt tattttttta
     3181 tagttatgtt agtattaaga acgttattta tatttcaaat ttttcttttt tttctgtaca
     3241 gacgcgtgta cgcatgtaac attatactga aaaccttgct tgagaaggtt ttgggacgct
     3301 cgaaggcttt aatttgcaag ctggagacca acatgtgagc aaaaggccag caaaaggcca
     3361 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     3421 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     3481 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     3541 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     3601 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     3661 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     3721 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     3781 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat
     3841 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     3901 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     3961 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     4021 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatc
//
