LOCUS       sequence-425636-        4254 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein expression..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4254)
  AUTHORS   Goulas T, Cuppari A, Garcia-Castellanos R, Snipas S, Glockshuber R, 
            Arolas JL, Gomis-Ruth FX
  TITLE     The pCri System: A Vector Collection for Recombinant Protein 
            Expression and Purification.
  JOURNAL   PLoS One. 2014 Nov 11;9(11):e112643. doi: 
            10.1371/journal.pone.0112643. eCollection 2014.
  PUBMED    25386923
REFERENCE   2  (bases 1 to 4254)
  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: "PLoS One.";
            date: "2014-11-11"; volume: "9(11):e112643. doi"; pages: " 
            10.1371/journal.pone.0112643. eCollection 2014"
COMMENT     Sequence Label: pCri-16a
FEATURES             Location/Qualifiers
     source          1..4254
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        40..978
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 promoter
                     /note="inducible promoter, regulated by methanol"
     CDS             join(979..1035,1036..1233,1234..1245)
                     /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="MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLE
                     GDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLEKREAMA"
     CDS             join(1249..1251,1252..1965)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1291..1312)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1552..1571)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1899..1920
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2018..2035
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      2115..2361
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     promoter        2376..2789
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     promoter        2796..2843
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             2862..3236
                     /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      3302..3549
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(3318..3336)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     rep_origin      complement(3624..4212)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3704..3723)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 aacgaaaact cacgttaagg gattttggtc atgagatcag atctaacatc caaagacgaa
       61 aggttgaatg aaaccttttt gccatccgac atccacaggt ccattctcac acataagtgc
      121 caaacgcaac aggaggggat acactagcag cagaccgttg caaacgcagg acctccactc
      181 ctcttctcct caacacccac ttttgccatc gaaaaaccag cccagttatt gggcttgatt
      241 ggagctcgct cattccaatt ccttctatta ggctactaac accatgactt tattagcctg
      301 tctatcctgg cccccctggc gaggttcatg tttgtttatt tccgaatgca acaagctccg
      361 cattacaccc gaacatcact ccagatgagg gctttctgag tgtggggtca aatagtttca
      421 tgttccccaa atggcccaaa actgacagtt taaacgctgt cttggaacct aatatgacaa
      481 aagcgtgatc tcatccaaga tgaactaagt ttggttcgtt gaaatgctaa cggccagttg
      541 gtcaaaaaga aacttccaaa agtcggcata ccgtttgtct tgtttggtat tgattgacga
      601 atgctcaaaa ataatctcat taatgcttag cgcagtctct ctatcgcttc tgaaccccgg
      661 tgcacctgtg ccgaaacgca aatggggaaa cacccgcttt ttggatgatt atgcattgtc
      721 tccacattgt atgcttccaa gattctggtg ggaatactgc tgatagccta acgttcatga
      781 tcaaaattta actgttctaa cccctacttg acagcaatat ataaacagaa ggaagctgcc
      841 ctgtcttaaa cctttttttt tatcatcatt attagcttac tttcataatt gcgactggtt
      901 ccaattgaca agcttttgat tttaacgact tttaacgaca acttgagaag atcaaaaaac
      961 aactaattat tcgaaacgat gagatttcct tcaattttta ctgctgtttt attcgcagca
     1021 tcctccgcat tagctgctcc agtcaacact acaacagaag atgaaacggc acaaattccg
     1081 gctgaagctg tcatcggtta ctcagattta gaaggggatt tcgatgttgc tgttttgcca
     1141 ttttccaaca gcacaaataa cgggttattg tttataaata ctactattgc cagcattgct
     1201 gctaaagaag aaggggtatc tctagagaaa agagaggcca tggccaaagt gagcaagggc
     1261 gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc
     1321 cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa gctgaccctg
     1381 aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccttc
     1441 ggctacggcc tgcagtgctt cgcccgctac cccgaccaca tgaagcagca cgacttcttc
     1501 aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa ggacgacggc
     1561 aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag
     1621 ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac
     1681 tacaacagcc acaacgtcta tatcatggcc gacaagcaga agaacggcat caaggtgaac
     1741 ttcaagatcc gccacaacat cgaggacggc agcgtgcagc tcgccgacca ctaccagcag
     1801 aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct gagcacccag
     1861 tccgccctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct ggagttcgtg
     1921 accgccgccg ggatcactct cggcatggac gagctgtaca agtaaggtac cggatccgaa
     1981 ttcgagctcc gtcgacaagc ttgcggccgc actcgagcat catcatcatc atcattgagt
     2041 ttgtagcctt agacatgact gttcctcagt tcaagttggg cacttacgag aagaccggtc
     2101 ttgctagatt ctaatcaaga ggatgtcaga atgccatttg cctgagagat gcaggcttca
     2161 tttttgatac ttttttattt gtaacctata tagtatagga ttttttttgt cattttgttt
     2221 cttctcgtac gagcttgctc ctgatcagcc tatctcgcag ctgatgaata tcttgtggta
     2281 ggggtttggg aaaatcattc gagtttgatg tttttcttgg tatttcccac tcctcttcag
     2341 agtacagaag attaagtgag accttcgttt gtgcggatcc cccacacacc atagcttcaa
     2401 aatgtttcta ctcctttttt actcttccag attttctcgg actccgcgca tcgccgtacc
     2461 acttcaaaac acccaagcac agcatactaa atttcccctc tttcttcctc tagggtgtcg
     2521 ttaattaccc gtactaaagg tttggaaaag aaaaaagaga ccgcctcgtt tctttttctt
     2581 cgtcgaaaaa ggcaataaaa atttttatca cgtttctttt tcttgaaaat tttttttttt
     2641 gatttttttc tctttcgatg acctcccatt gatatttaag ttaataaacg gtcttcaatt
     2701 tctcaagttt cagtttcatt tttcttgttc tattacaact ttttttactt cttgctcatt
     2761 agaaagaaag catagcaatc taatctaagg gcggtgttga caattaatca tcggcatagt
     2821 atatcggcat agtataatac gacaaggtga ggaactaaaa catggccaag ttgaccagtg
     2881 ccgttccggt gctcaccgcg cgcgacgtcg ccggagcggt cgagttctgg accgaccggc
     2941 tcgggttctc ccgggacttc gtggaggacg acttcgccgg tgtggtccgg gacgacgtga
     3001 ccctgttcat cagcgcggtc caggaccagg tggtgccgga caacaccctg gcctgggtgt
     3061 gggtgcgcgg cctggacgag ctgtacgccg agtggtcgga ggtcgtgtcc acgaacttcc
     3121 gggacgcctc cgggccggcc atgaccgaga tcggcgagca gccgtggggg cgggagttcg
     3181 ccctgcgcga cccggccggc aactgcgtgc acttcgtggc cgaggagcag gactgacacg
     3241 tccgacgcgg cccgacgggt ccgaggcctc ggagatccgt cccccttttc ctttgtcgat
     3301 atcatgtaat tagttatgtc acgcttacat tcacgccctc cccccacatc cgctctaacc
     3361 gaaaaggaag gagttagaca acctgaagtc taggtcccta tttatttttt tatagttatg
     3421 ttagtattaa gaacgttatt tatatttcaa atttttcttt tttttctgta cagacgcgtg
     3481 tacgcatgta acattatact gaaaaccttg cttgagaagg ttttgggacg ctcgaaggct
     3541 ttaatttgca agctggagac caacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     3601 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     3661 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     3721 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     3781 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     3841 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     3901 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     3961 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     4021 acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc
     4081 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     4141 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     4201 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtgg
//
